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Structural insights into emergent signaling modes of G protein–coupled receptors

  • Ieva Sutkeviciute
    Correspondence
    For correspondence: Ieva Sutkeviciute, ; Jean-Pierre Vilardaga,
    Affiliations
    Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
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  • Jean-Pierre Vilardaga
    Correspondence
    For correspondence: Ieva Sutkeviciute, ; Jean-Pierre Vilardaga,
    Affiliations
    Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
    Search for articles by this author
Open AccessPublished:June 22, 2020DOI:https://doi.org/10.1074/jbc.REV120.009348
      G protein–coupled receptors (GPCRs) represent the largest family of cell membrane proteins, with >800 GPCRs in humans alone, and recognize highly diverse ligands, ranging from photons to large protein molecules. Very important to human medicine, GPCRs are targeted by about 35% of prescription drugs. GPCRs are characterized by a seven-transmembrane α-helical structure, transmitting extracellular signals into cells to regulate major physiological processes via heterotrimeric G proteins and β-arrestins. Initially viewed as receptors whose signaling via G proteins is delimited to the plasma membrane, it is now recognized that GPCRs signal also at various intracellular locations, and the mechanisms and (patho)physiological relevance of such signaling modes are actively investigated. The propensity of GPCRs to adopt different signaling modes is largely encoded in the structural plasticity of the receptors themselves and of their signaling complexes. Here, we review emerging modes of GPCR signaling via endosomal membranes and the physiological implications of such signaling modes. We further summarize recent structural insights into mechanisms of GPCR activation and signaling. We particularly emphasize the structural mechanisms governing the continued GPCR signaling from endosomes and the structural aspects of the GPCR resensitization mechanism and discuss the recently uncovered and important roles of lipids in these processes.
      Cells sense their environment through the plasma membrane–embedded receptors, which capture external stimuli and convert them to intracellular signaling cascades leading to appropriate cellular responses (e.g. proliferation, differentiation, death) and subsequent physiological outcomes. G protein–coupled receptors (GPCRs) comprise the largest family of cell membrane receptors and recognize a broad spectrum of ligands ranging from photons to large protein molecules. GPCRs regulate many physiological processes in body systems, such as the skeletal, muscular, nervous, endocrine, urinary, and digestive systems among others. Due to their key role in human physiology, malfunctions of GPCRs cause severe diseases, and GPCRs are thus attractive pharmaceutical targets. They constitute the largest family of proteins targeted by currently approved drugs; ∼700 currently approved marketed drugs (or about 35%) target GPCRs (
      • Sriram K.
      • Insel P.A.
      G protein-coupled receptors as targets for approved drugs: how many targets and how many drugs?.
      ), and these numbers are likely to further increase because of intensive research efforts in GPCR druggability (
      • Hauser A.S.
      • Attwood M.M.
      • Rask-Andersen M.
      • Schiöth H.B.
      • Gloriam D.E.
      Trends in GPCR drug discovery: new agents, targets and indications.
      ). Signal transduction via a GPCR starts when ligand binding to the receptor triggers or stabilizes an active receptor conformation, permitting coupling to heterotrimeric guanine nucleotide–binding proteins (G proteins), which are composed of three distinct α, β, and γ subunits. There are five subtypes of Gβ subunits and 12 subtypes of Gγ subunits that form constitutive Gβγ heterodimers. Based on sequence homology of the Gα subunits, the G proteins are classified into four subfamilies: Gs (Gαs and Gαolf), Gi (Gαi1–3, Gαo, GαZ, and Gαt), Gq (Gαq, Gα11, and Gα14–15), and G12 (Gα12 and Gα13) (
      • Cabrera-Vera T.M.
      • Vanhauwe J.
      • Thomas T.O.
      • Medkova M.
      • Preininger A.
      • Mazzoni M.R.
      • Hamm H.E.
      Insights into G protein structure, function, and regulation.
      ), each of which affect distinct downstream effectors (Fig. 1). Binding with the active state of the receptor induces a rapid conformational change in G proteins catalyzing the exchange of GDP for GTP on the Gα subunit, resulting in the dissociation or rearrangement between the GTP-bound Gα and Gβγ subunits (
      • Neer E.J.
      • Clapham D.E.
      Roles of G protein subunits in transmembrane signalling.
      ,
      • Janetopoulos C.
      • Jin T.
      • Devreotes P.
      Receptor-mediated activation of heterotrimeric G-proteins in living cells.
      ,
      • Bünemann M.
      • Frank M.
      • Lohse M.J.
      Gi protein activation in intact cells involves subunit rearrangement rather than dissociation.
      ). Both Gα-GTP and Gβγ independently regulate the activity of diverse effectors (Fig. 1), such as transmembrane adenylate cyclases via Gαs to convert ATP into cAMP and then initiate protein kinase A (PKA)- and exchange protein directly activated by cAMP (Epac; a cAMP-regulated guanine nucleotide exchange protein for small GTPase Rap1)-dependent signaling pathways, phospholipase Cβ (PLCβ) via Gαq to hydrolyze phosphatidylinositol (4,5)-bisphosphate (PIP2) into diacylglycerol (DAG), and inositol (1,4,5)-trisphosphate (IP3) to release stored Ca2+ and activate protein kinase C (PKC), and/or channels such as G protein–activated inwardly rectifying K+ (GIRK) channels via Gβγ, resulting in cell membrane hyperpolarization. Once activated, the duration of G protein signaling depends on the intrinsic GTPase activity of Gα, which hydrolyzes GTP to GDP and leads to the recovery of the inactive form of the heterotrimer Gαβγ, prompting a new activation cycle. The time constant of the intrinsic GTPase reaction is slow (10–60 s) and accelerated by regulators of G protein signaling acting as GTPase-activating proteins (
      • De Vries L.
      • Zheng B.
      • Fischer T.
      • Elenko E.
      • Farquhar M.G.
      The regulator of G protein signaling family.
      ). Initially, GPCRs were viewed as signaling through G proteins exclusively at the plasma membrane. In this long-standing view, the agonist-induced receptor activation is rapidly terminated through receptor phosphorylation by GPCR kinases (GRKs), permitting recruitment of cytosolic β-arrestins and subsequent receptor internalization to endosomes for either redistribution of receptors to lysosomes for degradation or recycling to the cell surface, thus enabling receptor resensitization. During the last decade, this paradigm has been shifted by findings revealing an alternative mode of GPCR signaling and function via G proteins in various intracellular compartments, including early endosomes (Fig. 2A), Golgi, mitochondria, endoplasmic reticulum (ER), and nucleus (reviewed in Refs.
      • Jong Y.I.
      • Harmon S.K.
      • O'Malley K.L.
      GPCR signalling from within the cell.
      ,
      • Lobingier B.T.
      • von Zastrow M.
      When trafficking and signaling mix: how subcellular location shapes G protein-coupled receptor activation of heterotrimeric G proteins.
      ,
      • Calebiro D.
      • Godbole A.
      Internalization of G-protein-coupled receptors: implication in receptor function, physiology and diseases.
      ,
      • Eichel K.
      • von Zastrow M.
      Subcellular organization of GPCR signaling.
      ,
      • Plouffe B.
      • Thomsen A.R.B.
      • Irannejad R.
      Emerging role of compartmentalized G protein-coupled receptor signaling in the cardiovascular field.
      ,
      • Retamal J.S.
      • Ramirez-Garcia P.D.
      • Shenoy P.A.
      • Poole D.P.
      • Veldhuis N.A.
      Internalized GPCRs as potential therapeutic targets for the management of pain.
      ,
      • Hanyaloglu A.C.
      Advances in membrane trafficking and endosomal signaling of G protein-coupled receptors.
      ,
      • Jong Y.I.
      • Harmon S.K.
      • O'Malley K.L.
      Intracellular GPCRs play key roles in synaptic plasticity.
      ), whereas β-arrestins were discovered to have multiple functions that extend far beyond GPCR desensitization and internalization (reviewed in Refs.
      • Gurevich V.V.
      • Gurevich E.V.
      Plethora of functions packed into 45 kDa arrestins: biological implications and possible therapeutic strategies.
      and
      • Laporte S.A.
      • Scott M.G.H.
      β-Arrestins: multitask scaffolds orchestrating the where and when in cell signalling.
      ). Here we outline current knowledge of the structural basis for GPCR signaling, including the role of the lipids, with special emphasis on endosomal signaling determinants. We further discuss recent mechanistic findings on GPCR signaling from early endosomes and their (patho)physiological implications.
      Figure thumbnail gr1
      Figure 1Basic paradigm of GPCR signaling. Following ligand agonist binding, the receptor adopts an active-state conformation that couples to one subfamily or multiple subfamilies of heterotrimeric G proteins (Gαβγ). This interaction catalyzes the exchange of GDP for GTP on the Gα subunit. The GTP-bound Gα and Gβγ subunits dissociate and activate (→) or inhibit (—|) diverse effectors, which in turn regulate intracellular levels of second messengers, as well as activities of GIRK channels and phosphoinositide 3-kinases (PI3K) and recruitment of GRKs.
      Figure thumbnail gr2
      Figure 2General principle of GPCR signaling via cAMP. A, in the classical model, production of cAMP (1st pool) only takes place at the cell membrane after activation of Gs by the agonist-bound receptors (step 1). This cAMP response is usually short-lived due to the action of phosphodiesterases and rapid receptor desensitization initiated by recruitment of GRKs and receptor phosphorylation (step 2), followed by recruitment of β-arrestin (βarr; step 3) driving receptor endocytosis and eventually engaging β-arrestin-dependent mitogen-activated protein kinase signaling cascades (step 4). In the more recent model, agonists—usually peptide hormones—that interact tightly with receptor in a conformationally dependent rather than G-protein–dependent manner, also induce sustained cAMP that originated from ligand–GPCR complexes in endosomes (2nd pool) (step 4). Endosomal cAMP production continues until endosomal acidification induces the release of the agonist from the receptor (step 5) and receptor dephosphorylation (step 6), allowing receptor degradation (step 7), receptor transfer to the Golgi apparatus (step 8), and/or the receptor recycling (step 9). B, examples of cAMP time-course profiles mediated by PTH in live cells expressing PTHR and showing plasma membrane (PM; light orange) and endosomal (Endosomes; light blue) response phases for control (black), when the receptor internalization (step 4) is blocked by either dynasore or a dominant negative mutant of dynamin (purple), or when the endosomal acidification (step 5) is blocked by bafilomycin (blue). Data represent the mean ± S.E.M.

      Brief history of the concept of endosomal GPCR signaling via G proteins

      The concept that GPCRs are capable of sustaining G protein activity during and after their internalization into endosomes stems from studies comparing how the class B parathyroid hormone (PTH) type 1 receptor (PTH1R) transmits signals into cells in response to its two agonists, PTH and PTH-related peptide (PTHrP). Back in the year 2005, it was unclear why these two peptide hormones or their bioactive and structurally similar synthetic N-terminal analogs, PTH1-34 and PTHrP1-36, bind and activate the same receptor with identical pharmacological properties but display distinct responses in clinical testing: PTH1-34 stimulates more prolonged increases in serum levels of active vitamin D (i.e. 1,25-dihydroxyvitamin D3), calcium, and bone resorption markers than does PTHrP1-36 (
      • Horwitz M.J.
      • Tedesco M.B.
      • Sereika S.M.
      • Hollis B.W.
      • Garcia-Ocaña A.
      • Stewart A.F.
      Direct comparison of sustained infusion of human parathyroid hormone-related protein-(1-36) [hPTHrP-(1-36)] versus hPTH-(1-34) on serum calcium, plasma 1,25-dihydroxyvitamin D concentrations, and fractional calcium excretion in healthy human volunteers.
      ). Initial studies show that PTH1-34 differentiates itself from PTHrP1-36 by inducing prolonged cAMP responses in cultured cells expressing either recombinant or endogenous PTH1R, which are mediated at the receptor level and not by extended bioavailability of ligands (
      • Horwitz M.J.
      • Tedesco M.B.
      • Sereika S.M.
      • Syed M.A.
      • Garcia-Ocaña A.
      • Bisello A.
      • Hollis B.W.
      • Rosen C.J.
      • Wysolmerski J.J.
      • Dann P.
      • Gundberg C.
      • Stewart A.F.
      Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)2 vitamin D.
      ). Biophysical and microscopy studies in live cells showed that during the time frame of cAMP production, PTHrP1-36 action is restricted to the cell membrane, whereas the PTH1-34-bound PTH1R complex internalizes and redistributes into early endosomes where the active state of Gαs, adenylate cyclases, and cAMP production can be detected (
      • Ferrandon S.
      • Feinstein T.N.
      • Castro M.
      • Wang B.
      • Bouley R.
      • Potts J.T.
      • Gardella T.J.
      • Vilardaga J.P.
      Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.
      ,
      • Jean-Alphonse F.G.
      • Wehbi V.L.
      • Chen J.
      • Noda M.
      • Taboas J.M.
      • Xiao K.
      • Vilardaga J.P.
      β2-Adrenergic receptor control of endosomal PTH receptor signaling via Gβγ.
      ). These observations coupled to the finding that blocking PTH1R internalization prevents the sustained cAMP response mediated by PTH raised the new paradigm that PTH1R can generate cAMP from intracellular membranes and that early endosomes serve as a platform for PTH1R-mediated sustained cAMP production (Fig. 2B). The marked differences between PTH and PTHrP-mediated cAMP signaling provided a mechanistic understanding of differential biological responses induced by these two hormones.
      The same conclusion was concomitantly and independently reached by signaling studies of the thyroid-stimulating hormone receptor (TSHR), a Gs-coupled receptor regulating thyroid functions (
      • Calebiro D.
      • Nikolaev V.O.
      • Gagliani M.C.
      • de Filippis T.
      • Dees C.
      • Tacchetti C.
      • Persani L.
      • Lohse M.J.
      Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.
      ), and the sphingolipid S1P receptor (S1P1R) that internalizes and traffics in the trans-Golgi network to sustain Gi-dependent signaling mediated by FTY720, a S1P1R agonist (
      • Mullershausen F.
      • Zecri F.
      • Cetin C.
      • Billich A.
      • Guerini D.
      • Seuwen K.
      Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors.
      ). Reported in 2009, these findings led to the revision of the classical paradigm proposing that GPCRs are only active and signal via G proteins at the plasma membrane and internalize to be degraded or recycled, and they laid the foundation for a new mode of GPCR signaling via G proteins that is maintained after internalization and relocation of ligand–GPCR complexes to intracellular membranes. A growing number of studies confirmed this paradigm for additional receptor activating the Gs or Gi pathways (Table 1) and including class A (β-adrenergic receptors, vasopressin type 2 receptor) and class B (glucagon-like peptide 1 receptor, calcitonin receptor-like receptor, and pituitary adenylate cyclase–activating polypeptide type 1 receptor) GPCRs among others. In addition, several Gq-coupled GPCRs were reported to display extended Gq/PKC signaling after internalization (
      • Jensen D.D.
      • Lieu T.
      • Halls M.L.
      • Veldhuis N.A.
      • Imlach W.L.
      • Mai Q.N.
      • Poole D.P.
      • Quach T.
      • Aurelio L.
      • Conner J.
      • Herenbrink C.K.
      • Barlow N.
      • Simpson J.S.
      • Scanlon M.J.
      • Graham B.
      • et al.
      Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.
      ,
      • Yarwood R.E.
      • Imlach W.L.
      • Lieu T.
      • Veldhuis N.A.
      • Jensen D.D.
      • Klein Herenbrink C.
      • Aurelio L.
      • Cai Z.
      • Christie M.J.
      • Poole D.P.
      • Porter C.J.H.
      • McLean P.
      • Hicks G.A.
      • Geppetti P.
      • Halls M.L.
      • et al.
      Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.
      ,
      • Jimenez-Vargas N.N.
      • Pattison L.A.
      • Zhao P.
      • Lieu T.
      • Latorre R.
      • Jensen D.D.
      • Castro J.
      • Aurelio L.
      • Le G.T.
      • Flynn B.
      • Herenbrink C.K.
      • Yeatman H.R.
      • Edgington-Mitchell L.
      • Porter C.J.H.
      • Halls M.L.
      • et al.
      Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome.
      ,
      • Gorvin C.M.
      • Rogers A.
      • Hastoy B.
      • Tarasov A.I.
      • Frost M.
      • Sposini S.
      • Inoue A.
      • Whyte M.P.
      • Rorsman P.
      • Hanyaloglu A.C.
      • Breitwieser G.E.
      • Thakker R.V.
      AP2σ mutations impair calcium-sensing receptor trafficking and signaling, and show an endosomal pathway to spatially direct G-protein selectivity.
      ); however, the endosomes contain virtually no PIP2 that is necessary for production of DAG and IP3 to activate PKC, and there is no clear evidence of active Gq in endosomes so far, prompting the necessity to confirm these observations by future studies.
      Table 1GPCRs that display endosomal signaling via heterotrimeric G proteins
      GPCRClassEndosomal signaling pathwayPhysiological outcomeReference
      PTH1RBGsCa2+ homeostasis (calcemic effects)
      • Ferrandon S.
      • Feinstein T.N.
      • Castro M.
      • Wang B.
      • Bouley R.
      • Potts J.T.
      • Gardella T.J.
      • Vilardaga J.P.
      Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.
      ,
      • White A.D.
      • Fang F.
      • Jean-Alphonse F.G.
      • Clark L.J.
      • An H.J.
      • Liu H.
      • Zhao Y.
      • Reynolds S.L.
      • Lee S.
      • Xiao K.
      • Sutkeviciute I.
      • Vilardaga J.P.
      Ca2+ allostery in PTH-receptor signaling.
      TSHRAGsThyroid function
      • Calebiro D.
      • Nikolaev V.O.
      • Gagliani M.C.
      • de Filippis T.
      • Dees C.
      • Tacchetti C.
      • Persani L.
      • Lohse M.J.
      Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.
      S1P1RAGiIncreased chemokinetic migration of primary human umbilical vein endothelial cells
      • Mullershausen F.
      • Zecri F.
      • Cetin C.
      • Billich A.
      • Guerini D.
      • Seuwen K.
      Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors.
      D1RAGsND
      • Kotowski S.J.
      • Hopf F.W.
      • Seif T.
      • Bonci A.
      • von Zastrow M.
      Endocytosis promotes rapid dopaminergic signaling.
      PAC1RBGsCardiac neuron excitability, chronic pain, and anxiety-like responses
      • Merriam L.A.
      • Baran C.N.
      • Girard B.M.
      • Hardwick J.C.
      • May V.
      • Parsons R.L.
      Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.
      ,
      • May V.
      • Parsons R.L.
      G protein-coupled receptor endosomal signaling and regulation of neuronal excitability and stress responses: signaling options and lessons from the PAC1 receptor.
      GLP1RBGsPromotion of glucose-stimulated insulin secretion
      • Kuna R.S.
      • Girada S.B.
      • Asalla S.
      • Vallentyne J.
      • Maddika S.
      • Patterson J.T.
      • Smiley D.L.
      • DiMarchi R.D.
      • Mitra P.
      Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic β-cells.
      V2RAGsPotentially strong antidiuretic and antinatriuretic effects
      • Feinstein T.N.
      • Yui N.
      • Webber M.J.
      • Wehbi V.L.
      • Stevenson H.P.
      • King Jr., J.D.
      • Hallows K.R.
      • Brown D.
      • Bouley R.
      • Vilardaga J.P.
      Noncanonical control of vasopressin receptor type 2 signaling by retromer and arrestin.
      CTRBGs (in response to salmon calcitonin)ND
      • Andreassen K.V.
      • Hjuler S.T.
      • Furness S.G.
      • Sexton P.M.
      • Christopoulos A.
      • Nosjean O.
      • Karsdal M.A.
      • Henriksen K.
      Prolonged calcitonin receptor signaling by salmon, but not human calcitonin, reveals ligand bias.
      β2ARAShort GsND
      • Tsvetanova N.G.
      • von Zastrow M.
      Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis.
      LHRAGsFinal maturation of a fertilizable egg in females
      • Lyga S.
      • Volpe S.
      • Werthmann R.C.
      • Götz K.
      • Sungkaworn T.
      • Lohse M.J.
      • Calebiro D.
      Persistent cAMP signaling by internalized LH receptors in ovarian follicles.
      NK1RAGqNociception
      • Jensen D.D.
      • Lieu T.
      • Halls M.L.
      • Veldhuis N.A.
      • Imlach W.L.
      • Mai Q.N.
      • Poole D.P.
      • Quach T.
      • Aurelio L.
      • Conner J.
      • Herenbrink C.K.
      • Barlow N.
      • Simpson J.S.
      • Scanlon M.J.
      • Graham B.
      • et al.
      Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.
      CLRBGs and GqNociception
      • Yarwood R.E.
      • Imlach W.L.
      • Lieu T.
      • Veldhuis N.A.
      • Jensen D.D.
      • Klein Herenbrink C.
      • Aurelio L.
      • Cai Z.
      • Christie M.J.
      • Poole D.P.
      • Porter C.J.H.
      • McLean P.
      • Hicks G.A.
      • Geppetti P.
      • Halls M.L.
      • et al.
      Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.
      PAR2AGqColonic nociception
      • Jimenez-Vargas N.N.
      • Pattison L.A.
      • Zhao P.
      • Lieu T.
      • Latorre R.
      • Jensen D.D.
      • Castro J.
      • Aurelio L.
      • Le G.T.
      • Flynn B.
      • Herenbrink C.K.
      • Yeatman H.R.
      • Edgington-Mitchell L.
      • Porter C.J.H.
      • Halls M.L.
      • et al.
      Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome.
      CXCR4AGiCXCL12-instigated suppression of anoikis/apoptosis
      • English E.J.
      • Mahn S.A.
      • Marchese A.
      Endocytosis is required for CXC chemokine receptor type 4 (CXCR4)-mediated Akt activation and antiapoptotic signaling.
      MORAGiND
      • Stoeber M.
      • Jullie D.
      • Lobingier B.T.
      • Laeremans T.
      • Steyaert J.
      • Schiller P.W.
      • Manglik A.
      • von Zastrow M.
      A genetically encoded biosensor reveals location bias of opioid drug action.
      DORAGiND
      • Stoeber M.
      • Jullie D.
      • Lobingier B.T.
      • Laeremans T.
      • Steyaert J.
      • Schiller P.W.
      • Manglik A.
      • von Zastrow M.
      A genetically encoded biosensor reveals location bias of opioid drug action.
      ADGRG2AdhesionGsND
      • Azimzadeh P.
      • Talamantez-Lyburn S.C.
      • Chang K.T.
      • Inoue A.
      • Balenga N.
      Spatial regulation of GPR64/ADGRG2 signaling by β-arrestins and GPCR kinases.
      Where does the ability of certain GPCRs to sustain G protein signaling from endosomes come from? Despite the capacity of PTH to prolong endosomal cAMP signaling via a unique receptor conformation named R0 (reviewed in Ref.
      • Sutkeviciute I.
      • Clark L.J.
      • White A.D.
      • Gardella T.J.
      • Vilardaga J.P.
      PTH/PTHrP receptor signaling, allostery, and structures.
      ), our structural understanding of endosomal GPCR signaling via G proteins is incomplete. The recent development of structural approaches to study mechanisms of GPCR activation (
      • Thal D.M.
      • Vuckovic Z.
      • Draper-Joyce C.J.
      • Liang Y.L.
      • Glukhova A.
      • Christopoulos A.
      • Sexton P.M.
      Recent advances in the determination of G protein-coupled receptor structures.
      ) via cryo-EM, which will be discussed in the following paragraphs, however, opens new possibilities to unveil the exact underpinnings of endosomal GPCR signaling.

