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Mechanisms of adhesion G protein–coupled receptor activation

Open AccessPublished:August 06, 2020DOI:https://doi.org/10.1074/jbc.REV120.007423
      Adhesion G protein–coupled receptors (AGPCRs) are a thirty-three-member subfamily of Class B GPCRs that control a wide array of physiological processes and are implicated in disease. AGPCRs uniquely contain large, self-proteolyzing extracellular regions that range from hundreds to thousands of residues in length. AGPCR autoproteolysis occurs within the extracellular GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the N terminus of the G protein–coupling seven-transmembrane–spanning bundle. GAIN domain–mediated self-cleavage is constitutive and produces two-fragment holoreceptors that remain bound at the cell surface. It has been of recent interest to understand how AGPCRs are activated in relation to their two-fragment topologies. Dissociation of the AGPCR fragments stimulates G protein signaling through the action of the tethered-peptide agonist stalk that is occluded within the GAIN domain in the holoreceptor form. AGPCRs can also signal independently of fragment dissociation, and a few receptors possess GAIN domains incapable of self-proteolysis. This has resulted in complex theories as to how these receptors are activated in vivo, complicating pharmacological advances. Currently, there is no existing structure of an activated AGPCR to support any of the theories. Further confounding AGPCR research is that many of the receptors remain orphans and lack identified activating ligands. In this review, we provide a detailed layout of the current theorized modes of AGPCR activation with discussion of potential parallels to mechanisms used by other GPCR classes. We provide a classification means for the ligands that have been identified and discuss how these ligands may activate AGPCRs in physiological contexts.
      G protein–coupled receptors (GPCRs) are the largest class of membrane receptors, comprising over 800 members in humans. The GPCR seven-transmembrane helical bundle (7TM) allows for regulation of distinct G protein signaling cascades in response to diverse extracellular stimuli. Due to a broad influence on health and disease, GPCRs are heavily investigated for pharmacological intervention and are the targets of many approved drugs (
      • Rask-Andersen M.
      • Masuram S.
      • Schiöth H.B.
      The druggable genome: evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication.
      ). Consequently, study of each individual GPCR subclass will provide unique angles that are beneficial for the development of therapeutics. Canonically, GPCR signaling is initiated by agonist binding to its orthosteric site, which results in rearrangements of the transmembrane helices of the 7TM bundle to allow efficient heterotrimeric G protein coupling and activation. G protein α subunits exchange GDP for GTP, allowing for functional dissociation of Gβγ and activation of downstream effectors. GPCRs are divided into six classes as follows with in-class examples: Class A (rhodopsin-like), Class B (secretin), Class C (metabotropic glutamate), Class D (pheromone), Class E (cAMP), and Class F (Frizzled) (
      • Attwood T.K.
      • Findlay J.B.
      Fingerprinting G-protein-coupled receptors.
      ,
      • Krishnan A.
      • Almén M.S.
      • Fredriksson R.
      • Schiöth H.B.
      The origin of GPCRs: identification of mammalian like rhodopsin, adhesion, glutamate and frizzled GPCRs in fungi.
      ,
      • Fredriksson R.
      • Lagerström M.C.
      • Lundin L.G.
      • Schiöth H.B.
      The G-protein-coupled receptors in the human genome form five main families: phylogenetic analysis, paralogon groups, and fingerprints.
      ). Within this naming system, the adhesion GPCRs (AGPCRs) are family B members but have been more aptly termed subfamily B2, whereas the traditional Class B peptide hormone-binding GPCRs comprise subfamily B1.
      AGPCRs are distinguished not only by their large extracellular regions (ECRs) that contain a wide variety of adhesive subdomains, but also by the highly conserved GPCR autoproteolysis–inducing (GAIN) domain that constitutively self-cleaves the receptors into two fragments (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • Brunger A.T.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ). Whereas extensive work has been done to characterize AGPCRs, it is largely uncertain how they are activated in endogenous tissues. How protein ligand-ECR binding regulates the activation state of the 7TM bundle is arguably the most intensely studied problem in current AGPCR research. To date, mechanisms involving AGPCR fragment dissociation and modes of allosteric modulation in response to endogenous ligands have been proposed. Here, we sought to provide clarity to these activation mechanisms by detailing the structural topologies of AGPCRs, while examining the prospective actions of endogenous ligands. AGPCR activation models will be compared with established modes of activation of other GPCR classes. Select aspects of AGPCR physiological regulation will also be discussed as routes to receptor activation. The theories outlined in this review will provide a consistent framework for classification of endogenous AGPCR ligands as they are identified and develop thought of mechanistic considerations in advance of AGPCR structures that await solution. As knowledge of AGPCR function increases, the realization that this class of receptors are untapped therapeutic targets will increase, prompting efforts to target them.

      The unique structural topology of adhesion GPCRs

      The 33 human adhesion GPCRs (ADGRs) are divided among nine subfamilies, ADGRA–G, -L, and -V, based on sequence similarity (
      • Bjarnadóttir T.K.
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      The human and mouse repertoire of the adhesion family of G-protein-coupled receptors.
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      • Hamann J.
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      • Lin H.-H.
      • Martinelli D.C.
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      • et al.
      International union of basic and clinical pharmacology. XCIV. Adhesion G protein–coupled receptors.
      ). AGPCRs possess 7TM domains that are known to signal through heterotrimeric G proteins in many cases. The N-terminal ECRs range from hundreds to thousands of residues and often share common characteristics among receptors in the same subfamily. The ECRs contain a variety of adhesion related subdomains that are often repeated (Fig. 1A, multicolored nodules). For example, group E AGPCRs, which include the ADGRE (EMR (EGF-like module–containing mucin-like hormone receptor)) receptors, contain epidermal growth factor–like repeats that are found in many types of proteins that mediate cell-adhesive interactions (
      • McKnight A.J.