      Structural modes of GPCR signaling

      Based on amino acid sequence and functional similarities, the mammalian GPCRs are classified into five families, namely A (rhodopsin-like), B (secretin receptor-like), C (metabotropic glutamate receptor-like), F (frizzled-like), and adhesion GPCRs (aGPCRs) (Fig. 3A). All of the GPCRs share a common characteristic architecture of their transmembrane domain (TMD), which is composed of seven α-helices linked by three extracellular and three intracellular loops that form a compact bundle, and N and C termini located extracellularly and intracellularly, respectively. Owing to the technological advances of X-ray crystallography and cryo-EM, during the last 2 decades, much has been learned about the structural mechanisms underlying the GPCR signaling. Our initial perception of these receptors as simple ON/OFF switches has evolved to understanding them as highly dynamic structures capable of sampling and adopting diverse conformations, each of which mediate distinct signaling outputs. GPCRs are activated by agonist ligand binding in the orthosteric binding pocket located within the upper half of the TMD core. This binding event is relayed to cytosolic side of the receptor through allosteric interaction networks that are distinct for each GPCR class but converge in a common GPCR activation hallmark—the mobilization and outward movement of transmembrane helix 6 (TM6). The outward movement of TM6 leads to the opening of the cytosolic cavity of the GPCRs, allowing the subsequent binding and activation of the heterotrimeric G proteins. Below we summarize the recent structural and dynamic insights into activation mechanisms of members of all GPCR classes, except aGPCRs, whose structures remain unknown. We also overview structural and dynamic aspects of G protein activation and the emerging structural determinants of endosomal GPCR signaling.
      Figure thumbnail gr3
      Figure 3Structural dynamics of GPCR signaling. A, structural features and activation hallmarks of GPCR classes. Left, comparison of inactive→active state transition between representative members of class A, B, and F GPCRs: the common activation hallmark is an outward movement of TM6. The inactive- and active-state structures are shown as semitransparent light violet and orange cartoons, respectively, with TM6 helices highlighted as an opaque cartoon and dashed lines connected with an arrow depicting transition from the inactive to the active state. The superimposed structures are as follows: inactive-state (PDB entry 2R4R) and active-state (PDB entry 3SN6) β2AR; inactive-state (PDB entry 6FJ3) and active-state (PDB entry 6NBF) PTH1R; inactive-state (PDB entry 4N4W) and active-state (PDB 6OT0) SMO. Middle, the cryo-EM structures of the mGlu5 receptor unveil the class C GPCR activation mechanism: the ligand binding to ECD induces a substantial reorganization of the ECDs, leading to repositioning of TMDs in close proximity. However, the TMDs of agonist-bound mGlu5 receptors are nearly identical to those of apo-receptors, indicating that TM6 opening likely requires the G protein presence. The protomers of mGlu5 homodimers are shown as green and wheat cartoons. The apo-receptor (PDB entry 6N52) structure is superimposed onto agonist-bound mGlu5 (PDB code 6N51) by structural alignment of green protomers. The agonist l-quisqualate is shown in magenta spheres, and the ECD-bound nanobody Nb43 in the active state is omitted for clarity. Right, putative model of an aGPCR. In addition to a GPCR-characteristic TMD (wheat cartoon, TMD of inactive glucagon receptor, PDB entry 4L6R), which has phylogenetic relation to class B GPCRs, a common feature of aGPCRs is a large, mostly multidomain ECD. With a single exception of ADGRA1, all of the aGPCRs contain a GPCR autoproteolysis–inducing (GAIN) domain N-terminal to their TMDs (the green cartoon shows the X-ray structure of a GAIN domain of latrophilin 1, PDB entry 4DLQ), which harbors a GPCR proteolysis site (GPS) with a consensus cleavage sequence H(L/I)↓(S/T). The autoproteolysis takes place in the ER, and the ECDs remain noncovalently attached to the rest of the receptor through the tight interaction of the peptide C-terminal to the GPS (called the Stachel peptide, shown in red). For many aGPCRs, the disruption of Stachel peptide interaction with the ECD results in receptor activation. B, cryo-EM structure of PTH1R in complex with LA-PTH and Gs. Two conformational states of LA-PTH binding to PTH1R were detected: state 1 (green) forms a continuous interaction with PTH1R, and in state 2 (magenta), the C-terminal tip of LA-PTH is dissociated from the receptor's ECD (LA-PTH of state 2 (PDB entry 6NBI) is superimposed onto the state 1 structure (PDB entry 6NBF)). TMD-surrounding lipids are shown as sticks; a rectangle delimits the activating portion of the LA-PTH N-terminal part inserted into the receptor's TMD core, and the interaction network in this region between LA-PTH (green) and PTH1R (orange) residues is shown on the right (polar, nonpolar, and mixed interactions are shown as yellow, blue, and green lines, respectively). C, GPCR/β-arrestin interaction plasticity. The cryo-EM structure of NTS(8-13)-bound NTS1R/β-arrestin1 complex (PDB entry 6UP7) shows β-arrestin1 binding in a tilted conformation, possibly stabilized by interaction of PIP2 (black sticks) with the concave surface of the C-domain of β-arrestin1; the right panel shows a bottom view of a superposition of β-arrestin1 conformations bound to NTS1R (light pink) and M2R (purple; PDB entry 6U1N). D, G protein and β-arrestin can bind a GPCR simultaneously. Shown is a cryo-EM structure of a β2V2R chimera in complex with Gs (same color scheme as in A) and β-arrestin1 (combined PDB files 6NI3 and 6NI2). The flexible unresolved part of V2Rpp is shown as an orange dashed line. Gray rectangles in A–C denote putative lipid bilayer boundaries.

      Structural insights into GPCR activation

      GPCRs are allosterically regulated proteins, whereby the extracellular agonist binding is allosterically coupled to the G protein–binding site at the cytosolic receptor side through amino acid–, ion–, and water–mediated interaction networks. The GPCRs respond to a wide range of molecular cues, the diversity of which is particularly high for class A GPCRs, with agonists ranging from photons, protons, small soluble molecules, and lipids to peptides and large proteins. Other members of this family lack soluble ligands, such as protease-activated receptors (PARs) that are activated by the extracellular matrix proteases whose cleavage exposes a receptor-tethered agonist peptide. A considerable number of class A GPCRs have yet unidentified endogenous ligands and are called “orphan” receptors. Whereas intense research efforts are seeking the discovery of these ligands, a recent structural study of the orphan receptor GPR52 has revealed a remarkable self-activation mechanism that might be shared by other orphan GPCRs (
      • Lin X.
      • Li M.
      • Wang N.
      • Wu Y.
      • Luo Z.
      • Guo S.
      • Han G.W.
      • Li S.
      • Yue Y.
      • Wei X.
      • Xie X.
      • Chen Y.
      • Zhao S.
      • Wu J.
      • Lei M.
      • et al.
      Structural basis of ligand recognition and self-activation of orphan GPR52.
      ). The second extracellular loop (ECL2) of the GPR52 folds into a small module that is inserted into an orthosteric (i.e. active) binding site of the receptor, where it engages in interactions with the side chains commonly found to interact with orthosteric agonists within class A GPCRs. The elucidation of GPR52 structure thus explains the lack of a soluble endogenous ligand for this receptor and provides the structural basis for the observed constitutive activity of GPR52. Despite the high physicochemical diversity of class A GPCR agonists, the activation of the receptors follows a common mechanism delineated recently by Zhou and colleagues (
      • Zhou Q.
      • Yang D.
      • Wu M.
      • Guo Y.
      • Guo W.
      • Zhong L.
      • Cai X.
      • Dai A.
      • Jang W.
      • Shakhnovich E.I.
      • Liu Z.J.
      • Stevens R.C.
      • Lambert N.A.
      • Babu M.M.
      • Wang M.W.
      • et al.
      Common activation mechanism of class A GPCRs.
      ), whereby previously identified conserved receptor motifs CWXP (
      • Eddy M.T.
      • Lee M.Y.
      • Gao Z.G.
      • White K.L.
      • Didenko T.
      • Horst R.
      • Audet M.
      • Stanczak P.
      • McClary K.M.
      • Han G.W.
      • Jacobson K.A.
      • Stevens R.C.
      • Wuthrich K.
      Allosteric coupling of drug binding and intracellular signaling in the A2A adenosine receptor.
      ,
      • Filipek S.
      Molecular switches in GPCRs.
      ,
      • Wescott M.P.
      • Kufareva I.
      • Paes C.
      • Goodman J.R.
      • Thaker Y.
      • Puffer B.A.
      • Berdougo E.
      • Rucker J.B.
      • Handel T.M.
      • Doranz B.J.
      Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices.
      ,
      • Tehan B.G.
      • Bortolato A.
      • Blaney F.E.
      • Weir M.P.
      • Mason J.S.
      Unifying family A GPCR theories of activation.
      ,
      • Holst B.
      • Nygaard R.
      • Valentin-Hansen L.
      • Bach A.
      • Engelstoft M.S.
      • Petersen P.S.
      • Frimurer T.M.
      • Schwartz T.W.
      A conserved aromatic lock for the tryptophan rotameric switch in TM-VI of seven-transmembrane receptors.
      ,
      • Nygaard R.
      • Frimurer T.M.
      • Holst B.
      • Rosenkilde M.M.
      • Schwartz T.W.
      Ligand binding and micro-switches in 7TM receptor structures.
      ,
      • Hofmann K.P.
      • Scheerer P.
      • Hildebrand P.W.
      • Choe H.W.
      • Park J.H.
      • Heck M.
      • Ernst O.P.
      A G protein-coupled receptor at work: the rhodopsin model.
      ,
      • Trzaskowski B.
      • Latek D.
      • Yuan S.
      • Ghoshdastider U.
      • Debinski A.
      • Filipek S.
      Action of molecular switches in GPCRs—theoretical and experimental studies.
      ), PIF or PAF (
      • Ballesteros J.A.
      • Weinstein H.
      Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors.
      ,
      • Ishchenko A.
      • Wacker D.
      • Kapoor M.
      • Zhang A.
      • Han G.W.
      • Basu S.
      • Patel N.
      • Messerschmidt M.
      • Weierstall U.
      • Liu W.
      • Katritch V.
      • Roth B.L.
      • Stevens R.C.
      • Cherezov V.
      Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody.
      ,
      • Schönegge A.M.
      • Gallion J.
      • Picard L.P.
      • Wilkins A.D.
      • Le Gouill C.
      • Audet M.
      • Stallaert W.
      • Lohse M.J.
      • Kimmel M.
      • Lichtarge O.
      • Bouvier M.
      Evolutionary action and structural basis of the allosteric switch controlling β2AR functional selectivity.
      ,
      • Kato H.E.
      • Zhang Y.
      • Hu H.
      • Suomivuori C.M.
      • Kadji F.M.N.
      • Aoki J.
      • Krishna Kumar K.
      • Fonseca R.
      • Hilger D.
      • Huang W.
      • Latorraca N.R.
      • Inoue A.
      • Dror R.O.
      • Kobilka B.K.
      • Skiniotis G.
      Conformational transitions of a neurotensin receptor 1-Gi1 complex.
      ), Na+ pocket (
      • Eddy M.T.
      • Lee M.Y.
      • Gao Z.G.
      • White K.L.
      • Didenko T.
      • Horst R.
      • Audet M.
      • Stanczak P.
      • McClary K.M.
      • Han G.W.
      • Jacobson K.A.
      • Stevens R.C.
      • Wuthrich K.
      Allosteric coupling of drug binding and intracellular signaling in the A2A adenosine receptor.
      ,
      • Filipek S.
      Molecular switches in GPCRs.
      ,
      • Liu W.
      • Chun E.
      • Thompson A.A.
      • Chubukov P.
      • Xu F.
      • Katritch V.
      • Han G.W.
      • Roth C.B.
      • Heitman L.H.
      • IJzerman A.P.
      • Cherezov V.
      • Stevens R.C.
      Structural basis for allosteric regulation of GPCRs by sodium ions.
      ,
      • Yuan S.
      • Vogel H.
      • Filipek S.
      The role of water and sodium ions in the activation of the μ-opioid receptor.
      ,
      • Fenalti G.
      • Giguere P.M.
      • Katritch V.
      • Huang X.P.
      • Thompson A.A.
      • Cherezov V.
      • Roth B.L.
      • Stevens R.C.
      Molecular control of δ-opioid receptor signalling.
      ,
      • Katritch V.
      • Fenalti G.
      • Abola E.E.
      • Roth B.L.
      • Cherezov V.
      • Stevens R.C.
      Allosteric sodium in class A GPCR signaling.
      ,
      • Vickery O.N.
      • Carvalheda C.A.
      • Zaidi S.A.
      • Pisliakov A.V.
      • Katritch V.
      • Zachariae U.
      Intracellular transfer of Na+ in an active-state G-protein-coupled receptor.
      ,
      • White K.L.
      • Eddy M.T.
      • Gao Z.G.
      • Han G.W.
      • Lian T.
      • Deary A.
      • Patel N.
      • Jacobson K.A.
      • Katritch V.
      • Stevens R.C.
      Structural connection between activation microswitch and allosteric sodium site in GPCR signaling.
      ,
      • Ye L.
      • Neale C.
      • Sljoka A.
      • Lyda B.
      • Pichugin D.
      • Tsuchimura N.
      • Larda S.T.
      • Pomès R.
      • García A.E.
      • Ernst O.P.
      • Sunahara R.K.
      • Prosser R.S.
      Mechanistic insights into allosteric regulation of the A2A adenosine G protein-coupled receptor by physiological cations.
      ,
      • Chen S.
      • Lu M.
      • Liu D.
      • Yang L.
      • Yi C.
      • Ma L.
      • Zhang H.
      • Liu Q.
      • Frimurer T.M.
      • Wang M.W.
      • Schwartz T.W.
      • Stevens R.C.
      • Wu B.
      • Wüthrich K.
      • Zhao Q.
      Human substance P receptor binding mode of the antagonist drug aprepitant by NMR and crystallography.
      ), NPXXY (
      • Filipek S.
      Molecular switches in GPCRs.
      ,
      • Wescott M.P.
      • Kufareva I.
      • Paes C.
      • Goodman J.R.
      • Thaker Y.
      • Puffer B.A.
      • Berdougo E.
      • Rucker J.B.
      • Handel T.M.
      • Doranz B.J.
      Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices.
      ,
      • Nygaard R.
      • Frimurer T.M.
      • Holst B.
      • Rosenkilde M.M.
      • Schwartz T.W.
      Ligand binding and micro-switches in 7TM receptor structures.
      ,
      • Hofmann K.P.
      • Scheerer P.
      • Hildebrand P.W.
      • Choe H.W.
      • Park J.H.
      • Heck M.
      • Ernst O.P.
      A G protein-coupled receptor at work: the rhodopsin model.
      ,
      • Trzaskowski B.
      • Latek D.
      • Yuan S.
      • Ghoshdastider U.
      • Debinski A.
      • Filipek S.
      Action of molecular switches in GPCRs—theoretical and experimental studies.
      ,
      • Schönegge A.M.
      • Gallion J.
      • Picard L.P.
      • Wilkins A.D.
      • Le Gouill C.
      • Audet M.
      • Stallaert W.
      • Lohse M.J.
      • Kimmel M.
      • Lichtarge O.
      • Bouvier M.
      Evolutionary action and structural basis of the allosteric switch controlling β2AR functional selectivity.
      ,
      • Chen S.
      • Lu M.
      • Liu D.
      • Yang L.
      • Yi C.
      • Ma L.
      • Zhang H.
      • Liu Q.
      • Frimurer T.M.
      • Wang M.W.
      • Schwartz T.W.
      • Stevens R.C.
      • Wu B.
      • Wüthrich K.
      • Zhao Q.
      Human substance P receptor binding mode of the antagonist drug aprepitant by NMR and crystallography.
      ,
      • Rasmussen S.G.
      • DeVree B.T.
      • Zou Y.
      • Kruse A.C.
      • Chung K.Y.
      • Kobilka T.S.
      • Thian F.S.
      • Chae P.S.
      • Pardon E.
      • Calinski D.
      • Mathiesen J.M.
      • Shah S.T.
      • Lyons J.A.
      • Caffrey M.
      • Gellman S.H.
      • et al.
      Crystal structure of the β2 adrenergic receptor-Gs protein complex.
      ,
      • Venkatakrishnan A.J.
      • Deupi X.
      • Lebon G.
      • Heydenreich F.M.
      • Flock T.
      • Miljus T.
      • Balaji S.
      • Bouvier M.
      • Veprintsev D.B.
      • Tate C.G.
      • Schertler G.F.
      • Babu M.M.
      Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region.
      ), and DRY (
      • Schönegge A.M.
      • Gallion J.
      • Picard L.P.
      • Wilkins A.D.
      • Le Gouill C.
      • Audet M.
      • Stallaert W.
      • Lohse M.J.
      • Kimmel M.
      • Lichtarge O.
      • Bouvier M.
      Evolutionary action and structural basis of the allosteric switch controlling β2AR functional selectivity.
      ,
      • Alhadeff R.
      • Vorobyov I.
      • Yoon H.W.
      • Warshel A.
      Exploring the free-energy landscape of GPCR activation.
      ,
      • Jacobson K.A.
      • Costanzi S.
      • Paoletta S.
      Computational studies to predict or explain G protein coupled receptor polypharmacology.
      ,
      • Feng X.
      • Ambia J.
      • Chen K.M.
      • Young M.
      • Barth P.
      Computational design of ligand-binding membrane receptors with high selectivity.
      ,
      • Roth B.L.
      • Irwin J.J.
      • Shoichet B.K.
      Discovery of new GPCR ligands to illuminate new biology.
      ,
      • Shihoya W.
      • Nishizawa T.
      • Yamashita K.
      • Inoue A.
      • Hirata K.
      • Kadji F.M.N.
      • Okuta A.
      • Tani K.
      • Aoki J.
      • Fujiyoshi Y.
      • Doi T.
      • Nureki O.
      X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog.
      ,
      • Yuan S.
      • Filipek S.
      • Palczewski K.
      • Vogel H.
      Activation of G-protein-coupled receptors correlates with the formation of a continuous internal water pathway.
      ) were linked into a concerted sequence of events leading to interaction network reorganization within the core of the TMD that results in increased mobility and consequent outward movement of TM6 (Fig. 3A) with other TMs also undergoing rearrangement but to a much slighter extent.
      The structural trait unique to family B GPCRs is a well-characterized two-step peptide hormone-binding process. The first step involves a rapid bimolecular interaction between the N-terminal extracellular domain of the receptor (ECD) and the C-terminal part of the peptide, which strictly depends on the hormone (H) concentration following the pseudo-first-order equation, kobs=kon×[H]+koff, where kon and koff are the association and dissociation rate constants. The second step is slower and more complex, involving insertion of the N-terminal peptide hormone portion into the receptor's TMD, which in turn shifts the receptor to its active conformation and engages intracellular signaling events via G proteins and β-arrestins. With recently solved new structures of class B GPCRs, a comprehensive molecular mechanism of receptor activation is revealed, as detailed by Liang et al. (
      • Liang Y.L.
      • Belousoff M.J.
      • Zhao P.
      • Koole C.
      • Fletcher M.M.
      • Truong T.T.
      • Julita V.
      • Christopoulos G.
      • Xu H.E.
      • Zhang Y.
      • Khoshouei M.
      • Christopoulos A.
      • Danev R.
      • Sexton P.M.
      • Wootten D.
      Toward a structural understanding of class B GPCR peptide binding and activation.
      ). Globally, the peptide hormones bind their cognate receptors in an extended helical conformation, except for calcitonin (CT) receptor (CTR) and CTR-like receptor (CLR), where the C-terminal fragments of CT or CT gene–related peptide are unfolded. The ECDs compared with TMDs of all class B GPCRs have lower resolution owing to their higher mobility. The C-terminal peptide fragments bind the ECDs that are placed at varying positions with respect to TMDs. This interaction appears to have a dynamic nature exemplified by the cryo-EM structure of LA-PTH/PTH1R/Gs complex (
      • Zhao L.H.
      • Ma S.
      • Sutkeviciute I.
      • Shen D.D.
      • Zhou X.E.
      • de Waal P.W.
      • Li C.Y.
      • Kang Y.
      • Clark L.J.
      • Jean-Alphonse F.G.
      • White A.D.
      • Yang D.
      • Dai A.
      • Cai X.
      • Chen J.
      • et al.
      Structure and dynamics of the active human parathyroid hormone receptor-1.
      ). Two distinct conformational states were solved diverging by the mode of interaction between LA-PTH and the PTH1R's ECD, whereby the C-terminal part of the peptide either adopts an α-helical conformation tightly associated with or is partially dissociated from the ECD while the N-terminal part of the ligand retains its position (Fig. 3B). This finding illustrates the dynamics of peptide hormone interaction with class B GPCR and in addition provides a speculative mechanism for the peptide's ability to maintain the receptor in its active state for a prolonged time. Thereby, repeated cycles of association-dissociation between the ECD and the peptide's C terminus may occur before the complete ligand dissociation resulting in sustained receptor activation. Future structural dynamics studies will be required to verify this theory and fully elucidate structural mechanisms underlying short versus prolonged class B GPCR signaling. Despite the diverse spatial arrangement of peptide-bound ECD of class B GPCRs, the positions of N-terminal peptide tips converge deep within the TMD core of the receptors, indicating a conserved activation mechanism for class B GPCRs. Conserved motifs involved in receptor activation include the following: 1) the polar core mediated by T4106.42b interactions with H2.50b, E3.50b, and Y7.57b (except for F7.57b in calcitonin gene–related peptide 1 receptor) stabilizing the inactive state (
      • Yin Y.
      • de Waal P.W.
      • He Y.
      • Zhao L.H.
      • Yang D.
      • Cai X.
      • Jiang Y.
      • Melcher K.
      • Wang M.W.
      • Xu H.E.
      Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.
      ), and 2) N5.50b/P6.47b/G6.50b/Q7.49b (NPGQ) motif stabilizing the receptor's active state (
      • Zhao L.H.
      • Ma S.
      • Sutkeviciute I.
      • Shen D.D.
      • Zhou X.E.
      • de Waal P.W.
      • Li C.Y.
      • Kang Y.
      • Clark L.J.
      • Jean-Alphonse F.G.
      • White A.D.
      • Yang D.
      • Dai A.
      • Cai X.
      • Chen J.
      • et al.
      Structure and dynamics of the active human parathyroid hormone receptor-1.
      ,
      • Ma S.
      • Shen Q.
      • Zhao L.H.
      • Mao C.
      • Zhou X.E.
      • Shen D.D.
      • de Waal P.W.
      • Bi P.
      • Li C.
      • Jiang Y.
      • Wang M.W.
      • Sexton P.M.
      • Wootten D.
      • Melcher K.
      • Zhang Y.
      • et al.
      Molecular basis for hormone recognition and activation of corticotropin-releasing factor receptors.
      ). The hallmark of class B GPCR activation is a particularly large TM6 displacement—TM6 moves outward by ∼20 Å, compared with 6–14 Å in class A GPCRs, due to a sharp (∼90°) kink stabilized by a conserved NPGQ motif in the middle of TM6 (Fig. 3A).
      The aforementioned class B GPCR activation mechanism is derived from structural studies of receptors in complex with balanced agonist ligands (i.e. agonists that equally activate different downstream signaling pathways mediated by a given receptor (e.g. G protein and β-arrestin)). Whereas the structural understanding of the actions of biased agonists (selectively activating or favoring a particular signaling pathway such as Gs versus β-arrestins) is the subject of intense research efforts, recent studies of class B GPCRs revealed possible mechanisms of ligand-dependent signaling bias. The cryo-EM structure of GLP-1R bound to a nonpeptidic biased agonist TT-OAD2 and in complex with the Gs heterotrimer shows several unexpected features (
      • Zhao P.
      • Liang Y.L.
      • Belousoff M.J.
      • Deganutti G.
      • Fletcher M.M.
      • Willard F.S.
      • Bell M.G.
      • Christe M.E.
      • Sloop K.W.
      • Inoue A.
      • Truong T.T.
      • Clydesdale L.
      • Furness S.G.B.
      • Christopoulos A.
      • Wang M.W.
      • et al.
      Activation of the GLP-1 receptor by a non-peptidic agonist.
      ): 1) TT-OAD2 binds the receptor's TMD at a higher level than peptide agonists; 2) it engages in interactions with a distinct set of the receptor's amino acid side chains with only a small number of the side chains overlapping with those that peptide agonists interact with; 3) in contrast to peptide agonists, TT-OAD2 engages the conserved polar core responsible for the receptor activation indirectly through a network of structural water molecules; 4) TT-OAD2 assumes a boomerang shape with one end protruding through the GLP-1R TMD core to interact with detergent micelle or lipid bilayer; and 5) the receptor's ECD is more mobile and assumes a distinct averaged position relative to peptide-bound structures. Despite these striking differences and distinct pharmacological properties of TT-OAD2 as a weak Gs-biased agonist, the conformation of the helical bundle is highly similar to peptide agonist–bound GLP-1R/Gs structures (
      • Zhang Y.
      • Sun B.
      • Feng D.
      • Hu H.
      • Chu M.
      • Qu Q.
      • Tarrasch J.T.
      • Li S.
      • Sun Kobilka T.
      • Kobilka B.K.
      • Skiniotis G.
      Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein.
      ,
      • Liang Y.L.
      • Khoshouei M.
      • Glukhova A.
      • Furness S.G.B.
      • Zhao P.
      • Clydesdale L.
      • Koole C.
      • Truong T.T.
      • Thal D.M.
      • Lei S.
      • Radjainia M.
      • Danev R.
      • Baumeister W.
      • Wang M.W.
      • Miller L.J.
      • et al.
      Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex.
      ), likely owing to artificial stabilization of receptor/Gs interaction and reflecting a limitation of such an approach to detect physiologically relevant conformational states of GPCRs. The distinct positioning of the ECD in TT-OAD2–bound GLP-1R structure likely supports the earlier prediction that ECD mobility together with interactions with ECLs determines the ligand bias (
      • Liang Y.L.
      • Khoshouei M.
      • Glukhova A.
      • Furness S.G.B.
      • Zhao P.
      • Clydesdale L.
      • Koole C.
      • Truong T.T.
      • Thal D.M.
      • Lei S.
      • Radjainia M.
      • Danev R.
      • Baumeister W.
      • Wang M.W.
      • Miller L.J.
      • et al.
      Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex.
      ,
      • Lei S.
      • Clydesdale L.
      • Dai A.
      • Cai X.
      • Feng Y.
      • Yang D.
      • Liang Y.L.
      • Koole C.
      • Zhao P.
      • Coudrat T.
      • Christopoulos A.
      • Wang M.W.
      • Wootten D.
      • Sexton P.M.
      Two distinct domains of the glucagon-like peptide-1 receptor control peptide-mediated biased agonism.
      ). This mechanism of biased signaling controlled by receptor's ECD/ECLs conformations might be extended to PTH1R, given that the ECD-binding antibody fragment, which presumably stabilizes a distinct conformation of the ECD/ECLs, abolishes β-arrestin recruitment to PTH-stimulated PTH1R without affecting the Gs signaling (
      • Sarkar K.
      • Joedicke L.
      • Westwood M.
      • Burnley R.
      • Wright M.
      • McMillan D.
      • Byrne B.
      Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of β-arrestin 2 coupling.
      ). In line with these results, a recent study involving NMR analysis combined with MS, signaling, and computational approaches on PTH1R demonstrates how the receptor's ECD works to favor the formation of the PTH α-helical structure and reveals the existence of structure-encoded allosteric coupling between the receptor's ECL2 and residue His-9 in PTH that establishes direct and key interactions between PTH1R's ICL3 and β-arrestins. The assembly of the PTH1R–arrestin complex via cooperative fluctuations between the receptor and its ligand might cover a general mechanism for class B GPCRs (
      • Clark L.J.
      • Krieger J.
      • White A.D.
      • Bondarenko V.
      • Lei S.
      • Fang F.
      • Lee J.Y.
      • Doruker P.
      • Böttke T.
      • Jean-Alphonse F.
      • Tang P.
      • Gardella T.J.
      • Xiao K.
      • Sutkeviciute I.
      • Coin I.
      • et al.
      Allosteric interactions in the parathyroid hormone GPCR–arrestin complex formation.
      ).
      Insights into the activation mechanism of class C GPCRs begin to emerge. Class C GPCRs exist as obligate homo- or heteromers and regulate major biological functions as diverse as mineral and skeletal metabolism (
      • Chang W.
      • Tu C.-L.
      • Jean-Alphonse F.
      • Herberger A.
      • Cheng Z.
      • Hwong J.
      • Ho H.
      • Li A.
      • Wang D.
      • Liu H.
      • White A.D.
      • Suh I.
      • Shen W.
      • Duh Q.-Y.
      • Khanafshar E.
      • et al.
      PTH hypersecretion triggered by a GABAB1 and Ca2+-sensing receptor heterocomplex in hyperparathyroidism.
      ) and neurological function (
      • Pin J.P.
      • Bettler B.
      Organization and functions of mGlu and GABAB receptor complexes.
      ). Earlier structural studies of isolated ECDs of several class C GPCRs demonstrated that agonist binding induces the closure of the VFT (Venus flytrap) domain of their ECDs, leading to rearrangement of the interface between the dimer ECDs and bringing them into close proximity—a hallmark of class C GPCR activation (
      • Kunishima N.
      • Shimada Y.
      • Tsuji Y.
      • Sato T.
      • Yamamoto M.
      • Kumasaka T.
      • Nakanishi S.
      • Jingami H.
      • Morikawa K.
      Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor.
      ,
      • Tsuchiya D.
      • Kunishima N.
      • Kamiya N.
      • Jingami H.
      • Morikawa K.
      Structural views of the ligand-binding cores of a metabotropic glutamate receptor complexed with an antagonist and both glutamate and Gd3+.
      ,
      • Muto T.
      • Tsuchiya D.
      • Morikawa K.
      • Jingami H.
      Structures of the extracellular regions of the group II/III metabotropic glutamate receptors.
      ,
      • Geng Y.
      • Bush M.
      • Mosyak L.
      • Wang F.
      • Fan Q.R.
      Structural mechanism of ligand activation in human GABA(B) receptor.
      ,
      • Geng Y.
      • Mosyak L.
      • Kurinov I.
      • Zuo H.
      • Sturchler E.
      • Cheng T.C.
      • Subramanyam P.
      • Brown A.P.
      • Brennan S.C.
      • Mun H.C.
      • Bush M.
      • Chen Y.
      • Nguyen T.X.
      • Cao B.
      • Chang D.D.
      • et al.
      Structural mechanism of ligand activation in human calcium-sensing receptor.
      ). The recent X-ray crystallographic and cryo-EM studies of full-length metabotropic glutamate receptor 5 (mGlu5) revealed that agonist binding within the ECDs leads to profound movement of the TMDs within a dimer (
      • Koehl A.
      • Hu H.
      • Feng D.
      • Sun B.
      • Zhang Y.
      • Robertson M.J.
      • Chu M.
      • Kobilka T.S.
      • Laeremans T.
      • Steyaert J.
      • Tarrasch J.
      • Dutta S.
      • Fonseca R.
      • Weis W.I.
      • Mathiesen J.M.
      • et al.
      Structural insights into the activation of metabotropic glutamate receptors.
      ). Whereas in the inactive state, the TMD regions are apart from each other, the structural rearrangements of the ECDs upon agonist binding induce the rotation of the TMDs that brings them close to each other with the TM6 helices forming a common interface, a second class C GPCR activation hallmark that was depicted by earlier biochemical studies (
      • Xue L.
      • Rovira X.
      • Scholler P.
      • Zhao H.
      • Liu J.
      • Pin J.P.
      • Rondard P.
      Major ligand-induced rearrangement of the heptahelical domain interface in a GPCR dimer.
      ). Importantly, the ECL2 was found to serve as a rigid fulcrum that allosterically relays the conformational changes within ECDs to the TMDs. Despite the observed structural rearrangements at the extracellular part following the mGlu5 activation, the conformation of agonist-bound mGlu5 TMD does not display the outward movement of TM6, characteristic of GPCR activation. Instead, the TMD overlaps with the apo- or thermostabilized inactive-state mGlu5 structures (
      • Christopher J.A.
      • Aves S.J.
      • Bennett K.A.
      • Doré A.S.
      • Errey J.C.
      • Jazayeri A.
      • Marshall F.H.
      • Okrasa K.
      • Serrano-Vega M.J.
      • Tehan B.G.
      • Wiggin G.R.
      • Congreve M.
      Fragment and structure-based drug discovery for a class C GPCR: discovery of the mGlu5 negative allosteric modulator HTL14242 (3-chloro-5-[6-(5-fluoropyridin-2-yl)pyrimidin-4-yl]benzonitrile).
      ,
      • Christopher J.A.
      • Orgován Z.
      • Congreve M.
      • Doré A.S.
      • Errey J.C.
      • Marshall F.H.
      • Mason J.S.
      • Okrasa K.
      • Rucktooa P.
      • Serrano-Vega M.J.
      • Ferenczy G.G.
      • Keserű G.M.
      Structure-based optimization strategies for G protein-coupled receptor (GPCR) allosteric modulators: a case study from analyses of new metabotropic glutamate receptor 5 (mGlu5) X-ray structures.
      ,
      • Doré A.S.
      • Okrasa K.
      • Patel J.C.
      • Serrano-Vega M.
      • Bennett K.
      • Cooke R.M.
      • Errey J.C.
      • Jazayeri A.
      • Khan S.
      • Tehan B.
      • Weir M.
      • Wiggin G.R.
      • Marshall F.H.
      Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain.
      ), likely indicating that the transition to the fully active state requires G protein binding. Therefore, more studies are needed to fully elucidate the mechanism of class C GPCR activation.
      The first insights into the class F GPCR activation mechanism came from solved structures of active-state Smoothened receptor (SMO), either activated by 25(S),25-epoxycholesterol and in complex with Gi heterotrimer (
      • Qi X.
      • Liu H.
      • Thompson B.
      • McDonald J.
      • Zhang C.
      • Li X.
      Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi.
      ) or bound to agonist SAG21k and stabilized by active-state selective nanobody NbSmo8 (
      • Deshpande I.
      • Liang J.
      • Hedeen D.
      • Roberts K.J.
      • Zhang Y.
      • Ha B.
      • Latorraca N.R.
      • Faust B.
      • Dror R.O.
      • Beachy P.A.
      • Myers B.R.
      • Manglik A.
      Smoothened stimulation by membrane sterols drives Hedgehog pathway activity.
      ), and the details of the state-of-the-art understanding of class F GPCRs have been recently reviewed by Kozielewicz et al. (
      • Kozielewicz P.
      • Turku A.
      • Schulte G.
      Molecular pharmacology of class F receptor activation.
      ). Similarly to class A GPCRs, activation of SMO induces outward and inward movements of TM6 and TM5, respectively. The outward movement of TM6 disrupts polar interactions of the conserved basic R/K6.32 residue with TM7 backbone that was recently identified as a part of a conserved molecular switch stabilizing inactive-state class F GPCRs (
      • Wright S.C.
      • Kozielewicz P.
      • Kowalski-Jahn M.
      • Petersen J.
      • Bowin C.F.
      • Slodkowicz G.
      • Marti-Solano M.
      • Rodríguez D.
      • Hot B.
      • Okashah N.
      • Strakova K.
      • Valnohova J.
      • Babu M.M.
      • Lambert N.A.
      • Carlsson J.
      • et al.
      A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection.
      ). Interestingly, the Gαi binds SMO in a distinct manner compared with Gi-coupled class A GPCRs: the C-terminal α5 helix of Gαi is inserted to cytosolic core of SMO in an angle parallel to the TMD, which is ∼5° tilted compared with class A GPCR/Gi structures. Although these findings shed light on class F GPCR activation, much remains to be learned about structural mechanisms governing the function of class F GPCRs, whereas structural aspects of the function of aGPCRs (
      • Hamann J.
      • Aust G.
      • Araç D.
      • Engel F.B.
      • Formstone C.
      • Fredriksson R.
      • Hall R.A.
      • Harty B.L.
      • Kirchhoff C.
      • Knapp B.
      • Krishnan A.
      • Liebscher I.
      • Lin H.H.
      • Martinelli D.C.
      • Monk K.R.
      • et al.
      International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors.
      ) remain to be determined, as no member of this family has a solved structure so far.