      • Gordon S.
      EGF-TM7: a novel subfamily of seven-transmembrane-region leukocyte cell-surface molecules.
      ,
      • Lin H.-H.
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      Human EMR2, a novel EGF-TM7 molecule on chromosome 19p13.1, is closely related to CD97.
      ). Twelve AGPCRs contain a ∼70-residue hormone-binding (HormR) domain located N-terminally to their GAIN domain. There has yet to be a report of a hormone that binds to an AGPCR, leading many to believe that the HormR domain has additional functions beyond hormone binding (Fig. 1E) (
      • Arac D.
      • Strater N.
      • Seiradake E.
      Understanding the structural basis of adhesion GPCR functions.
      ). Another interesting motif found in select adhesion GPCR ECRs is the sperm protein, enterokinase, and agrin (SEA) domain. This domain is found in ADGRF1 (GPR110), ADGRF5 (GPR116), and ADGRG6 (GPR126) (
      • Lum A.M.
      • Wang B.B.
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      Orphan receptor GPR110, an oncogene overexpressed in lung and prostate cancer.
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      • Abe J.
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      ,
      • Leon K.
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      • Li J.
      • Sosnick T.R.
      • Zhao M.
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      Structural basis for adhesion G protein-coupled receptor Gpr126 function.
      ). SEA domains mediate a second autoproteolytic cleavage event that is distinct from GAIN domain self-cleavage. Not much is known about the role of the SEA domain, but its function leaves open the possibility that these particular receptors have alternative modes of signaling regulation.
      Figure thumbnail gr1
      Figure 1Structural topology of adhesion GPCRs. A, adhesion GPCRs are two fragment receptors that arise from a constitutive autoproteolytic cleavage event at the conserved GPS within the central core of the membrane-proximal, ∼320-amino acid GAIN domain. In the holoreceptor form, the two AGPCR fragments are noncovalently bound. In the dissociated form, the NTF or ECR is released extracellularly, whereas the freed CTF or GPCR domain remains in the plasma membrane. The ∼20-residue stalk (orange) that is exposed following NTF/CTF dissociation is termed the tethered-peptide agonist. B, the GAIN domain is a fully self-sufficient protease that constitutively cleaves the internal His Leu/Thr consensus site (GPS) via a nucleophilic attack mechanism, as shown for ADGRL1 (latrophilin-1) (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • Brunger A.T.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ). C, ribbon representation of the β-strand 12–GPS proteolyzed loop–β-strand 13 orientation within the GAIN domain in the holoreceptor state. D and E, space-filled models of the ADGRG1 (GPR56) NTF (PDB entry 5KVM) with stabilizer antibody and the GAIN domain plus adjacent HormR domain from ADGRL1 (PDB entry 4DLQ). The residues of the tethered-peptide agonists and remainder of the stalks are colored orange and depict the degree of concealment within the interior core of the GAIN domain in the holoreceptor state.
      At the C-terminal end of nearly every AGPCR ECR, ending 7–18 residues prior to the start of the first transmembrane span of the 7TM bundle, is the GAIN domain (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • Brunger A.T.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ) (Fig. 1A). A seminal finding in the AGPCR field was the X-ray crystallographic solution of GAIN domain structures (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • Brunger A.T.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ). GAIN domains are divided into two subdomains: an α-helix–rich GAINA and β-sandwich GAINB. Complete GAIN domains are ∼320 amino acids with variability typically observed within the GAINA subdomain. As indicated by its name, the GAIN domain is a fully self-sufficient protease that catalyzes constitutive autoproteolysis that splits the receptors into two fragments. Autoproteolytic cleavage is believed to be constitutive in most cases, but some studies have raised the possibility that cleavage might be regulated, although consideration of overexpression artifacts is also merited (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • Brunger A.T.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ,
      • Hsiao C.C.
      • Cheng K.F.
      • Chen H.Y.
      • Chou Y.H.
      • Stacey M.
      • Chang G.W.
      • Lin H.H.
      Site-specific N-glycosylation regulates the GPS auto-proteolysis of CD97.
      ,
      • Wei W.
      • Hackmann K.
      • Xu H.
      • Germino G.
      • Qian F.
      Characterization of cis-autoproteolysis of polycystin-1, the product of human polycystic kidney disease 1 gene.
      ). The receptor fragments remaining after self-cleavage are the extracellular N-terminal fragment (NTF) or ECR and the membrane-intercalated C-terminal fragment (CTF), which is also referred to as the GPCR or 7TM domain. A dense network of hydrogen bonds within the GAIN domain allows the NTF and CTF to remain noncovalently bound after self-proteolysis, which is considered to occur early during receptor biosynthesis in an intracellular compartment (
      • Nieberler M.
      • Kittel R.J.
      • Petrenko A.G.
      • Lin H.H.
      • Langenhan T.
      Control of adhesion GPCR function through proteolytic processing.
      ). The two-fragment holoreceptor is trafficked to the plasma membrane where it resides, poised for signaling.

      The self-proteolytic reaction of adhesion GPCRs

      The AGPCR self-proteolysis reaction requires proper folding of the GAIN domain and occurs within GAINB when a conserved basic residue at the P2 position of the cleavage site abstracts a hydrogen from the side chain of the conserved, polar threonine (or serine) at the P1′ position (Fig. 1B) (
      • Lin H.H.
      • Chang G.W.
      • Davies J.Q.
      • Stacey M.
      • Harris J.
      • Gordon S.
      Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein-coupled receptor proteolytic site motif.