      Structural dynamics of G protein activation and GPCR/G protein signaling

      The recent surge of near-atomic structures of GPCRs in complex with heterotrimeric Gαβγ proteins freed of guanosine nucleotide revealed interaction sites in the receptor/G protein interface and the G protein activation mechanism, whereby the C-terminal α5 helix of the Gα subunit is inserted into the cytosolic cavity of the receptor. The α5 helix is allosterically coupled to the nucleotide-binding site, and thus the rotational translation of the α5 helix induced by the interaction with the GPCR leads to conformational changes within the nucleotide-binding site, resulting in the GDP/GTP exchange (reviewed in Ref.
      • Hilger D.
      • Masureel M.
      • Kobilka B.K.
      Structure and dynamics of GPCR signaling complexes.
      ). All of the solved GPCR/G protein structures display α5 helix insertion to the cytosolic core of the receptor, suggesting that this mode of engagement is universal among different GPCR families and G protein subtypes that have solved structures. However, these structures represent only a snapshot of the G protein activation process, especially considering that the Gα subunit in a nucleotide-free state may exist in cells only for a brief period of time, given the high concentration (∼400 μm) of free GTP in cells (
      • Traut T.W.
      Physiological concentrations of purines and pyrimidines.
      ). Indeed, recent spectroscopic studies raised the possibility that GPCR/G protein complexes may adopt multiple conformations (
      • Sounier R.
      • Mas C.
      • Steyaert J.
      • Laeremans T.
      • Manglik A.
      • Huang W.
      • Kobilka B.K.
      • Déméné H.
      • Granier S.
      Propagation of conformational changes during μ-opioid receptor activation.
      ,
      • Gregorio G.G.
      • Masureel M.
      • Hilger D.
      • Terry D.S.
      • Juette M.
      • Zhao H.
      • Zhou Z.
      • Perez-Aguilar J.M.
      • Hauge M.
      • Mathiasen S.
      • Javitch J.A.
      • Weinstein H.
      • Kobilka B.K.
      • Blanchard S.C.
      Single-molecule analysis of ligand efficacy in β2AR-G-protein activation.
      ,
      • Van Eps N.
      • Altenbach C.
      • Caro L.N.
      • Latorraca N.R.
      • Hollingsworth S.A.
      • Dror R.O.
      • Ernst O.P.
      • Hubbell W.L.
      Gi- and Gs-coupled GPCRs show different modes of G-protein binding.
      ), engaging more research to understand further structural insights into the initial steps and dynamics of the G protein activation process (
      • Liu X.
      • Xu X.
      • Hilger D.
      • Aschauer P.
      • Tiemann J.K.S.
      • Du Y.
      • Liu H.
      • Hirata K.
      • Sun X.
      • Guixa-Gonzalez R.
      • Mathiesen J.M.
      • Hildebrand P.W.
      • Kobilka B.K.
      Structural insights into the process of GPCR-G protein complex formation.
      ,
      • Du Y.
      • Duc N.M.
      • Rasmussen S.G.F.
      • Hilger D.
      • Kubiak X.
      • Wang L.
      • Bohon J.
      • Kim H.R.
      • Wegrecki M.
      • Asuru A.
      • Jeong K.M.
      • Lee J.
      • Chance M.R.
      • Lodowski D.T.
      • Kobilka B.K.
      • et al.
      Assembly of a GPCR-G protein complex.
      ,
      • Gao Y.
      • Hu H.
      • Ramachandran S.
      • Erickson J.W.
      • Cerione R.A.
      • Skiniotis G.
      Structures of the rhodopsin-transducin complex: insights into G-protein activation.
      ). Other studies employing hydrogen/deuterium exchange MS and hydroxyl radical–mediated protein footprinting coupled with MS delineated time-resolved conformational changes when the Gs protein interacts with β2AR or A2AR (
      • Du Y.
      • Duc N.M.
      • Rasmussen S.G.F.
      • Hilger D.
      • Kubiak X.
      • Wang L.
      • Bohon J.
      • Kim H.R.
      • Wegrecki M.
      • Asuru A.
      • Jeong K.M.
      • Lee J.
      • Chance M.R.
      • Lodowski D.T.
      • Kobilka B.K.
      • et al.
      Assembly of a GPCR-G protein complex.
      ). Most notably, the release of GDP happens long before the α5 helix fully enters the cytosolic cavity of the agonist-bound GPCR, as it is observed in the crystal structure of the β2AR/Gs complex (
      • Rasmussen S.G.
      • DeVree B.T.
      • Zou Y.
      • Kruse A.C.
      • Chung K.Y.
      • Kobilka T.S.
      • Thian F.S.
      • Chae P.S.
      • Pardon E.
      • Calinski D.
      • Mathiesen J.M.
      • Shah S.T.
      • Lyons J.A.
      • Caffrey M.
      • Gellman S.H.
      • et al.
      Crystal structure of the β2 adrenergic receptor-Gs protein complex.
      ). This result indicates that the initial contacts of Gs with the active GPCR are responsible for GDP release and distinct from those observed in structures of GPCR/Gs complexes. The latter suggestion was further corroborated by the crystal structure of the β2AR in complex with the last 14 amino acids of the Gαs α5 helix (
      • Liu X.
      • Xu X.
      • Hilger D.
      • Aschauer P.
      • Tiemann J.K.S.
      • Du Y.
      • Liu H.
      • Hirata K.
      • Sun X.
      • Guixa-Gonzalez R.
      • Mathiesen J.M.
      • Hildebrand P.W.
      • Kobilka B.K.
      Structural insights into the process of GPCR-G protein complex formation.
      ) that showed a distinct interaction mode compared with the β2AR/Gs complex. Furthermore, the recent cryo-EM structure of human neurotensin 1 receptor (NTS1R) in complex with the Gi1 heterotrimer revealed two conformational states of the complex, termed canonical and noncanonical, that differed in the positioning and conformation of the Gi1 protein (
      • Kato H.E.
      • Zhang Y.
      • Hu H.
      • Suomivuori C.M.
      • Kadji F.M.N.
      • Aoki J.
      • Krishna Kumar K.
      • Fonseca R.
      • Hilger D.
      • Huang W.
      • Latorraca N.R.
      • Inoue A.
      • Dror R.O.
      • Kobilka B.K.
      • Skiniotis G.
      Conformational transitions of a neurotensin receptor 1-Gi1 complex.
      ). In the canonical state, the NTS1R/Gi1 engagement is similar to other recent GPCR/Gi/o complex structures, whereas in the noncanonical state, the G protein is twisted by ∼45° and the nucleotide-binding pocket of the Gα subunit displays higher rigidity compared with the canonical state, precluding efficient nucleotide exchange. Interestingly, the NTS1R conformation in the noncanonical state exhibits features characteristic to both active- and inactive-state NTS1R structures, suggesting that the noncanonical state complex represents an intermediate state of GPCR activation/G protein engagement/activation process. The dynamics of the G protein engagement to the GPCR was further detailed by the recent cryo-EM structures of rhodopsin (Rho) in complex with transducin (GT) and in the presence or absence of an engineered stabilizing nanobody that binds at the interface of Gα and β subunits but does not preclude nucleotide exchange (
      • Gao Y.
      • Hu H.
      • Ramachandran S.
      • Erickson J.W.
      • Cerione R.A.
      • Skiniotis G.
      Structures of the rhodopsin-transducin complex: insights into G-protein activation.
      ). In the absence of the nanobody, the Ras domain of GαT moves further away from Gβ1γ1, giving an insight into nucleotide exchange–driven dissociation of the Gα and β1γ1 subunits. Notably, this study revealed the putative role of the Gβ1 and the α-helical domain (αHD) of the Gα subunit in the G protein activation process. Whereas αHD is highly flexible (
      • Westfield G.H.
      • Rasmussen S.G.
      • Su M.
      • Dutta S.
      • DeVree B.T.
      • Chung K.Y.
      • Calinski D.
      • Velez-Ruiz G.
      • Oleskie A.N.
      • Pardon E.
      • Chae P.S.
      • Liu T.
      • Li S.
      • Woods Jr., V.L.
      • Steyaert J.
      • et al.
      Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex.
      ,
      • Gao Y.
      • Westfield G.
      • Erickson J.W.
      • Cerione R.A.
      • Skiniotis G.
      • Ramachandran S.
      Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex.
      ,
      • Van Eps N.
      • Preininger A.M.
      • Alexander N.
      • Kaya A.I.
      • Meier S.
      • Meiler J.
      • Hamm H.E.
      • Hubbell W.L.
      Interaction of a G protein with an activated receptor opens the interdomain interface in the α subunit.
      ) and therefore it remains unresolved in other GPCR/G protein cryo-EM structures, Gao et al. (
      • Gao Y.
      • Westfield G.
      • Erickson J.W.
      • Cerione R.A.
      • Skiniotis G.
      • Ramachandran S.
      Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex.
      ) solved the structures of three Rho/GT complex conformers (at 4.5 Å) that differed in degrees of αHD opening (66°, 68°, and 91° compared with GDP-bound GT, but none as extensively displaced (132°) as αHD in the β2AR/Gs crystal structure). Most notably, in all of the three structures, αHD appears to make polar contacts with the Gβ1 subunit, which were found to be crucial to support optimal GDP→GTP exchange rate. Thus, this study unveiled that Gβ1 likely plays a role of a “latching switch” by capturing and separating αHD from the Gα Ras domain and consequently freeing the path for nucleotide exchange.
      The structural dynamics of GPCR signaling are further confirmed by the ever-growing evidence that these receptors exist in multiple conformational states, opposed to their initial understanding as simple ON/OFF molecular switches. As such, distinct conformational states of the receptor can be stabilized by different agonists or allosteric modulators, leading to signaling bias toward a certain G protein subtype or β-arrestin–mediated signaling pathways (reviewed in Refs.
      • Zhao L.H.
      • Ma S.
      • Sutkeviciute I.
      • Shen D.D.
      • Zhou X.E.
      • de Waal P.W.
      • Li C.Y.
      • Kang Y.
      • Clark L.J.
      • Jean-Alphonse F.G.
      • White A.D.
      • Yang D.
      • Dai A.
      • Cai X.
      • Chen J.
      • et al.
      Structure and dynamics of the active human parathyroid hormone receptor-1.
      and
      • Wootten D.
      • Christopoulos A.
      • Marti-Solano M.
      • Babu M.M.
      • Sexton P.M.
      Mechanisms of signalling and biased agonism in G protein-coupled receptors.
      ). In addition, a ligand-dependent stabilized conformational state may modulate the downstream G protein activation efficacy (
      • Furness S.G.B.
      • Liang Y.L.
      • Nowell C.J.
      • Halls M.L.
      • Wookey P.J.
      • Dal Maso E.
      • Inoue A.
      • Christopoulos A.
      • Wootten D.
      • Sexton P.M.
      Ligand-dependent modulation of G protein conformation alters drug efficacy.
      ).
      An additional layer of GPCR signaling complexity is contributed by their ability to continue signaling after internalization to the early endosomes (Fig. 2A). The structural/molecular basis of such a signaling mode is not well-understood. However, one of the molecular prerequisites of endosomal signaling can be anticipated: given the inevitable endosomal pH acidification, the stability of the ligand/receptor complex to acidic pH must be a key factor to maintain the receptor in its active signaling state. The PTH1R is a prototypical example of a GPCR with ligand-dependent spatiotemporal signaling bias: PTH mediates extended PTH1R signaling through Gs/cAMP after internalization in endosomes as opposed to PTHrP that only signals at the plasma membrane (
      • Ferrandon S.
      • Feinstein T.N.
      • Castro M.
      • Wang B.
      • Bouley R.
      • Potts J.T.
      • Gardella T.J.
      • Vilardaga J.P.
      Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.
      ). Remarkably, LA-PTH prolongs endosomal PTH1R/Gs/cAMP signaling even more than PTH, a feature that is considered as a form of ligand bias as it relates to location and duration of signaling. The recently solved cryo-EM structure of LA-PTH-bound PTH1R in complex with Gs (
      • Zhao L.H.
      • Ma S.
      • Sutkeviciute I.
      • Shen D.D.
      • Zhou X.E.
      • de Waal P.W.
      • Li C.Y.
      • Kang Y.
      • Clark L.J.
      • Jean-Alphonse F.G.
      • White A.D.
      • Yang D.
      • Dai A.
      • Cai X.
      • Chen J.
      • et al.
      Structure and dynamics of the active human parathyroid hormone receptor-1.
      ) revealed an extensive interaction network between LA-PTH and the receptor (Fig. 3B), giving the first insight into structural basis of endosomal GPCR signaling. Thereby, the strong interaction between the peptide ligand and the receptor likely increases resistance to acidic endosomal pH and consequently the ligand residence time (1/koff, where koff is the dissociation rate constant), prevents recycling (
      • White A.D.
      • Fang F.
      • Jean-Alphonse F.G.
      • Clark L.J.
      • An H.J.
      • Liu H.
      • Zhao Y.
      • Reynolds S.L.
      • Lee S.
      • Xiao K.
      • Sutkeviciute I.
      • Vilardaga J.P.
      Ca2+ allostery in PTH-receptor signaling.
      ), and thus sustains the receptor in its active state, leading to prolonged signaling after internalization. A second determinant for prolonged GPCR signaling after internalization is suggested by a profound structural rearrangement of the active-state class B GPCRs, as discussed above. It can be anticipated that the time course of receptor deactivation takes considerably longer than agonist dissociation, thus extending the number of rounds of G protein coupling/activation as observed for the PTH1R (
      • Ferrandon S.
      • Feinstein T.N.
      • Castro M.
      • Wang B.
      • Bouley R.
      • Potts J.T.
      • Gardella T.J.
      • Vilardaga J.P.
      Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.
      ). As such, the sustained active state of the receptor leads to prolonged signaling. Whether such prolonged signaling takes place at the plasma membrane or in early endosomes depends on ligand-induced structural features of the receptor that determine whether or not it will be endocytosed, a process most often mediated by β-arrestins.

      Paradoxical actions of β-arrestin as terminator and promoter of GPCR signaling via Gs

      Activated GPCRs are rapidly phosphorylated by GRKs, consequently increasing binding affinity with β-arrestins to stabilize receptor–arrestin complexes, which in turn prevents further cycles of G protein activation (
      • Kühn H.
      • Hall S.W.
      • Wilden U.
      Light-induced binding of 48-kDa protein to photoreceptor membranes is highly enhanced by phosphorylation of rhodopsin.
      ,
      • Lohse M.J.
      • Andexinger S.
      • Pitcher J.
      • Trukawinski S.
      • Codina J.
      • Faure J.P.
      • Caron M.G.
      • Lefkowitz R.J.
      Receptor-specific desensitization with purified proteins: kinase dependence and receptor specificity of β-arrestin and arrestin in the β2-adrenergic receptor and rhodopsin systems.
      ,
      • Wilden U.
      Duration and amplitude of the light-induced cGMP hydrolysis in vertebrate photoreceptors are regulated by multiple phosphorylation of rhodopsin and by arrestin binding.
      ,
      • Krupnick J.G.
      • Gurevich V.V.
      • Benovic J.L.
      Mechanism of quenching of phototransduction: binding competition between arrestin and transducin for phosphorhodopsin.
      ). Initial structural insights into the mechanism of arrestin and GPCR interaction have been unveiled with the structure of β-arrestin1 in complex with the vasopressin type 2 receptor C-tail phosphopeptide (V2Rpp), which engages electrostatic interactions between phosphorylated and negatively charged amino acids on the V2Rpp with positively charged side chains lining the concave surface of the N-domain of β-arrestin1 (
      • Shukla A.K.
      • Manglik A.
      • Kruse A.C.
      • Xiao K.
      • Reis R.I.
      • Tseng W.C.
      • Staus D.P.
      • Hilger D.
      • Uysal S.
      • Huang L.Y.
      • Paduch M.
      • Tripathi-Shukla P.
      • Koide A.
      • Koide S.
      • Weis W.I.
      • et al.
      Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.
      ). A peptide corresponding to the fingerloop (arr-FL) of visual arrestin was then found to bind to the cytosolic core of the light-activated rhodopsin (
      • Szczepek M.
      • Beyrière F.
      • Hofmann K.P.
      • Elgeti M.
      • Kazmin R.
      • Rose A.
      • Bartl F.J.
      • von Stetten D.
      • Heck M.
      • Sommer M.E.
      • Hildebrand P.W.
      • Scheerer P.
      Crystal structure of a common GPCR-binding interface for G protein and arrestin.
      ). These first insights were recapitulated in a crystal structure of rhodopsin–arrestin complex (
      • Kang Y.
      • Zhou X.E.
      • Gao X.
      • He Y.
      • Liu W.
      • Ishchenko A.
      • Barty A.
      • White T.A.
      • Yefanov O.
      • Han G.W.
      • Xu Q.
      • de Waal P.W.
      • Ke J.
      • Tan M.H.
      • Zhang C.
      • et al.
      Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.
      ) and in recent cryo-EM structures of β-arrestin1 in complex with NTS1R (
      • Huang W.
      • Masureel M.
      • Qianhui Q.
      • Janetzko J.
      • Inoue A.
      • Kato H.E.
      • Robertson M.J.
      • Nguyen K.C.
      • Glenn J.S.
      • Skiniotis G.
      • Kobilka B.K.
      Structure of the neurotensin receptor 1 in complex with β-arrestin 1.
      ,
      • Yin W.
      • Li Z.
      • Jin M.
      • Yin Y.L.
      • de Waal P.W.
      • Pal K.
      • Yin Y.
      • Gao X.
      • He Y.
      • Gao J.
      • Wang X.
      • Zhang Y.
      • Zhou H.
      • Melcher K.
      • Jiang Y.
      • et al.
      A complex structure of arrestin-2 bound to a G protein-coupled receptor.
      ) or the muscarinic acetylcholine-2 receptor (M2R) (
      • Staus D.P.
      • Hu H.
      • Robertson M.J.
      • Kleinhenz A.L.W.
      • Wingler L.M.
      • Capel W.D.
      • Latorraca N.R.
      • Lefkowitz R.J.
      • Skiniotis G.
      Structure of the M2 muscarinic receptor-β-arrestin complex in a lipid nanodisc.
      ). These studies confirmed that the arr-FL is inserted into receptor's cytosolic cavity, whereas the phosphorylated receptor C-tail (ppCt) binds to arrestin's N-domain. Such a mode of interaction explains how arrestins block the access to the cytosolic cavity of GPCRs, thus sterically interrupting G protein coupling, and might be a conserved mechanism by which arrestins control GPCR signaling via G proteins.
      Furthermore, these structural studies revealed the plasticity of GPCR/arrestin interactions. Whereas the overall structural organization of the M2R/β-arrestin1 and rhodopsin/arrestin complexes is similar, β-arrestin1 is rotated by 90° along the axis running through the core of the receptor in the NTS1R/β-arrestin1 complex (Fig. 3C). The arr-FL of arrestin-1 assumes a helical structure in both the rhodopsin/arr-FL and rhodopsin/arrestin-1 crystal structures, but positioning of this motif differs significantly, suggesting that arrestin-1 may engage the rhodopsin in different conformations. Such conformational plasticity is further supported by a recent study using unnatural amino acid p-azido-l-phenylalanine incorporation into 25 different positions of cytosolic domains of AngII (angiotensin II) type 1 receptor (AT1R) and UV-induced photocross-linking with β-arrestins (
      • Gagnon L.
      • Cao Y.
      • Cho A.
      • Sedki D.
      • Huber T.
      • Sakmar T.P.
      • Laporte S.A.
      Genetic code expansion and photocross-linking identify different β-arrestin binding modes to the angiotensin II type 1 receptor.
      ). The study revealed different interaction patterns between β-arrestins and AT1R, suggesting that AT1R/β-arrestin complexes of different conformations may form. All of these studies point to structural plasticity of arrestin/GPCR interaction. As a result, only four arrestins, of which only two are nonvisual, are able to function as GPCR/G protein interaction terminators at over 800 GPCRs in humans due to their structural plasticity that enables them to adapt to numerous cytosolic cavity conformations of GPCRs.
      The binding to GPCRs leads to activation of arrestins that is signified by ∼20° twist of their N- and C-domains with respect to each other (detailed in Ref.
      • Gurevich V.V.
      • Gurevich E.V.
      The structural basis of the arrestin binding to GPCRs.
      ). Initially, it was thought that full activation requires arrestins to engage both the ppCt and the core of GPCRs (
      • Gurevich V.V.
      • Benovic J.L.
      Visual arrestin interaction with rhodopsin. Sequential multisite binding ensures strict selectivity toward light-activated phosphorylated rhodopsin.
      ,
      • Gurevich V.V.
      • Gurevich E.V.
      The structural basis of arrestin-mediated regulation of G-protein-coupled receptors.
      ); however, later studies indicated that interaction with ppCt is sufficient to activate β-arrestins (
      • Shukla A.K.
      • Manglik A.
      • Kruse A.C.
      • Xiao K.
      • Reis R.I.
      • Tseng W.C.
      • Staus D.P.
      • Hilger D.
      • Uysal S.
      • Huang L.Y.
      • Paduch M.
      • Tripathi-Shukla P.
      • Koide A.
      • Koide S.
      • Weis W.I.
      • et al.
      Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.
      ,
      • Nobles K.N.
      • Guan Z.
      • Xiao K.
      • Oas T.G.
      • Lefkowitz R.J.
      The active conformation of β-arrestin1: direct evidence for the phosphate sensor in the N-domain and conformational differences in the active states of β-arrestins1 and -2.
      ,
      • Kumari P.
      • Srivastava A.
      • Banerjee R.
      • Ghosh E.
      • Gupta P.
      • Ranjan R.
      • Chen X.
      • Gupta B.
      • Gupta C.
      • Jaiman D.
      • Shukla A.K.
      Functional competence of a partially engaged GPCR-β-arrestin complex.
      ,
      • Thomsen A.R.B.
      • Plouffe B.
      • Cahill 3rd, T.J.
      • Shukla A.K.
      • Tarrasch J.T.
      • Dosey A.M.
      • Kahsai A.W.
      • Strachan R.T.
      • Pani B.
      • Mahoney J.P.
      • Huang L.
      • Breton B.
      • Heydenreich F.M.
      • Sunahara R.K.
      • Skiniotis G.
      • et al.
      GPCR-G protein-β-arrestin super-complex mediates sustained G protein signaling.
      ). A recent computational investigation of the arrestin-1 activation mechanism suggested that the core and ppCt of GPCRs may have equal potential to activate arrestins, although the sequence of events remains to be determined (
      • Latorraca N.R.
      • Wang J.K.
      • Bauer B.
      • Townshend R.J.L.
      • Hollingsworth S.A.
      • Olivieri J.E.
      • Xu H.E.
      • Sommer M.E.
      • Dror R.O.
      Molecular mechanism of GPCR-mediated arrestin activation.
      ). In either case, activated β-arrestins can mediate a plethora of regulatory functions (
      • Gurevich V.V.
      • Gurevich E.V.
      Plethora of functions packed into 45 kDa arrestins: biological implications and possible therapeutic strategies.
      ), and, importantly, they bind clathrin, AP2 (adaptor protein 2), and phosphoinositides (
      • Tian X.
      • Kang D.S.
      • Benovic J.L.
      β-Arrestins and G protein-coupled receptor trafficking.
      ) to initiate the internalization of GPCRs to early endosomes.
      GPCR desensitization mediated by β-arrestins has two well-established layers: 1) the steric blockade of the G protein–binding site by β-arrestin interaction with the receptor's cytosolic core and 2) the removal of receptors from the cell surface via clathrin-coated pit endocytosis. Unexpectedly, this general mechanism of GPCR desensitization by β-arrestins is needed to promote endosomal cAMP production in the cases of PTH1R and V2R (
      • Feinstein T.N.
      • Yui N.
      • Webber M.J.
      • Wehbi V.L.
      • Stevenson H.P.
      • King Jr., J.D.
      • Hallows K.R.
      • Brown D.
      • Bouley R.
      • Vilardaga J.P.
      Noncanonical control of vasopressin receptor type 2 signaling by retromer and arrestin.
      ,
      • Feinstein T.N.
      • Wehbi V.L.
      • Ardura J.A.
      • Wheeler D.S.
      • Ferrandon S.
      • Gardella T.J.
      • Vilardaga J.P.
      Retromer terminates the generation of cAMP by internalized PTH receptors.
      ,
      • Wehbi V.L.
      • Stevenson H.P.
      • Feinstein T.N.
      • Calero G.
      • Romero G.
      • Vilardaga J.P.
      Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gβγ complex.
      ). Notwithstanding the paradoxical action of β-arrestins, few studies shed light on a plausible structural mechanism by which β-arrestins can promote rather than terminate endosomal GPCR signaling through G protein. Biochemical and cellular analyses demonstrated that PTH bound to a ternary PTH1R–Gβγ–arrestin complex can accelerate the rate of Gs activation and increase the steady-state levels of activated Gs, thus suggesting that the ternary complex permits multiple cycles of Gs activation/deactivation or stabilizes the active state of Gαs, thus extending the generation of cAMP (
      • Wehbi V.L.
      • Stevenson H.P.
      • Feinstein T.N.
      • Calero G.
      • Romero G.
      • Vilardaga J.P.
      Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gβγ complex.
      ). The existence of a ternary GPCR–G protein–arrestin complex (referred to as megaplex (
      • Thomsen A.R.B.
      • Plouffe B.
      • Cahill 3rd, T.J.
      • Shukla A.K.
      • Tarrasch J.T.
      • Dosey A.M.
      • Kahsai A.W.
      • Strachan R.T.
      • Pani B.
      • Mahoney J.P.
      • Huang L.
      • Breton B.
      • Heydenreich F.M.
      • Sunahara R.K.
      • Skiniotis G.
      • et al.
      GPCR-G protein-β-arrestin super-complex mediates sustained G protein signaling.
      )) was further supported by negative-staining transmission EM studies revealing the complex of β2AR fused to V2Rpp (β2V2R) simultaneously bound to β-arrestin1 and Gs (
      • Thomsen A.R.B.
      • Plouffe B.
      • Cahill 3rd, T.J.
      • Shukla A.K.
      • Tarrasch J.T.
      • Dosey A.M.
      • Kahsai A.W.
      • Strachan R.T.
      • Pani B.
      • Mahoney J.P.
      • Huang L.
      • Breton B.
      • Heydenreich F.M.
      • Sunahara R.K.
      • Skiniotis G.
      • et al.
      GPCR-G protein-β-arrestin super-complex mediates sustained G protein signaling.
      ). The more recent cryo-EM structure of β2V2R bound to both β-arrestin1 and Gs (
      • Nguyen A.H.
      • Thomsen A.R.B.
      • Cahill 3rd, T.J.
      • Huang R.
      • Huang L.Y.
      • Marcink T.
      • Clarke O.B.
      • Heissel S.
      • Masoudi A.
      • Ben-Hail D.
      • Samaan F.
      • Dandey V.P.
      • Tan Y.Z.
      • Hong C.
      • Mahoney J.P.
      • et al.
      Structure of an endosomal signaling GPCR-G protein-β-arrestin megacomplex.
      ) allows envisioning that the ppCt strongly binds β-arrestin, triggering its activation and subsequent assembly and internalization of the receptor/β-arrestin complex. The inherent flexibility of the ppCt, and possibly the lower affinity of arr-FL interaction with the cytosolic core of the receptor, leads to displacement of the β-arrestin from the core of the receptor, thus freeing space for G protein coupling (Fig. 3D). Nonetheless, β2V2R/Gs/β-arrestin1 megacomplex, solved in a membrane-free environment, represents an artificial system where interaction of a chimeric GPCR with Gs and with β-arrestin1 is stabilized by two nanobodies and by an antibody fragment. As such, it poses a question of how well such a structure reflects naturally in cells forming complexes, such as ternary PTH1R/Gβγ/β-arrestin1 signaling complexes. Specifically, it is not clear how the membrane presence would affect the ppCt flexibility and thus the arrangement of β-arrestin and G protein and what would be the arrangement of such a megacomplex for the receptors with long ppCts (e.g. PTH1R) in the presence of the endosomal lipid membrane. These exciting questions remain to be elucidated by future studies.