      ). AGPCR P2 site basic residues are most commonly histidines, such as His-836 in ADGRL1 (latrophilin (LPHN1)), but can be arginine, such as Arg-855 in ADGRB5 (BAI3) (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • Brunger A.T.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ). The proton abstraction initiates a nucleophilic attack of the carbonyl group of the P1 residue, which is most commonly leucine. The resulting ester intermediate is resolved by a final nucleophilic attack of a water molecule. The consensus self-cleavage site within the GAIN domains of most AGPCRs is HL/T. Prior to solution of the GAIN domain structure, the HL/T site and surrounding sequence was termed the GPCR proteolysis site (GPS), reflecting the idea that the minimal sequence was sufficient for proteolysis rather than the larger structure of the GAIN domain (
      • Krasnoperov V.
      • Lu Y.
      • Buryanovsky L.
      • Neubert T.A.
      • Ichtchenko K.
      • Petrenko A.G.
      Post-translational proteolytic processing of the calcium-independent receptor of α-latrotoxin (CIRL), a natural chimera of the cell adhesion protein and the G protein-coupled receptor: role of the G protein-coupled receptor proteolysis site (GPS) motif.
      ,
      • Prömel S.
      • Langenhan T.
      • Araç D.
      Matching structure with function: the GAIN domain of adhesion-GPCR and PKD1-like proteins.
      ,
      • Krasnoperov V.
      • Bittner M.A.
      • Holz R.W.
      • Chepurny O.
      • Petrenko A.G.
      Structural requirements for α-latrotoxin binding and α-latrotoxin-stimulated secretion: a study with calcium-independent receptor of α-latrotoxin (CIRL) deletion mutants.
      ). Given that this original definition has changed, GPS has now commonly come to mean the HL/T consensus site within the GAIN domain.
      The GPS is located 14–25 residues N-terminal to the start of the first transmembrane span (TM1). The start of the CTF, the P1′ threonine, is also immediately N-terminal to the first residue of the final (13th) β-strand of the β-sandwich structure that comprises the GAINB subdomain. Therefore, β-strand 13 is part of the CTF, but it is embedded within GAINB, and the GPS (i.e. HL/T) is essentially the loop that links β-strands 12 and 13 (Fig. 1C). β-Strand 13 sequences of adhesion GPCRs are highly conserved and very hydrophobic, which aligns with their location within the interior core of GAINB subdomain (Fig. 1 (C–E) and Table 1). β-Strand 13 is noncovalently bound by a dense network of ∼20 hydrogen bonds that hold it firmly within the GAINB subdomain (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • Brunger A.T.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ).
      Table 1Adhesion GPCR GPS and tethered agonist/β-Strand 13 sequences
      * ADGRA1 (GPR123) does not have a GAIN domain and thus does not have a GPS.
      Within recent years, the stalk that connects TM1 to the GAIN domain and includes β-strand 13 has been named the adhesion GPCR tethered-peptide agonist (also referred to as the tethered agonist). This conserved sequence within multiple AGPCRs was shown independently by two groups to play a pivotal role in mediating receptor activation (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ,
      • Liebscher I.
      • Schön J.
      • Petersen S.C.
      • Fischer L.
      • Auerbach N.
      • Demberg L.M.
      • Mogha A.
      • Cöster M.
      • Simon K.-U.
      • Rothemund S.
      • Monk K.R.
      • Schöneberg T.
      A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.
      ). One feature that seems to be an obvious requirement for tethered-peptide agonism is that the NTF and CTF of the receptor must become dissociated to liberate the agonist peptide from the interior core of the GAIN domain. Many within the field are currently deciphering exactly how AGPCR tethered agonism intersects with the varied AGPCR activation mechanisms, which are discussed below. Interestingly, not all AGPCRs undergo autoproteolysis; some receptors, including ADGRC1 (cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1)), ADGRA2 (GPR124), ADGRF2 (GPR111), ADGRA3 (GPR125), and ADGRF4 (GPR115), may be activated by alternative modes that do not involve fragment dissociation and tethered-peptide agonism (
      • Formstone C.J.
      • Moxon C.
      • Murdoch J.
      • Little P.
      • Mason I.
      Basal enrichment within neuroepithelia suggests novel function(s) for Celsr1 protein.
      ,
      • Vallon M.
      • Essler M.
      Proteolytically processed soluble tumor endothelial marker (TEM) 5 mediates endothelial cell survival during angiogenesis by linking integrin αvβ3 to glycosaminoglycans.
      ,
      • Prömel S.
      • Waller-Evans H.
      • Dixon J.
      • Zahn D.
      • Colledge W.H.
      • Doran J.
      • Carlton M.B.L.
      • Grosse J.
      • Schöneberg T.
      • Russ A.P.
      • Langenhan T.
      Characterization and functional study of a cluster of four highly conserved orphan adhesion-GPCR in mouse.
      ). Impaired self-cleavage is typically attributed to alterations of the GPS; receptors lacking a basic residue at the P2 position (e.g. ADGRF2 or ADGRF4) or a polar residue at the P1′ position. (e.g. ADGRC1) demonstrate minimal or no autoproteolysis (Table 1) (
      • Formstone C.J.
      • Moxon C.
      • Murdoch J.
      • Little P.
      • Mason I.
      Basal enrichment within neuroepithelia suggests novel function(s) for Celsr1 protein.
      ,
      • Vallon M.
      • Essler M.
      Proteolytically processed soluble tumor endothelial marker (TEM) 5 mediates endothelial cell survival during angiogenesis by linking integrin αvβ3 to glycosaminoglycans.
      ,
      • Prömel S.
      • Waller-Evans H.
      • Dixon J.
      • Zahn D.
      • Colledge W.H.
      • Doran J.
      • Carlton M.B.L.
      • Grosse J.
      • Schöneberg T.
      • Russ A.P.
      • Langenhan T.
      Characterization and functional study of a cluster of four highly conserved orphan adhesion-GPCR in mouse.
      ). Differences in post-translational modifications have also been proposed to regulate GAIN-mediated cleavage, such as N-linked glycosylation events within the GAIN domain (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • Brunger A.T.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ,
      • Hsiao C.C.