      The role of lipids in endosomal GPCR signaling

      As transmembrane proteins, GPCRs constantly and dynamically interact with surrounding lipids of the membrane bilayer of the cell surface or intracellular compartments. The differences in lipid composition in different cell types, physiological states (i.e. healthy versus diseased), and diverse intracellular compartments provide a means of GPCR function modulation. As such, lipids control the GPCR structure and function either through mechanical effects imposed by the physical properties of the bilayer (curvature, thickness, or surface tension) or through specific binding of lipids to GPCRs resulting in allosteric modulation of their function. In addition, the fluidity of the membrane bilayer determined by the nature of the lipid tails may also contribute to the regulation of GPCR signaling and function (
      • Yoshida K.
      • Nagatoishi S.
      • Kuroda D.
      • Suzuki N.
      • Murata T.
      • Tsumoto K.
      Phospholipid membrane fluidity alters ligand binding activity of a G protein-coupled receptor by shifting the conformational equilibrium.
      ).
      Lipids can modulate GPCR function at three levels: 1) modulation of orthosteric ligand binding, 2) modulation of GPCRs oligomerization, and 3) modulation of efficacy/selectivity of downstream effectors' coupling. Such regulation affects the subsequent signaling and trafficking of the receptors. Cholesterol is the most studied lipid with a prominent role for GPCR structure and function, and it variably modulates GPCR activity on all three levels, indirectly by changing membrane physical properties or through direct interaction, in a receptor-dependent manner (see recent reviews (
      • Sarkar P.
      • Chattopadhyay A.
      Cholesterol interaction motifs in G protein-coupled receptors: slippery hot spots?.
      ,
      • Kiriakidi S.
      • Kolocouris A.
      • Liapakis G.
      • Ikram S.
      • Durdagi S.
      • Mavromoustakos T.
      Effects of cholesterol on GPCR function: insights from computational and experimental studies.
      )). The studies of GPCRs reconstituted in a lipidic environment, such as nanodiscs with variable lipid composition, have demonstrated that phospholipids may play a key role in GPCR function. For instance, adding phospholipids to purified light-activated rhodpsin/transducin complex significantly improves its stability (
      • Jastrzebska B.
      • Goc A.
      • Golczak M.
      • Palczewski K.
      Phospholipids are needed for the proper formation, stability, and function of the photoactivated rhodopsin-transducin complex.
      ), the presence of negatively charged POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)) is required for efficient coupling and activation of Gq by NTS1R (
      • Inagaki S.
      • Ghirlando R.
      • White J.F.
      • Gvozdenovic-Jeremic J.
      • Northup J.K.
      • Grisshammer R.
      Modulation of the interaction between neurotensin receptor NTS1 and Gq protein by lipid.
      ), whereas distinct headgroups of the phospholipids differentially modulate ligand binding and the conformational state of the β2AR, with phosphatidylglycerol and phosphatidylethanolamine favoring agonist- and antagonist-bound receptor states, respectively (
      • Dawaliby R.
      • Trubbia C.
      • Delporte C.
      • Masureel M.
      • Van Antwerpen P.
      • Kobilka B.K.
      • Govaerts C.
      Allosteric regulation of G protein-coupled receptor activity by phospholipids.
      ). Recent studies provided evidence that the phospholipids, in particular phosphoinositides, can directly bind GPCRs and potently modulate their function (for a comprehensive overview of the role and cellular distribution of phosphoinositides, see Ref.
      • Hammond G.R.V.
      • Burke J.E.
      Novel roles of phosphoinositides in signaling, lipid transport, and disease.
      ). Yen et al. (
      • Yen H.Y.
      • Hoi K.K.
      • Liko I.
      • Hedger G.
      • Horrell M.R.
      • Song W.
      • Wu D.
      • Heine P.
      • Warne T.
      • Lee Y.
      • Carpenter B.
      • Plückthun A.
      • Tate C.G.
      • Sansom M.S.P.
      • Robinson C.V.
      PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling.
      ) demonstrated that PIP2 specifically binds class A GPCRs, β1AR, NTS1R, and A2AAR, and stabilizes their active state, by promoting coupling to and activation of G proteins, thus suggesting a possible conserved mechanism for class A GPCRs. A subsequent computational study of A2AAR embedded into in vivo–mimetic membrane bilayer corroborated the findings of Yen et al. and provided further structural insights into PIP2 interaction with class A GPCRs and its potential to bridge receptor/G protein interactions (
      • Song W.
      • Yen H.Y.
      • Robinson C.V.
      • Sansom M.S.P.
      State-dependent lipid interactions with the A2a receptor revealed by MD simulations using in vivo-mimetic membranes.
      ).
      The importance of PIP2 is further extended by the findings of its requirement for β-arrestin–mediated internalization of GPCRs. The efficient binding to and phosphorylation of agonist-bound β2AR by GRK5 is achieved only in the presence of PIP2 (
      • Komolov K.E.
      • Du Y.
      • Duc N.M.
      • Betz R.M.
      • Rodrigues J.
      • Leib R.D.
      • Patra D.
      • Skiniotis G.
      • Adams C.M.
      • Dror R.O.
      • Chung K.Y.
      • Kobilka B.K.
      • Benovic J.L.
      Structural and functional analysis of a β2-adrenergic receptor complex with GRK5.
      ), whereas β-arrestins themselves require phosphoinositides for proper endocytosis (
      • Gaidarov I.
      • Krupnick J.G.
      • Falck J.R.
      • Benovic J.L.
      • Keen J.H.
      Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.
      ). The recent structure of NTS1R/β-arrestin1 complex revealed the PIP2 binding site within the concave surface of the C-domain of β-arrestin1 (
      • Huang W.
      • Masureel M.
      • Qianhui Q.
      • Janetzko J.
      • Inoue A.
      • Kato H.E.
      • Robertson M.J.
      • Nguyen K.C.
      • Glenn J.S.
      • Skiniotis G.
      • Kobilka B.K.
      Structure of the neurotensin receptor 1 in complex with β-arrestin 1.
      ). In this complex, PIP2 binding to β-arrestin1 appears to stabilize the tilting of β-arrestin1, which is primarily mediated by the interaction of the loops at the edge of the C-domain with the lipid bilayer. The latter interaction is observed in all three structures of GPCR/arrestin complexes solved so far (
      • Kang Y.
      • Zhou X.E.
      • Gao X.
      • He Y.
      • Liu W.
      • Ishchenko A.
      • Barty A.
      • White T.A.
      • Yefanov O.
      • Han G.W.
      • Xu Q.
      • de Waal P.W.
      • Ke J.
      • Tan M.H.
      • Zhang C.
      • et al.
      Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.
      ,
      • Huang W.
      • Masureel M.
      • Qianhui Q.
      • Janetzko J.
      • Inoue A.
      • Kato H.E.
      • Robertson M.J.
      • Nguyen K.C.
      • Glenn J.S.
      • Skiniotis G.
      • Kobilka B.K.
      Structure of the neurotensin receptor 1 in complex with β-arrestin 1.
      ,
      • Staus D.P.
      • Hu H.
      • Robertson M.J.
      • Kleinhenz A.L.W.
      • Wingler L.M.
      • Capel W.D.
      • Latorraca N.R.
      • Lefkowitz R.J.
      • Skiniotis G.
      Structure of the M2 muscarinic receptor-β-arrestin complex in a lipid nanodisc.
      ), suggesting that such an arrangement of GPCR/arrestin complexes may play a role in initiation of the endocytic vesicle formation by inducing and/or stabilizing membrane curvature.
      These findings unveil a lipid role in class A GPCR function that might also apply to other GPCRs that are able to continue signaling from endosomes. For instance, the structures of PTH1R in its inactive (
      • Ehrenmann J.
      • Schöppe J.
      • Klenk C.
      • Rappas M.
      • Kummer L.
      • Doré A.S.
      • Plückthun A.
      High-resolution crystal structure of parathyroid hormone 1 receptor in complex with a peptide agonist.
      ) and active (
      • Zhao L.H.
      • Ma S.
      • Sutkeviciute I.
      • Shen D.D.
      • Zhou X.E.
      • de Waal P.W.
      • Li C.Y.
      • Kang Y.
      • Clark L.J.
      • Jean-Alphonse F.G.
      • White A.D.
      • Yang D.
      • Dai A.
      • Cai X.
      • Chen J.
      • et al.
      Structure and dynamics of the active human parathyroid hormone receptor-1.
      ) states revealed multiple lipids bound to the periphery of the TMD, suggesting that lipids may also play an important role to the function of this GPCR. As illustrated in our current operational model for PTH-mediated endosomal cAMP (Fig. 4), recent observations unveil that phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) is a critical determinant of PTH1R endosomal signaling by promoting recruitment of β-arrestin and the formation of a PTH1R–Gβγ–βarr complex (
      • White A.D.
      • Jean-Alphonse F.G.
      • Fang F.
      • Peña K.A.
      • Liu S.
      • Konig G.M.
      • Inoue A.
      • Aslanoglou D.
      • Gellman S.H.
      • Kostenis E.
      • Xiao K.
      • Vilardaga J.P.
      Gq/11-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR.
      ) (Fig. 4B).
      Figure thumbnail gr4
      Figure 4Mechanism of PTH1R signaling via Gs and Gq proteins. A, upon PTH binding, PTH1R couples and activates heterotrimeric Gs and Gq proteins at the plasma membrane (steps 1 and 2). Gs activates adenylate cyclases (AC), leading to acute cAMP synthesis and activation of PKA. The time course of cAMP is short due to the action of phosphodiesterases (PDE) (pink box). Gq activates PLCβ, which cleaves phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) into IP3, which diffuses through the cytosol to activate IP3-gated Ca2+ channels, releasing stored Ca2+. B, liberation of Gβγ subunits from Gq promotes PI3Kβ-dependent generation of PI(3,4,5)P3 (step 3), which in turn promotes β-arrestin (βarr) recruitment to the PTH1R (step 4) and formation of ternary PTH1R–βarr–Gβγ complex that remains active following internalization and redistribution to early endosomes (step 5). Reassembly of the ternary PTH1R complex with Gαs is thought to be dependent on Gαs diffusion. C, the sustained phase of cAMP production (step 6) is due to the inhibitory action of extracellular signal-regulated protein kinase 1/2 (ERK1/2) on PDE4 (step 7) and can efficiently diffuse to the nucleus to activate nuclear PKA (step 8). D, termination of endosomal cAMP signaling is initiated by a negative feedback loop, where PKA-dependent activation of the H+ pump v-ATPase increases endosome acidification (step 9), which sequentially disassembles the ternary PTHR–arrestin–Gβγ signaling complex and engages retromer coupling to PTHR (step 10) and its recycling to the cell surface or redistribution in the Golgi apparatus. Adapted from Refs.
      • Kuna R.S.
      • Girada S.B.
      • Asalla S.
      • Vallentyne J.
      • Maddika S.
      • Patterson J.T.
      • Smiley D.L.
      • DiMarchi R.D.
      • Mitra P.
      Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic β-cells.
      and
      • Kiriakidi S.
      • Kolocouris A.
      • Liapakis G.
      • Ikram S.
      • Durdagi S.
      • Mavromoustakos T.
      Effects of cholesterol on GPCR function: insights from computational and experimental studies.
      . This research was originally published in Trends in Endocrinology and Metabolism. Sutkeviciute, I., Clark, L. J., White, A. D., Gardella, T. J., and Vilardaga, J. P. PTH/PTHrP receptor signaling, allostery, and structures. Trends in Endocrinology and Metabolism. 2019; 30:860–874. © Cell Press; Proceedings of the National Academy of Sciences of the United States of America. White, A. D., Jean-Alphonse, F. G., Fang, F., Peña, K. A., Liu, S., Konig, G. M., Inoue, A., Aslanoglou, D., Gellman, S. H., Kostenis, E., Xiao, K., and Vilardaga, J. P. Gq/11-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR. Proceedings of the National Academy of Sciences of the United States of America. 2020; 117:7455–7460. © United States National Academy of Sciences.
      Taken together, the membrane lipids may promote endosomal GPCR signaling by enhancing the recruitment of β-arrestins and subsequent internalization. Once receptors are internalized to the endosomes, the specific endosomal lipids may stabilize the active state of the receptors and promote G protein coupling and activation, whereas the changes in endosomal lipid composition or receptor trafficking to microdomains containing lipids that stabilize the inactive state may contribute to signaling termination. For instance, sorting nexin (Snx) proteins that are involved in GPCR recycling as described below associate with membranes through the interaction of their PX (Phox homology) domains with phosphatidylinositol 3-phosphate, which is the main endosomal lipid (
      • Trousdale C.
      • Kim K.
      Retromer: structure, function, and roles in mammalian disease.
      ). This may imply that dephosphorylation of phosphoinositols that stabilize active-state GPCRs to phosphatidylinositol 3-phosphate may promote their uncoupling from G proteins and transition to an inactive state leading to recycling or retrograde trafficking.

      Structural insights into the resensitization of the GPCR signaling

      The termination of endosomal cAMP signaling in the case of the PTH1R and its resensitization proceeds via two sequential processes (Fig. 4D). The first involves the PKA-dependent phosphorylation and activation of the v-ATPase, resulting in endosomal acidification that causes ligand dissociation from the receptor and disassembly of the ternary PTH1R–Gβγ–βarr complex (
      • Gidon A.
      • Al-Bataineh M.M.
      • Jean-Alphonse F.G.
      • Stevenson H.P.
      • Watanabe T.
      • Louet C.
      • Khatri A.
      • Calero G.
      • Pastor-Soler N.M.
      • Gardella T.J.
      • Vilardaga J.P.
      Endosomal GPCR signaling turned off by negative feedback actions of PKA and v-ATPase.
      ). The second engages receptor coupling with the retromer complex (
      • Feinstein T.N.
      • Wehbi V.L.
      • Ardura J.A.
      • Wheeler D.S.
      • Ferrandon S.
      • Gardella T.J.
      • Vilardaga J.P.
      Retromer terminates the generation of cAMP by internalized PTH receptors.
      ), an elongated 150-kDa heterotrimer composed of vesicle protein sorting 35 (Vps35), an α-helical solenoid, and Vps29 and Vps26, which have structural similarities with phosphoesterases and β-arrestins, respectively. Given that the engagement of β-arrestins and retromer to endosomal PTH1R was found to be mutually exclusive (
      • Feinstein T.N.
      • Wehbi V.L.
      • Ardura J.A.
      • Wheeler D.S.
      • Ferrandon S.
      • Gardella T.J.
      • Vilardaga J.P.
      Retromer terminates the generation of cAMP by internalized PTH receptors.
      ), the retromer complex likely engages with receptors freed of ligand and β-arrestins after they return to an inactive state. The coupling of PTH1R to the retromer can be mediated by Snx27, a PDZ (postsynaptic density protein/Drosophila discs large tumor suppressor/zonula occludens-1 protein, also referred to as PSD95/Dlg/ZO1 domain) ligand-binding adaptor protein that interacts with the PTH1R's C-tail PDZ-binding motif, ETVM, or by direct interaction with Vps26, thus bypassing the requirement of Snx27 (
      • McGarvey J.C.
      • Xiao K.
      • Bowman S.L.
      • Mamonova T.
      • Zhang Q.
      • Bisello A.
      • Sneddon W.B.
      • Ardura J.A.
      • Jean-Alphonse F.
      • Vilardaga J.P.
      • Puthenveedu M.A.
      • Friedman P.A.
      Actin-sorting nexin 27 (SNX27)-retromer complex mediates rapid parathyroid hormone receptor recycling.
      ). The retromer-captured receptor then takes two possible routes: it recycles back to the cell surface, where another cycle of signaling can be initiated, and/or it moves to the Golgi apparatus for functional consequences that remain to be determined. The structural mechanisms of retromer-driven cargo trafficking are just emerging. Recent structural studies of the retromer revealed that it assembles as dimers of the Vps35/Vps29/Vps26 heterotrimers (
      • Lucas M.
      • Gershlick D.C.
      • Vidaurrazaga A.
      • Rojas A.L.
      • Bonifacino J.S.
      • Hierro A.
      Structural mechanism for cargo recognition by the retromer complex.
      ,
      • Kovtun O.
      • Leneva N.
      • Bykov Y.S.
      • Ariotti N.
      • Teasdale R.D.
      • Schaffer M.
      • Engel B.D.
      • Owen D.J.
      • Briggs J.A.G.
      • Collins B.M.
      Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.
      ,
      • Kendall A.K.
      • Xie B.
      • Xu P.
      • Wang J.
      • Burcham R.
      • Frazier M.N.
      • Binshtein E.
      • Wei H.
      • Graham T.R.
      • Nakagawa T.
      • Jackson L.P.
      Mammalian retromer is an adaptable scaffold for cargo sorting from endosomes.
      ). The dimers assume an arch-like shape with the dimer interface located at the apex and formed by electrostatic interactions between the two Vps35 C-terminal domains. The Vps29 subunits decorate both sides of the arch's apex, whereas Vps26 proteins are connected to each Vps35 N-terminal domain tip, forming the feet that engage in interactions with the endosomal membrane surface via adaptor proteins (Fig. 5). This arch-shaped retromer structure has a high degree of plasticity (
      • Kendall A.K.
      • Xie B.
      • Xu P.
      • Wang J.
      • Burcham R.
      • Frazier M.N.
      • Binshtein E.
      • Wei H.
      • Graham T.R.
      • Nakagawa T.
      • Jackson L.P.
      Mammalian retromer is an adaptable scaffold for cargo sorting from endosomes.
      ): the angle at the apex may vary such that Vps35 C termini and Vps29 subunits might be brought close to or pulled away from the membrane surface, whereas Vps26 binds the Vps35 in a particularly flexible manner (
      • Kendall A.K.
      • Xie B.
      • Xu P.
      • Wang J.
      • Burcham R.
      • Frazier M.N.
      • Binshtein E.
      • Wei H.
      • Graham T.R.
      • Nakagawa T.
      • Jackson L.P.
      Mammalian retromer is an adaptable scaffold for cargo sorting from endosomes.
      ). Such inherent plasticity of the retromer likely allows adapting to different membrane curvatures and binding various adaptor proteins. The finding that retromer heterotrimers in vitro form dimers or even higher-order oligomers (
      • Kendall A.K.
      • Xie B.
      • Xu P.
      • Wang J.
      • Burcham R.
      • Frazier M.N.
      • Binshtein E.
      • Wei H.
      • Graham T.R.
      • Nakagawa T.
      • Jackson L.P.
      Mammalian retromer is an adaptable scaffold for cargo sorting from endosomes.
      ) suggests that such preformed oligomers are recruited to the endosomal membrane surfaces. Recent cryo-electron tomography studies of thermophilic fungus Chaetomium thermophilum retromer revealed that the dimers of Vps35/Vps29/Vps26 heterotrimers assemble on Vps5-coated membrane tubules in a semiregular manner (
      • Kovtun O.
      • Leneva N.
      • Bykov Y.S.
      • Ariotti N.
      • Teasdale R.D.
      • Schaffer M.
      • Engel B.D.
      • Owen D.J.
      • Briggs J.A.G.
      • Collins B.M.
      Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.
      ). In such assemblies, Vps29 and Vps35 subunits are exposed at the surface and are available for the interaction with multiple regulatory proteins to modulate the captured cargos and to allow for further tubule trafficking. Despite these recent insights, the specific structural details of GPCR recruitment to the retromer system remain to be elucidated.
      Figure thumbnail gr5
      Figure 5Structural view of retromer recruitment to endosomes. The putative engagement of the inactive PTH1R (PDB entry 6FJ3) to the retromer complex (cryo-ET structure, PDB 6H7W) through Snx27 on the endosomal membrane bilayer surface. The complex of Snx27 PDZ/PTH1R C-tail (PDB 4Z8J) was superimposed onto Vps26A/Snx27 PDZ complex (PDB entry 4P2A), which was then superimposed onto Vps26 in the retromer dimer complex (PDB entry 6H7W); the second Vps26 subunit (right side) in the retromer dimer complex was placed in a putative positioning.

      Pathophysiological implications of endosomal GPCR signaling via G proteins

      The spatiotemporal bias of GPCR signaling via G proteins has physiological outcomes, disease relevance, and therapeutic potential. It appears that endosomal cAMP signaling entails multiple aspects relevant to cellular physiology: 1) it prolongs the duration of the cAMP signaling cascade in response to low extracellular concentrations of circulating hormones, such as PTH; 2) it provides specialized platforms for efficient coupling to PKA-mediated signal compartmentalization (
      • Tsvetanova N.G.
      • von Zastrow M.
      Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis.
      ,
      • Stoeber M.
      • Jullie D.
      • Lobingier B.T.
      • Laeremans T.
      • Steyaert J.
      • Schiller P.W.
      • Manglik A.
      • von Zastrow M.
      A genetically encoded biosensor reveals location bias of opioid drug action.
      ); and/or 3) it facilitates the diffusion of cAMP into the nucleus, a rate-limiting process for nuclear PKA activation that in turn regulates CREB (cAMP-response element–binding protein), a determinant transcription factor (
      • Jean-Alphonse F.G.
      • Wehbi V.L.
      • Chen J.
      • Noda M.
      • Taboas J.M.
      • Xiao K.
      • Vilardaga J.P.
      β2-Adrenergic receptor control of endosomal PTH receptor signaling via Gβγ.
      ,
      • Nigg E.A.
      • Hilz H.
      • Eppenberger H.M.
      • Dutly F.
      Rapid and reversible translocation of the catalytic subunit of cAMP-dependent protein kinase type II from the Golgi complex to the nucleus.
      ). In the case of the PTH1R, transient plasma membrane cAMP response–inducing ligands, such as PTHrP or ABL (abaloparatide), demonstrate bone formation (
      • Bhattacharyya S.
      • Pal S.
      • Chattopadhyay N.
      Abaloparatide, the second generation osteoanabolic drug: Molecular mechanisms underlying its advantages over the first-in-class teriparatide.
      ,
      • Miao D.
      • He B.
      • Jiang Y.
      • Kobayashi T.
      • Sorocéanu M.A.
      • Zhao J.
      • Su H.
      • Tong X.
      • Amizuka N.
      • Gupta A.
      • Genant H.K.
      • Kronenberg H.M.
      • Goltzman D.
      • Karaplis A.C.
      Osteoblast-derived PTHrP is a potent endogenous bone anabolic agent that modifies the therapeutic efficacy of administered PTH 1-34.
      ), whereas LA-PTH, a long-acting chimera of PTH and PTHrP, which triggers substantially more prolonged endosomal cAMP response than PTH, also leads to more enhanced and prolonged blood Ca2+ elevation in mice and in monkeys compared with PTH (
      • Okazaki M.
      • Ferrandon S.
      • Vilardaga J.P.
      • Bouxsein M.L.
      • Potts Jr., J.T.
      • Gardella T.J.
      Prolonged signaling at the parathyroid hormone receptor by peptide ligands targeted to a specific receptor conformation.
      ,
      • Shimizu M.
      • Joyashiki E.
      • Noda H.
      • Watanabe T.
      • Okazaki M.
      • Nagayasu M.
      • Adachi K.
      • Tamura T.
      • Potts Jr., J.T.
      • Gardella T.J.
      • Kawabe Y.
      Pharmacodynamic actions of a long-acting PTH analog (LA-PTH) in thyroparathyroidectomized (TPTX) rats and normal monkeys.
      ). A new form of hypocalcemia has been reported in patients carrying a PTH mutant (Arg at position 25 is substituted by Cys) that appeared to be defective in engaging endosomal PTH1R signaling via cAMP as well as blood Ca2+ elevation in mice (
      • White A.D.
      • Fang F.
      • Jean-Alphonse F.G.
      • Clark L.J.
      • An H.J.
      • Liu H.
      • Zhao Y.
      • Reynolds S.L.
      • Lee S.
      • Xiao K.
      • Sutkeviciute I.
      • Vilardaga J.P.
      Ca2+ allostery in PTH-receptor signaling.
      ,
      • Lee S.
      • Mannstadt M.
      • Guo J.
      • Kim S.M.
      • Yi H.S.
      • Khatri A.
      • Dean T.
      • Okazaki M.
      • Gardella T.J.
      • Jüppner H.
      A homozygous [Cys25]PTH(1-84) mutation that impairs PTH/PTHrP receptor activation defines a novel form of hypoparathyroidism.
      ). These observations link the duration and location of cAMP signaling mediated by PTH1R activation with distinct physiological outcomes. Studies with the neurokinin 1 receptor (NK1R) evidenced that substance P can mediate endosomal NK1R signaling via cAMP and PKC as possible pathways promoting pain transmission by neurons of the spinal cord (
      • Jensen D.D.
      • Lieu T.
      • Halls M.L.
      • Veldhuis N.A.
      • Imlach W.L.
      • Mai Q.N.
      • Poole D.P.
      • Quach T.
      • Aurelio L.
      • Conner J.
      • Herenbrink C.K.
      • Barlow N.
      • Simpson J.S.
      • Scanlon M.J.
      • Graham B.
      • et al.
      Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.
      ). Despite our gap in understanding how endosomal Gq can stimulate PKC activation, elegant studies demonstrated that NK1R antagonists either conjugated to cholestanol or encapsulated in polymeric pH-sensitive nanoparticles block selectively endosomal NK1R signaling in cells and substance P–induced activation of pain transmission in the spine in rodents (
      • Jensen D.D.
      • Lieu T.
      • Halls M.L.
      • Veldhuis N.A.
      • Imlach W.L.
      • Mai Q.N.
      • Poole D.P.
      • Quach T.
      • Aurelio L.
      • Conner J.
      • Herenbrink C.K.
      • Barlow N.
      • Simpson J.S.
      • Scanlon M.J.
      • Graham B.
      • et al.
      Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.
      ,
      • Ramírez-García P.D.
      • Retamal J.S.
      • Shenoy P.
      • Imlach W.
      • Sykes M.
      • Truong N.
      • Constandil L.
      • Pelissier T.
      • Nowell C.J.
      • Khor S.Y.
      • Layani L.M.
      • Lumb C.
      • Poole D.P.
      • Lieu T.
      • Stewart G.D.
      • et al.
      A pH-responsive nanoparticle targets the neurokinin 1 receptor in endosomes to prevent chronic pain.
      ). Collectively, these observations link endosomal G protein signaling with distinct physiological outcomes, such as Ca2+ metabolism for PTH1R and chronic pain for NK1R. The development of novel ligands capable of modulating endosomal GPCR/G protein signaling might present valuable therapeutic tools. For example, PTH analogs that favor endosomal cAMP signaling may present drug candidates for treatment of hypoparathyroidism, and endosomal delivery of NK1R antagonist would provide an alternative to opioids for chronic pain treatment.

      Departure for future GPCR research

      In humans there are over 800 GPCRs that couple to only four major types of G proteins, giving rise to a limited number of second messenger responses (Fig. 1). Yet GPCR activation is translated to a surprising diversity of cellular and physiological responses. The mechanisms underlying such a high diversity of signaling outputs are being actively pursued. In this regard, GPCRs are viewed as an integral part of a highly complex functional network within the cells, where their signaling cross-talks with other signaling networks. The GPCR signaling itself falls under multilevel regulation mechanisms, including 1) homo- and/or heteromerization (reviewed in Ref.
      • Ferré S.
      • Casadó V.
      • Devi L.A.
      • Filizola M.
      • Jockers R.
      • Lohse M.J.
      • Milligan G.
      • Pin J.P.
      • Guitart X.
      G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives.
      ) resulting in altered GPCR function as it pertains to G protein coupling selectivity/efficacy or trafficking; 2) regulation of function by accessory proteins such as RAMPs (receptor activity–regulating proteins) for class B and other GPCRs (
      • Serafin D.S.
      • Harris N.R.
      • Nielsen N.R.
      • Mackie D.I.
      • Caron K.M.
      Dawn of a new RAMPage.
      ) or co-receptors such as LRP5/6 (lipoprotein receptor-related protein 5 or 6) for Frizzled receptor signaling in response to WNT (
      • Bilic J.
      • Huang Y.L.
      • Davidson G.
      • Zimmermann T.
      • Cruciat C.M.
      • Bienz M.
      • Niehrs C.
      Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation.
      ); and 3) regulation of trafficking contributing to spatiotemporal GPCR signaling. Thus, the spatiotemporal control of GPCR signaling only partially explains the high diversity of GPCR signaling outcomes. As such, the localization of GPCR signaling complexes in various intracellular compartments allows signal generation at distinct intracellular locations allowing the activation of local effectors, which in turn translates to distinct cellular responses. This review discussed the implications of endosomal GPCR signaling; however, GPCRs can also signal from other intracellular compartments with distinct physiological outputs, including Golgi, ER, nucleus, and mitochondria (reviewed in Refs.
      • Jong Y.I.
      • Harmon S.K.
      • O'Malley K.L.
      GPCR signalling from within the cell.
      ,
      • Lobingier B.T.
      • von Zastrow M.
      When trafficking and signaling mix: how subcellular location shapes G protein-coupled receptor activation of heterotrimeric G proteins.
      ,
      • Calebiro D.
      • Godbole A.
      Internalization of G-protein-coupled receptors: implication in receptor function, physiology and diseases.
      ,
      • Eichel K.
      • von Zastrow M.
      Subcellular organization of GPCR signaling.
      ,
      • Plouffe B.
      • Thomsen A.R.B.
      • Irannejad R.
      Emerging role of compartmentalized G protein-coupled receptor signaling in the cardiovascular field.
      ,
      • Retamal J.S.
      • Ramirez-Garcia P.D.
      • Shenoy P.A.
      • Poole D.P.
      • Veldhuis N.A.
      Internalized GPCRs as potential therapeutic targets for the management of pain.
      ,
      • Hanyaloglu A.C.
      Advances in membrane trafficking and endosomal signaling of G protein-coupled receptors.
      ,
      • Jong Y.I.
      • Harmon S.K.
      • O'Malley K.L.
      Intracellular GPCRs play key roles in synaptic plasticity.
      ). For example, mouse brain nonsynaptosomal mitochondria express a functional melatonin type 1 receptor (MT1) in their outer membranes as well as Gi proteins and β-arrestins in their intermembrane spaces (
      • Suofu Y.
      • Li W.
      • Jean-Alphonse F.G.
      • Jia J.
      • Khattar N.K.
      • Li J.
      • Baranov S.V.
      • Leronni D.
      • Mihalik A.C.
      • He Y.
      • Cecon E.
      • Wehbi V.L.
      • Kim J.
      • Heath B.E.
      • Baranova O.V.
      • et al.
      Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.
      ). The mitochondrial MT1 mediates melatonin-dependent inhibition of stress-mediated cytochrome c release and caspase activation as possible means of ischemic brain injury reduction. Whereas melatonin activates mitochondrial Gi protein and inhibits forskolin-mediated cAMP production in purified brain mitochondria, more studies are needed to determine the role of the mitochondrial Gi signaling pathway in the neuroprotective action of melatonin. Remarkably, melatonin itself is synthesized from serotonin in the mitochondrial matrix and released upon Ca2+-induced stress, after which it likely binds and activates the mitochondrial MT1, a process referred to as automitocrine. Given the progressive and unavoidable age-associated loss of melatonin (
      • Karasek M.
      Melatonin, human aging, and age-related diseases.
      ), the automitocrine action of melatonin supports a tantalizing new way to rethink aging: if mitochondrial MT1 signaling is a determinant of cell death prevention, then the development and optimization of cell-permeant melatonin analogs (
      • Gbahou F.
      • Cecon E.
      • Viault G.
      • Gerbier R.
      • Jean-Alphonse F.
      • Karamitri A.
      • Guillaumet G.
      • Delagrange P.
      • Friedlander R.M.
      • Vilardaga J.P.
      • Suzenet F.
      • Jockers R.
      Design and validation of the first cell-impermeant melatonin receptor agonist.
      ) might lead to novel therapies for treating or preventing age-associated neurodegeneration. Overall, these insights are changing our classical understanding of the biological functions of GPCRs and exemplify new signaling schemes for regulation of cell functions in tissues, which hopefully will spur new and exciting research avenues.