      • Cheng K.F.
      • Chen H.Y.
      • Chou Y.H.
      • Stacey M.
      • Chang G.W.
      • Lin H.H.
      Site-specific N-glycosylation regulates the GPS auto-proteolysis of CD97.
      ,
      • Wei W.
      • Hackmann K.
      • Xu H.
      • Germino G.
      • Qian F.
      Characterization of cis-autoproteolysis of polycystin-1, the product of human polycystic kidney disease 1 gene.
      ). However, observations of inefficient cleavage in these instances may be manifestations of experimental receptor overexpression that impart improper receptor trafficking or processing. Noncleaved AGPCRs are still capable of signaling, leaving open the question of how these receptors become activated.

      Adhesion GPCR activation mechanisms

      GPCRs exhibit different basal activity levels that depend on the individual characteristics of each receptor. Basal activity is one state that GPCRs occupy within a dynamic energy landscape of active and inactive conformations (
      • Manglik A.
      • Kobilka B.
      The role of protein dynamics in GPCR function: insights from the β2AR and rhodopsin.
      ,
      • Hilger D.
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      Structure and dynamics of GPCR signaling complexes.
      ,
      • Xu J.
      • Hu Y.
      • Kaindl J.
      • Risel P.
      • Hubner H.
      • Maeda S.
      • Niu X.
      • Li H.
      • Gmeiner P.
      • Jin C.
      • Kobilka B.K.
      Conformational complexity and dynamics in a muscarinic receptor revealed by NMR spectroscopy.
      ). Fig. 2 depicts four proposed activity states of adhesion GPCRs with cartoon diagrams that the field has used with representation of G protein–binding site dynamism as a function of receptor activation. Accompanying the diagrams are activity profiles of relative signaling strength. An understanding of the ways that AGPCRs become activated is emerging. There has been a broad and imaginative variety of proposed AGPCR activation schemes (
      • Arac D.
      • Strater N.
      • Seiradake E.
      Understanding the structural basis of adhesion GPCR functions.
      ,
      • Purcell R.H.
      • Hall R.A.
      Adhesion G protein–coupled receptors as drug targets.
      ,
      • Langenhan T.
      Adhesion G protein-coupled receptors—candidate metabotropic mechanosensors and novel drug targets.
      ,
      • Kishore A.
      • Hall R.A.
      Versatile signaling activity of adhesion GPCRs.
      ,
      • Paavola K.J.
      • Hall R.A.
      Adhesion G protein-coupled receptors: signaling, pharmacology, and mechanisms of activation.
      ,
      • Folts C.J.
      • Giera S.
      • Li T.
      • Piao X.
      Adhesion G protein-coupled receptors as drug targets for neurological diseases.
      ,
      • Liebscher I.
      • Schoneberg T.
      Tethered agonism: a common activation mechanism of adhesion GPCRs.
      ). The current evidence supports two fundamental modes of AGPCR modulation: orthosteric agonism (i.e. tethered-peptide agonism), in which NTF/CTF dissociation is required, and allosteric regulation, which has also been termed the tunable model and does not require receptor subunit dissociation (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ,
      • Langenhan T.
      Adhesion G protein-coupled receptors—candidate metabotropic mechanosensors and novel drug targets.
      ,
      • Kishore A.
      • Hall R.A.
      Versatile signaling activity of adhesion GPCRs.
      ,
      • Folts C.J.
      • Giera S.
      • Li T.
      • Piao X.
      Adhesion G protein-coupled receptors as drug targets for neurological diseases.
      ,
      • Langenhan T.
      • Aust G.
      • Hamann J.
      Sticky signaling–adhesion class G protein-coupled receptors take the stage.
      ). Both fundamental activation modes are supported through several lines of evidence, and it is likely that individual AGPCRs can be activated in both manners.
      Figure thumbnail gr2
      Figure 2Models of adhesion GPCR activation. A, adhesion GPCRs, like other GPCRs, occupy a range of activated and inhibited states. Consequently, AGPCRs possess varying levels of basal G protein signaling. The adhesion GPCR N-terminal subdomains (dark green, yellow, and brown modules) are portrayed to reflect the potential variety within adhesion GPCR ECRs. B, in the orthosteric agonism model of activation, NTF/CTF dissociation via an anchored ligand (depicted by the green star) results in exposure of the tethered-peptide agonist (orange), allowing it to bind to an orthosteric site that is predicted to lie within the 7TM helical bundle. Orthosteric agonism is proposed to be a threshold response (all or none) due to forced NTF dissociation, which results in stabilization of highly active states of the receptors and maximal signaling. C and D, in allosteric modes of AGPCR regulation, ligands (indicated with a blue or red star) can interact with various AGPCR N-terminal adhesive motifs to stabilize active (activation, C) or inactive states (inhibition, D), respectively. Allosteric activation and inhibition mechanisms are unknown but may be mediated by GAIN-7TM interactions that favor stabilization of specific receptor conformations. E, relative signaling strength outputs in response to stimulus for each of the receptor modulation modes.

      Orthosteric agonism (tethered-peptide agonism)

      Orthosteric agonism is a receptor activation model that depends on the action of the AGPCR tethered agonist. The most important residues, or the core of the tethered agonist, are the seven residues located immediately C-terminal to the GPS. These residues mostly overlap with β-strand 13, the conformation the sequence adopts in the holoreceptor form. The core residues share the consensus sequence TXFAVLM, with the T, F, and M residues showing the highest conversation across all AGPCRs (Table 1). Prior to understanding that this sequence was a tethered-peptide agonist, a study by Hall and colleagues (
      • Paavola K.J.
      • Stephenson J.R.
      • Ritter S.L.
      • Alter S.P.
      • Hall R.A.
      The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity.