      References

        • Sriram K.
        • Insel P.A.
        G protein-coupled receptors as targets for approved drugs: how many targets and how many drugs?.
        Mol. Pharmacol. 2018; 93 (29298813): 251-258
        • Hauser A.S.
        • Attwood M.M.
        • Rask-Andersen M.
        • Schiöth H.B.
        • Gloriam D.E.
        Trends in GPCR drug discovery: new agents, targets and indications.
        Nat. Rev. Drug Discov. 2017; 16 (29075003): 829-842
        • Cabrera-Vera T.M.
        • Vanhauwe J.
        • Thomas T.O.
        • Medkova M.
        • Preininger A.
        • Mazzoni M.R.
        • Hamm H.E.
        Insights into G protein structure, function, and regulation.
        Endocr. Rev. 2003; 24 (14671004): 765-781
        • Neer E.J.
        • Clapham D.E.
        Roles of G protein subunits in transmembrane signalling.
        Nature. 1988; 333 (3130578): 129-134
        • Janetopoulos C.
        • Jin T.
        • Devreotes P.
        Receptor-mediated activation of heterotrimeric G-proteins in living cells.
        Science. 2001; 291 (11264536): 2408-2411
        • Bünemann M.
        • Frank M.
        • Lohse M.J.
        Gi protein activation in intact cells involves subunit rearrangement rather than dissociation.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100 (14673086): 16077-16082
        • De Vries L.
        • Zheng B.
        • Fischer T.
        • Elenko E.
        • Farquhar M.G.
        The regulator of G protein signaling family.
        Annu. Rev. Pharmacol. Toxicol. 2000; 40 (10836135): 235-271
        • Jong Y.I.
        • Harmon S.K.
        • O'Malley K.L.
        GPCR signalling from within the cell.
        Br. J. Pharmacol. 2018; 175 (28872669): 4026-4035
        • Lobingier B.T.
        • von Zastrow M.
        When trafficking and signaling mix: how subcellular location shapes G protein-coupled receptor activation of heterotrimeric G proteins.
        Traffic. 2019; 20 (30578610): 130-136
        • Calebiro D.
        • Godbole A.
        Internalization of G-protein-coupled receptors: implication in receptor function, physiology and diseases.
        Best Pract. Res. Clin. Endocrinol. Metab. 2018; 32 (29678288): 83-91
        • Eichel K.
        • von Zastrow M.
        Subcellular organization of GPCR signaling.
        Trends Pharmacol. Sci. 2018; 39 (29478570): 200-208
        • Plouffe B.
        • Thomsen A.R.B.
        • Irannejad R.
        Emerging role of compartmentalized G protein-coupled receptor signaling in the cardiovascular field.
        ACS Pharmacol. Transl. Sci. 2020; 3 (32296764): 221-236
        • Retamal J.S.
        • Ramirez-Garcia P.D.
        • Shenoy P.A.
        • Poole D.P.
        • Veldhuis N.A.
        Internalized GPCRs as potential therapeutic targets for the management of pain.
        Front. Mol. Neurosci. 2019; 12 (31798411): 273
        • Hanyaloglu A.C.
        Advances in membrane trafficking and endosomal signaling of G protein-coupled receptors.
        Int. Rev. Cell Mol. Biol. 2018; 339 (29776606): 93-131
        • Jong Y.I.
        • Harmon S.K.
        • O'Malley K.L.
        Intracellular GPCRs play key roles in synaptic plasticity.
        ACS Chem. Neurosci. 2018; 9 (29409317): 2162-2172
        • Gurevich V.V.
        • Gurevich E.V.
        Plethora of functions packed into 45 kDa arrestins: biological implications and possible therapeutic strategies.
        Cell Mol. Life Sci. 2019; 76 (31422444): 4413-4421
        • Laporte S.A.
        • Scott M.G.H.
        β-Arrestins: multitask scaffolds orchestrating the where and when in cell signalling.
        Methods Mol. Biol. 2019; 1957 (30919345): 9-55
        • Horwitz M.J.
        • Tedesco M.B.
        • Sereika S.M.
        • Hollis B.W.
        • Garcia-Ocaña A.
        • Stewart A.F.
        Direct comparison of sustained infusion of human parathyroid hormone-related protein-(1-36) [hPTHrP-(1-36)] versus hPTH-(1-34) on serum calcium, plasma 1,25-dihydroxyvitamin D concentrations, and fractional calcium excretion in healthy human volunteers.
        J. Clin. Endocrinol. Metab. 2003; 88 (12679445): 1603-1609
        • Horwitz M.J.
        • Tedesco M.B.
        • Sereika S.M.
        • Syed M.A.
        • Garcia-Ocaña A.
        • Bisello A.
        • Hollis B.W.
        • Rosen C.J.
        • Wysolmerski J.J.
        • Dann P.
        • Gundberg C.
        • Stewart A.F.
        Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)2 vitamin D.
        J. Bone Miner. Res. 2005; 20 (16160737): 1792-1803
        • Ferrandon S.
        • Feinstein T.N.
        • Castro M.
        • Wang B.
        • Bouley R.
        • Potts J.T.
        • Gardella T.J.
        • Vilardaga J.P.
        Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.
        Nat. Chem. Biol. 2009; 5 (19701185): 734-742
        • Jean-Alphonse F.G.
        • Wehbi V.L.
        • Chen J.
        • Noda M.
        • Taboas J.M.
        • Xiao K.
        • Vilardaga J.P.
        β2-Adrenergic receptor control of endosomal PTH receptor signaling via Gβγ.
        Nat. Chem. Biol. 2017; 13 (28024151): 259-261
        • Calebiro D.
        • Nikolaev V.O.
        • Gagliani M.C.
        • de Filippis T.
        • Dees C.
        • Tacchetti C.
        • Persani L.
        • Lohse M.J.
        Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.
        PLoS Biol. 2009; 7 (19688034): e1000172
        • Mullershausen F.
        • Zecri F.
        • Cetin C.
        • Billich A.
        • Guerini D.
        • Seuwen K.
        Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors.
        Nat. Chem. Biol. 2009; 5 (19430484): 428-434
        • White A.D.
        • Fang F.
        • Jean-Alphonse F.G.
        • Clark L.J.
        • An H.J.
        • Liu H.
        • Zhao Y.
        • Reynolds S.L.
        • Lee S.
        • Xiao K.
        • Sutkeviciute I.
        • Vilardaga J.P.
        Ca2+ allostery in PTH-receptor signaling.
        Proc. Natl. Acad. Sci. U. S. A. 2019; 116 (30718391): 3294-3299
        • Kotowski S.J.
        • Hopf F.W.
        • Seif T.
        • Bonci A.
        • von Zastrow M.
        Endocytosis promotes rapid dopaminergic signaling.
        Neuron. 2011; 71 (21791287): 278-290
        • Merriam L.A.
        • Baran C.N.
        • Girard B.M.
        • Hardwick J.C.
        • May V.
        • Parsons R.L.
        Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.
        J. Neurosci. 2013; 33 (23467377): 4614-4622
        • May V.
        • Parsons R.L.
        G protein-coupled receptor endosomal signaling and regulation of neuronal excitability and stress responses: signaling options and lessons from the PAC1 receptor.
        J. Cell. Physiol. 2017; 232 (27661062): 698-706
        • Kuna R.S.
        • Girada S.B.
        • Asalla S.
        • Vallentyne J.
        • Maddika S.
        • Patterson J.T.
        • Smiley D.L.
        • DiMarchi R.D.
        • Mitra P.
        Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic β-cells.
        Am. J. Physiol. Endocrinol. Metab. 2013; 305 (23592482): E161-E170
        • Feinstein T.N.
        • Yui N.
        • Webber M.J.
        • Wehbi V.L.
        • Stevenson H.P.
        • King Jr., J.D.
        • Hallows K.R.
        • Brown D.
        • Bouley R.
        • Vilardaga J.P.
        Noncanonical control of vasopressin receptor type 2 signaling by retromer and arrestin.
        J. Biol. Chem. 2013; 288 (23935101): 27849-27860
        • Andreassen K.V.
        • Hjuler S.T.
        • Furness S.G.
        • Sexton P.M.
        • Christopoulos A.
        • Nosjean O.
        • Karsdal M.A.
        • Henriksen K.
        Prolonged calcitonin receptor signaling by salmon, but not human calcitonin, reveals ligand bias.
        PLoS ONE. 2014; 9: e92042
        • Tsvetanova N.G.
        • von Zastrow M.
        Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis.
        Nat. Chem. Biol. 2014; 10 (25362359): 1061-1065
        • Lyga S.
        • Volpe S.
        • Werthmann R.C.
        • Götz K.
        • Sungkaworn T.
        • Lohse M.J.
        • Calebiro D.
        Persistent cAMP signaling by internalized LH receptors in ovarian follicles.
        Endocrinology. 2016; 157 (26828746): 1613-1621
        • Jensen D.D.
        • Lieu T.
        • Halls M.L.
        • Veldhuis N.A.
        • Imlach W.L.
        • Mai Q.N.
        • Poole D.P.
        • Quach T.
        • Aurelio L.
        • Conner J.
        • Herenbrink C.K.
        • Barlow N.
        • Simpson J.S.
        • Scanlon M.J.
        • Graham B.
        • et al.
        Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.
        Sci. Transl. Med. 2017; 9 (28566424): eaal3447
        • Yarwood R.E.
        • Imlach W.L.
        • Lieu T.
        • Veldhuis N.A.
        • Jensen D.D.
        • Klein Herenbrink C.
        • Aurelio L.
        • Cai Z.
        • Christie M.J.
        • Poole D.P.
        • Porter C.J.H.
        • McLean P.
        • Hicks G.A.
        • Geppetti P.
        • Halls M.L.
        • et al.
        Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.
        Proc. Natl. Acad. Sci. U. S. A. 2017; 114 (29087309): 12309-12314
        • Jimenez-Vargas N.N.
        • Pattison L.A.
        • Zhao P.
        • Lieu T.
        • Latorre R.
        • Jensen D.D.
        • Castro J.
        • Aurelio L.
        • Le G.T.
        • Flynn B.
        • Herenbrink C.K.
        • Yeatman H.R.
        • Edgington-Mitchell L.
        • Porter C.J.H.
        • Halls M.L.
        • et al.
        Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome.
        Proc. Natl. Acad. Sci. U. S. A. 2018; 115 (30012612): E7438-E7447
        • English E.J.
        • Mahn S.A.
        • Marchese A.
        Endocytosis is required for CXC chemokine receptor type 4 (CXCR4)-mediated Akt activation and antiapoptotic signaling.
        J. Biol. Chem. 2018; 293 (29899118): 11470-11480
        • Stoeber M.
        • Jullie D.
        • Lobingier B.T.
        • Laeremans T.
        • Steyaert J.
        • Schiller P.W.
        • Manglik A.
        • von Zastrow M.
        A genetically encoded biosensor reveals location bias of opioid drug action.
        Neuron. 2018; 98 (29754753): 963-976.e5
        • Azimzadeh P.
        • Talamantez-Lyburn S.C.
        • Chang K.T.
        • Inoue A.
        • Balenga N.
        Spatial regulation of GPR64/ADGRG2 signaling by β-arrestins and GPCR kinases.
        Ann. N. Y. Acad. Sci. 2019; 1456 (31502283): 26-43
        • Gorvin C.M.
        • Rogers A.
        • Hastoy B.
        • Tarasov A.I.
        • Frost M.
        • Sposini S.
        • Inoue A.
        • Whyte M.P.
        • Rorsman P.
        • Hanyaloglu A.C.
        • Breitwieser G.E.
        • Thakker R.V.
        AP2σ mutations impair calcium-sensing receptor trafficking and signaling, and show an endosomal pathway to spatially direct G-protein selectivity.
        Cell Rep. 2018; 22 (29420171): 1054-1066
        • Sutkeviciute I.
        • Clark L.J.
        • White A.D.
        • Gardella T.J.
        • Vilardaga J.P.
        PTH/PTHrP receptor signaling, allostery, and structures.
        Trends Endocrinol. Metab. 2019; 30 (31699241): 860-874
        • Thal D.M.
        • Vuckovic Z.
        • Draper-Joyce C.J.
        • Liang Y.L.
        • Glukhova A.
        • Christopoulos A.
        • Sexton P.M.
        Recent advances in the determination of G protein-coupled receptor structures.
        Curr. Opin. Struct. Biol. 2018; 51 (29547818): 28-34
        • Lin X.
        • Li M.
        • Wang N.
        • Wu Y.
        • Luo Z.
        • Guo S.
        • Han G.W.
        • Li S.
        • Yue Y.
        • Wei X.
        • Xie X.
        • Chen Y.
        • Zhao S.
        • Wu J.
        • Lei M.
        • et al.
        Structural basis of ligand recognition and self-activation of orphan GPR52.
        Nature. 2020; 579 (32076264): 152-157
        • Zhou Q.
        • Yang D.
        • Wu M.
        • Guo Y.
        • Guo W.
        • Zhong L.
        • Cai X.
        • Dai A.
        • Jang W.
        • Shakhnovich E.I.
        • Liu Z.J.
        • Stevens R.C.
        • Lambert N.A.
        • Babu M.M.
        • Wang M.W.
        • et al.
        Common activation mechanism of class A GPCRs.
        Elife. 2019; 8 (31855179): e50279
        • Eddy M.T.
        • Lee M.Y.
        • Gao Z.G.
        • White K.L.
        • Didenko T.
        • Horst R.
        • Audet M.
        • Stanczak P.
        • McClary K.M.
        • Han G.W.
        • Jacobson K.A.
        • Stevens R.C.
        • Wuthrich K.
        Allosteric coupling of drug binding and intracellular signaling in the A2A adenosine receptor.
        Cell. 2018; 172 (29290469): 68-80.e12
        • Filipek S.
        Molecular switches in GPCRs.
        Curr. Opin. Struct. Biol. 2019; 55 (31082695): 114-120
        • Wescott M.P.
        • Kufareva I.
        • Paes C.
        • Goodman J.R.
        • Thaker Y.
        • Puffer B.A.
        • Berdougo E.
        • Rucker J.B.
        • Handel T.M.
        • Doranz B.J.
        Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices.
        Proc. Natl. Acad. Sci. U. S. A. 2016; 113 (27543332): 9928-9933
        • Tehan B.G.
        • Bortolato A.
        • Blaney F.E.
        • Weir M.P.
        • Mason J.S.
        Unifying family A GPCR theories of activation.
        Pharmacol. Ther. 2014; 143 (24561131): 51-60
        • Holst B.
        • Nygaard R.
        • Valentin-Hansen L.
        • Bach A.
        • Engelstoft M.S.
        • Petersen P.S.
        • Frimurer T.M.
        • Schwartz T.W.
        A conserved aromatic lock for the tryptophan rotameric switch in TM-VI of seven-transmembrane receptors.
        J. Biol. Chem. 2010; 285 (19920139): 3973-3985
        • Nygaard R.
        • Frimurer T.M.
        • Holst B.
        • Rosenkilde M.M.
        • Schwartz T.W.
        Ligand binding and micro-switches in 7TM receptor structures.
        Trends Pharmacol. Sci. 2009; 30 (19375807): 249-259
        • Hofmann K.P.
        • Scheerer P.
        • Hildebrand P.W.
        • Choe H.W.
        • Park J.H.
        • Heck M.
        • Ernst O.P.
        A G protein-coupled receptor at work: the rhodopsin model.
        Trends Biochem. Sci. 2009; 34 (19836958): 540-552
        • Trzaskowski B.
        • Latek D.
        • Yuan S.
        • Ghoshdastider U.
        • Debinski A.
        • Filipek S.
        Action of molecular switches in GPCRs—theoretical and experimental studies.
        Curr. Med. Chem. 2012; 19 (22300046): 1090-1109
        • Ballesteros J.A.
        • Weinstein H.
        Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors.
        Methods Neurosci. 1995; 25: 366-428
        • Ishchenko A.
        • Wacker D.
        • Kapoor M.
        • Zhang A.
        • Han G.W.
        • Basu S.
        • Patel N.
        • Messerschmidt M.
        • Weierstall U.
        • Liu W.
        • Katritch V.
        • Roth B.L.
        • Stevens R.C.
        • Cherezov V.
        Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody.
        Proc. Natl. Acad. Sci. U. S. A. 2017; 114 (28716900): 8223-8228
        • Schönegge A.M.
        • Gallion J.
        • Picard L.P.
        • Wilkins A.D.
        • Le Gouill C.
        • Audet M.
        • Stallaert W.
        • Lohse M.J.
        • Kimmel M.
        • Lichtarge O.
        • Bouvier M.
        Evolutionary action and structural basis of the allosteric switch controlling β2AR functional selectivity.
        Nat. Commun. 2017; 8 (29255305): 2169
        • Kato H.E.
        • Zhang Y.
        • Hu H.
        • Suomivuori C.M.
        • Kadji F.M.N.
        • Aoki J.
        • Krishna Kumar K.
        • Fonseca R.
        • Hilger D.
        • Huang W.
        • Latorraca N.R.
        • Inoue A.
        • Dror R.O.
        • Kobilka B.K.
        • Skiniotis G.
        Conformational transitions of a neurotensin receptor 1-Gi1 complex.
        Nature. 2019; 572 (31243364): 80-85
        • Liu W.
        • Chun E.
        • Thompson A.A.
        • Chubukov P.
        • Xu F.
        • Katritch V.
        • Han G.W.
        • Roth C.B.
        • Heitman L.H.
        • IJzerman A.P.
        • Cherezov V.
        • Stevens R.C.
        Structural basis for allosteric regulation of GPCRs by sodium ions.
        Science. 2012; 337 (22798613): 232-236
        • Yuan S.
        • Vogel H.
        • Filipek S.
        The role of water and sodium ions in the activation of the μ-opioid receptor.
        Angew. Chem. Int. Ed. Engl. 2013; 52 (23904331): 10112-10115
        • Fenalti G.
        • Giguere P.M.
        • Katritch V.
        • Huang X.P.
        • Thompson A.A.
        • Cherezov V.
        • Roth B.L.
        • Stevens R.C.
        Molecular control of δ-opioid receptor signalling.
        Nature. 2014; 506 (24413399): 191-196
        • Katritch V.
        • Fenalti G.
        • Abola E.E.
        • Roth B.L.
        • Cherezov V.
        • Stevens R.C.
        Allosteric sodium in class A GPCR signaling.
        Trends Biochem. Sci. 2014; 39 (24767681): 233-244
        • Vickery O.N.
        • Carvalheda C.A.
        • Zaidi S.A.
        • Pisliakov A.V.
        • Katritch V.
        • Zachariae U.
        Intracellular transfer of Na+ in an active-state G-protein-coupled receptor.
        Structure. 2018; 26 (29249607): 171-180.e2
        • White K.L.
        • Eddy M.T.
        • Gao Z.G.
        • Han G.W.
        • Lian T.
        • Deary A.
        • Patel N.
        • Jacobson K.A.
        • Katritch V.
        • Stevens R.C.
        Structural connection between activation microswitch and allosteric sodium site in GPCR signaling.
        Structure. 2018; 26 (29395784): 259-269.e5