      ) provided evidence that an isolated CTF was more active than its cognate holoreceptor. The signaling strengths of ADGRG1 (GPR56) and an engineered ADGRG1 construct in which the entirety of the NTF was deleted (ΔNTF, or CTF) were compared. The ΔNTF receptor exhibited substantially higher G protein–dependent signaling than the full-length receptor (
      • Paavola K.J.
      • Stephenson J.R.
      • Ritter S.L.
      • Alter S.P.
      • Hall R.A.
      The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity.
      ). Since then, ΔNTF variants of several AGPCRs were found to have increased signaling capacity (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ,
      • Paavola K.J.
      • Stephenson J.R.
      • Ritter S.L.
      • Alter S.P.
      • Hall R.A.
      The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity.
      ,
      • Okajima D.
      • Kudo G.
      • Yokota H.
      Brain-specific angiogenesis inhibitor 2 (BAI2) may be activated by proteolytic processing.
      ,
      • Ward Y.
      • Lake R.
      • Yin J.J.
      • Heger C.D.
      • Raffeld M.
      • Goldsmith P.K.
      • Merino M.
      • Kelly K.
      LPA receptor heterodimerizes with CD97 to amplify LPA-initiated RHO-dependent signaling and invasion in prostate cancer cells.
      ,
      • Paavola K.J.
      • Sidik H.
      • Zuchero J.B.
      • Eckart M.
      • Talbot W.S.
      Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126.
      ). A subsequent study used urea to operationally dissociate the two fragments of the AGDRG1 in membrane homogenates. The isolated CTF was markedly more efficacious at activating G proteins than the holoreceptor (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ). At this point, the aspect that rendered AGPCR CTFs to be activated was not clear. It was found that the TM1 N-terminal stalk sequence consisting of ∼20 residues behaved as a tethered-peptide agonist and dramatically activated AGPCRs. Deletion of tethered agonist residues dramatically lowered receptor activities in vitro, as shown for ADGRG1, ADGRG2 (GPR64), ADGRG6, ADGRD1 (GPR133), and ADGRF1 (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ,
      • Liebscher I.
      • Schön J.
      • Petersen S.C.
      • Fischer L.
      • Auerbach N.
      • Demberg L.M.
      • Mogha A.
      • Cöster M.
      • Simon K.-U.
      • Rothemund S.
      • Monk K.R.
      • Schöneberg T.
      A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.
      ,
      • Kishore A.
      • Purcell R.H.
      • Nassiri-Toosi Z.
      • Hall R.A.
      Stalk-dependent and stalk-independent signaling by the adhesion G protein-coupled receptors GPR56 (ADGRG1) and BAI1 (ADGRB1).
      ,
      • 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.
      ). Partial deletion of the zebrafish ADGRG6 tethered agonist resulted in a puffy ear phenotype and impairment of nerve cell myelination, essentially phenocopying the fish model deletion of the ADGRG6 CTF (
      • Liebscher I.
      • Schön J.
      • Petersen S.C.
      • Fischer L.
      • Auerbach N.
      • Demberg L.M.
      • Mogha A.
      • Cöster M.
      • Simon K.-U.
      • Rothemund S.
      • Monk K.R.
      • Schöneberg T.
      A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.
      ,
      • Monk K.R.
      • Naylor S.G.
      • Glenn T.D.
      • Mercurio S.
      • Perlin J.R.
      • Dominguez C.
      • Moens C.B.
      • Talbot W.S.
      A G protein-coupled receptor is essential for Schwann cells to initiate myelination.
      ). Sequential N-terminal truncations to the first residues of ΔNTF AGDGRG1 and ADGRF1 receptors resulted in incremental loss of G protein signaling activity in vitro (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ). AGPCRs with substitution mutations to critical residues within the tethered agonist also had reduced activities (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ,
      • Liebscher I.
      • Schön J.
      • Petersen S.C.
      • Fischer L.
      • Auerbach N.
      • Demberg L.M.
      • Mogha A.
      • Cöster M.
      • Simon K.-U.
      • Rothemund S.
      • Monk K.R.
      • Schöneberg T.
      A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.
      ,
      • Brown K.
      • Filuta A.
      • Ludwig M.-G.
      • Seuwen K.
      • Jaros J.
      • Vidal S.
      • Arora K.
      • Naren A.P.
      • Kandasamy K.
      • Parthasarathi K.
      • Offermanns S.
      • Mason R.J.
      • Miller W.E.
      • Whitsett J.A.
      • Bridges J.P.
      Epithelial Gpr116 regulates pulmonary alveolar homeostasis via Gq/11 signaling.
      ,
      • Wilde C.
      • Fischer L.
      • Lede V.
      • Kirchberger J.
      • Rothemund S.
      • Schöneberg T.
      • Liebscher I.
      The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist.
      ). In contrast, deletion of the entire stalk of a ΔNTF version of ADGRB1 surprisingly had little impact on signaling compared with a version with an intact stalk (
      • Kishore A.
      • Purcell R.H.
      • Nassiri-Toosi Z.
      • Hall R.A.
      Stalk-dependent and stalk-independent signaling by the adhesion G protein-coupled receptors GPR56 (ADGRG1) and BAI1 (ADGRB1).
      ). This led to the proposal of stalk-independent signaling, which may align with the concept that GPCRs are diverse and have a broad range of basal signaling capacities and that AGPCRs can be activated by means that do not always rely on the tethered agonist (
      • Kishore A.
      • Purcell R.H.
      • Nassiri-Toosi Z.
      • Hall R.A.
      Stalk-dependent and stalk-independent signaling by the adhesion G protein-coupled receptors GPR56 (ADGRG1) and BAI1 (ADGRB1).
      ,
      • Salzman G.S.
      • Zhang S.
      • Gupta A.
      • Koide A.
      • Koide S.
      • Araç D.
      Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region.
      ). Full-length, WT ADGRB1 did respond in vivo to synthetic peptide agonists, indicating the probability of bimodal ADGRB1 activation (
      • Tu Y.K.
      • Duman J.G.
      • Tolias K.F.
      The adhesion-GPCR BAI1 promotes excitatory synaptogenesis by coordinating bidirectional trans-synaptic signaling.
      ).
      Further evidence supporting AGPCR tethered agonism was demonstrated through the use of synthetic tethered agonist-mimetic peptides that activated their corresponding receptors in vitro and, in some cases, in vivo. ADGRG1, ADGRG2, ADGRG5 (GPR114), ADGRG6, ADGRD1, ADGRF1, ADGRF4, and ADGRF5 all demonstrated increases in signaling when exposed to these synthetic activating peptides (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ,
      • Liebscher I.
      • Schön J.
      • Petersen S.C.
      • Fischer L.
      • Auerbach N.
      • Demberg L.M.
      • Mogha A.
      • Cöster M.
      • Simon K.-U.
      • Rothemund S.
      • Monk K.R.
      • Schöneberg T.
      A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.
      ,
      • Brown K.
      • Filuta A.
      • Ludwig M.-G.
      • Seuwen K.
      • Jaros J.
      • Vidal S.
      • Arora K.
      • Naren A.P.
      • Kandasamy K.
      • Parthasarathi K.
      • Offermanns S.
      • Mason R.J.
      • Miller W.E.
      • Whitsett J.A.
      • Bridges J.P.
      Epithelial Gpr116 regulates pulmonary alveolar homeostasis via Gq/11 signaling.
      ,
      • Wilde C.
      • Fischer L.
      • Lede V.
      • Kirchberger J.
      • Rothemund S.
      • Schöneberg T.
      • Liebscher I.
      The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist.
      ,
      • Demberg L.M.
      • Winkler J.
      • Wilde C.
      • Simon K.U.
      • Schön J.
      • Rothemund S.
      • Schöneberg T.
      • Prömel S.
      • Liebscher I.
      Activation of adhesion G protein-coupled receptors: agonist specificity of Stachel sequence-derived peptides.
      ,
      • Demberg L.M.
      • Rothemund S.
      • Schöneberg T.
      • Liebscher I.
      Identification of the tethered peptide agonist of the adhesion G protein-coupled receptor GPR64/ADGRG2.
      ). As with the tethered agonist stalks from which they are derived, synthetic peptide agonists have a critical dependence on their N-terminal residues. Differences as small as single-residue substitutions or deletions at the N terminus can severely abrogate the agonistic properties of the peptides (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ,
      • Liebscher I.
      • Schön J.
      • Petersen S.C.
      • Fischer L.
      • Auerbach N.
      • Demberg L.M.
      • Mogha A.
      • Cöster M.
      • Simon K.-U.
      • Rothemund S.
      • Monk K.R.
      • Schöneberg T.
      A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.
      ). Receptor specificity of synthetic peptide agonists also depends on the sequence similarities shared among the tethered agonists. For example, synthetic peptide agonist cross-reactivity was exhibited among members of the Group VI (Group F) AGPCR subfamily that share a highly conserved tethered-peptide agonist and include ADGRF1, ADGRF4, and ADGRF5 (Table 1) (
      • Demberg L.M.
      • Winkler J.
      • Wilde C.
      • Simon K.U.
      • Schön J.
      • Rothemund S.
      • Schöneberg T.
      • Prömel S.
      • Liebscher I.
      Activation of adhesion G protein-coupled receptors: agonist specificity of Stachel sequence-derived peptides.
      ).
      The conformation that tethered-peptide agonists adopt once released from the GAIN domain core is not known, but it is interesting that many seven-residue tethered-peptide agonist sequences are followed C-terminally by predicted β-turn elements within the middle of the stalk regions (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ). Performing turn element predictions on all tethered agonist–containing AGPCRs via the Chou and Fasman Secondary Structure Prediction (CFSSP) server revealed that turn elements are common and often conserved among receptors within the same subfamily (Table 1, red) (
      • Ashok Kumar T.
      CFSSP: Chou and Fasman Secondary Structure Prediction server.
      ). The presence of the mid-stalk turns suggests that when the tethered agonist is exposed by NTF/CTF dissociation, it may serve as a flexible point to allow the tethered agonist to bind intramolecularly back toward its proposed orthosteric site within the CTF. Point mutations of synthetic peptide agonists near the prospective turn points alter their capacity to stimulate signaling. For example, a 13-residue synthetic peptide that was derived from the stalk sequence of ADGRF4, a noncleaved receptor with an unusually small CTF stalk (Table 1), was incapable of promoting signaling (
      • Demberg L.M.
      • Winkler J.
      • Wilde C.
      • Simon K.U.
      • Schön J.
      • Rothemund S.
      • Schöneberg T.
      • Prömel S.
      • Liebscher I.
      Activation of adhesion G protein-coupled receptors: agonist specificity of Stachel sequence-derived peptides.
      ). However, mutating 2–3 residues around the predicted turn element of the peptide, so that it now matched the ADGRF5 sequence, restored its ability to activate ADGRF4. This suggests that the turn regions of AGPCR tethered-peptide agonists may be critical for receptor activation.
      The presence of the turn elements may also help to account for the sprawling evidence that synthetic peptides modeled after AGPCR tethered agonists critically depend upon length. Most studies found that longer peptides with lengths of 12–20 residues, which in most cases include the predicted turn motifs, exhibit the highest efficacies (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ,
      • Liebscher I.
      • Schön J.
      • Petersen S.C.
      • Fischer L.
      • Auerbach N.
      • Demberg L.M.
      • Mogha A.
      • Cöster M.
      • Simon K.-U.
      • Rothemund S.
      • Monk K.R.
      • Schöneberg T.
      A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.
      ,
      • Demberg L.M.
      • Winkler J.
      • Wilde C.
      • Simon K.U.
      • Schön J.
      • Rothemund S.
      • Schöneberg T.
      • Prömel S.
      • Liebscher I.
      Activation of adhesion G protein-coupled receptors: agonist specificity of Stachel sequence-derived peptides.
      ,
      • Demberg L.M.
      • Rothemund S.
      • Schöneberg T.
      • Liebscher I.
      Identification of the tethered peptide agonist of the adhesion G protein-coupled receptor GPR64/ADGRG2.
      ). The lone reported exception to this is the 7-mer peptide derived from the ADGRG1 tethered agonist, which is the only ADGRG1-modeled peptide capable of activating the receptor in vitro (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ). Intriguingly, the ADGRG1 7-mer peptide does not activate ADGRG5, even though ADGRG5 has this identical sequence (Table 1). However, longer ADGRG5-mimetic peptides (18–20 residues, comprising the complete stalk) will activate both ADGRG1 and -G5 in vitro and in cells, even though, beyond the first seven residues, the sequences have no conservation to the ADGRG1 stalk (
      • Liebscher I.
      • Schön J.
      • Petersen S.C.
      • Fischer L.
      • Auerbach N.
      • Demberg L.M.
      • Mogha A.
      • Cöster M.
      • Simon K.-U.
      • Rothemund S.
      • Monk K.R.
      • Schöneberg T.
      A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.
      ,
      • Stoveken H.M.
      • Larsen S.D.
      • Smrcka A.V.
      • Tall G.G.
      Gedunin- and Khivorin-derivatives are small-molecule partial agonists for adhesion G protein-coupled receptors GPR56/ADGRG1 and GPR114/ADGRG5.
      ). The C-terminal ends of longer peptides may be necessary for proper folding or bending about the predicted turn element to accommodate binding to the AGPCR orthosteric site, whereas the 7-mer ADGRG1 peptide may be a rare perfect fit that requires only the core tethered agonist sequence. It is also plausible that the C-terminal ends of activating peptides help stabilize them in solution, as the C termini are far more hydrophilic than the hydrophobic 7-mer N termini.
      The current leading model of adhesion GPCR activation is that the tethered-peptide agonist binds intramolecularly to its orthosteric site following receptor NTF/CTF dissociation. Upon NTF dissociation, the hydrophobic agonist residues are exposed to the aqueous extracellular environment, resulting in a thermodynamically unfavored condition. Hydrophobic effects may be a driving force for the tethered agonist to rapidly bind to its orthosteric binding pocket within the 7TM bundle (Fig. 2B). Given that the core seven residues comprising the tethered agonist are completely embedded within the GAIN domain in the holo-receptor form, it is likely that orthosteric agonism is an abrupt, threshold-like response. In other words, receptor fragment dissociation is a binary off/on switch and results in full agonist-driven signaling once the NTF is removed (Fig. 2E). The rapid onset of signaling is predicted because tethered agonist binding to its orthosteric site is a first-order event; the agonist and receptor are tethered together in extreme proximity, and binding of the tethered agonist within the 7TM may be driven to overcome the disfavored hydrophilic environment. This mechanism has some parallels with and distinctions from protease-activated receptor (PAR) activation. PARs are Class A GPCRs and are activated when exogenous proteases (e.g. thrombin or trypsin) cleave their N-terminal stalk leader sequences. PARs are distinguished from AGPCRs in that they are proteolyzed in trans, rather than autoproteolytically. Following proteolysis, the new N terminus of the TM1 stalk is proposed to bind to an orthosteric site that includes extracellular loop 2 (
      • Vu T.K.
      • Hung D.T.
      • Wheaton V.I.
      • Coughlin S.R.
      Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.
      ,
      • Gerszten R.E.
      • Chen J.
      • Ishii M.
      • Ishii K.
      • Wang L.
      • Nanevicz T.
      • Turck C.W.
      • Vu T.K.
      • Coughlin S.R.
      Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface.
      ,
      • Lerner D.J.
      • Chen M.
      • Tram T.
      • Coughlin S.R.
      Agonist recognition by proteinase-activated receptor 2 and thrombin receptor: importance of extracellular loop interactions for receptor function.
      ). A fundamental difference between PARs and AGPCRs is that PAR stalk sequences are typically longer, and the tethered agonists are less hydrophobic than those of AGPCRs. Consequently, whereas PAR tethered agonists are proposed to bind to PAR 7TM extracellular loops, it would make sense that AGPCR tethered agonists might instead bind deeper within the hydrophobic core of the 7TM bundle. However, it is currently unsolved precisely where the tethered agonists of both receptor classes bind to their respective 7TMs.
      Given the strong noncovalent interactions that hold the tethered-peptide agonist (as β-strand 13) within the GAIN domain, it is expected that a substantial force would be needed to break them to liberate the agonist. However, the means by which force-mediated AGPCR fragment dissociation occurs are largely unknown and likely to vary. Many adhesion GPCRs have been observed to undergo fragment dissociation, which has been referred to as NTF shedding. Freed NTFs in various tissues or cell culture models were observed for ADGRG1, ADGRA2, ADGRB1 (BAI1), and ADGRE5 (CD97) (
      • Vallon M.
      • Essler M.
      Proteolytically processed soluble tumor endothelial marker (TEM) 5 mediates endothelial cell survival during angiogenesis by linking integrin αvβ3 to glycosaminoglycans.
      ,
      • Chiang N.Y.
      • Chang G.W.
      • Huang Y.S.
      • Peng Y.M.
      • Hsiao C.C.
      • Kuo M.L.
      • Lin H.H.
      Heparin interacts with the adhesion GPCR GPR56, reduces receptor shedding, and promotes cell adhesion and motility.
      ,
      • Kaur B.
      • Brat D.J.
      • Devi N.S.
      • Van Meir E.G.
      Vasculostatin, a proteolytic fragment of brain angiogenesis inhibitor 1, is an antiangiogenic and antitumorigenic factor.
      ,
      • de Groot D.M.
      • Vogel G.
      • Dulos J.
      • Teeuwen L.
      • Stebbins K.
      • Hamann J.
      • Owens B.M.
      • van Eenennaam H.
      • Bos E.
      • Boots A.M.
      Therapeutic antibody targeting of CD97 in experimental arthritis: the role of antigen expression, shedding, and internalization on the pharmacokinetics of anti-CD97 monoclonal antibody 1B2.
      ,
      • Chiang N.Y.
      • Hsiao C.C.
      • Huang Y.S.
      • Chen H.Y.
      • Hsieh I.J.
      • Chang G.W.
      • Lin H.H.
      Disease-associated GPR56 mutations cause bilateral frontoparietal polymicrogyria via multiple mechanisms.
      ). Whereas shedding may imply spontaneous dissociation of the NTFs, these observations could also be remnants of ligand-induced NTF dissociation events (
      • Vallon M.
      • Essler M.
      Proteolytically processed soluble tumor endothelial marker (TEM) 5 mediates endothelial cell survival during angiogenesis by linking integrin αvβ3 to glycosaminoglycans.
      ,
      • Luo R.
      • Jeong S.J.
      • Yang A.
      • Wen M.
      • Saslowsky D.E.
      • Lencer W.I.
      • Araç D.
      • Piao X.
      Mechanism for adhesion G protein-coupled receptor GPR56-mediated RhoA activation induced by collagen III stimulation.
      ). AGPCR fragment dissociation could be achieved by NTF binding to its ligands that are anchored to the extracellular matrix (ECM) or adjacent cells. Cell movement in relation to the anchored ligands would generate a sufficient shear force to dissociate the NTF from the CTF to initiate G protein signaling. In line with this, adhesion GPCRs were recently considered to be a group of metabotropic mechanosensors (
      • Langenhan T.
      Adhesion G protein-coupled receptors—candidate metabotropic mechanosensors and novel drug targets.
      ,
      • Scholz N.
      • Guan C.
      • Nieberler M.
      • Grotemeyer A.
      • Maiellaro I.
      • Gao S.
      • Beck S.
      • Pawlak M.
      • Sauer M.
      • Asan E.
      • Rothemund S.
      • Winkler J.
      • Prömel S.
      • Nagel G.
      • Langenhan T.
      • et al.
      Mechano-dependent signaling by latrophilin/CIRL quenches cAMP in proprioceptive neurons.
      ,
      • Scholz N.
      • Monk K.R.
      • Kittel R.J.
      • Langenhan T.
      Adhesion GPCRs as a putative class of metabotropic mechanosensors.
      ,
      • Scholz N.
      • Gehring J.
      • Guan C.
      • Ljaschenko D.
      • Fischer R.
      • Lakshmanan V.
      • Kittel R.J.
      • Langenhan T.
      The adhesion GPCR latrophilin/CIRL shapes mechanosensation.
      ). Ligand-mediated shear force dissociation of the NTF is discussed further under “How do AGPCR ligands modulate receptor activity?”
      Tethered agonist activation of AGPCRs does not account for all means of AGPCR activation, with the most obvious examples being the noncleaved receptors that are incapable of undergoing NTF/CTF dissociation. Noncleaved receptors and many engineered uncleavable mutants of cleaved receptors are still capable of signaling through G proteins (
      • Kishore A.
      • Purcell R.H.
      • Nassiri-Toosi Z.
      • Hall R.A.
      Stalk-dependent and stalk-independent signaling by the adhesion G protein-coupled receptors GPR56 (ADGRG1) and BAI1 (ADGRB1).
      ,
      • Wilde C.
      • Fischer L.
      • Lede V.
      • Kirchberger J.
      • Rothemund S.
      • Schöneberg T.
      • Liebscher I.
      The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist.
      ,
      • Prömel S.
      • Frickenhaus M.
      • Hughes S.
      • Mestek L.
      • Staunton D.
      • Woollard A.
      • Vakonakis I.
      • Schöneberg T.
      • Schnabel R.
      • Russ A.P.
      • Langenhan T.
      The GPS motif is a molecular switch for bimodal activities of adhesion class G protein-coupled receptors.
      ,
      • Zhu B.
      • Luo R.
      • Jin P.
      • Li T.
      • Oak H.C.
      • Giera S.
      • Monk K.R.
      • Lak P.
      • Shoichet B.K.
      • Piao X.
      GAIN domain-mediated cleavage is required for activation of G protein-coupled receptor 56 (GPR56) by its natural ligands and a small-molecule agonist.
      ). In these instances, it is unreasonable to predict that the tethered agonist directly regulates signaling as it is covalently bound to the NTF within the interior of the GAIN domain. Consequently, an alternative model has been proposed whereby AGPCR CTFs may be activated allosterically via ligand-induced structural changes of the NTFs.

      Allosteric activation and inhibition

      In allosteric models of adhesion GPCR activation, the NTF and CTF remain bound, and changes in signaling may be induced by receptor conformational changes upon ligand binding to the NTF, rather than orthosteric engagement by the tethered-peptide agonist. The exact mechanisms of allosteric activation or inhibition are not clear, as the N-terminal ligand-binding domains and the G protein-coupling 7TM domain are distal, separated by hundreds or even thousands of residues. Binding of an allosteric ligand must somehow transmit an activation signal over a large structural space. The GAIN domain, located immediately N-terminal to the 7TM domain, is the best candidate to transmit the allosteric signals. Following binding of an allosteric ligand or antagonist, the GAIN domain could help to stabilize active- or restricted-conformation states of the 7TM domain to promote or inhibit signaling, respectively (Fig. 2, C and D).
      Beyond the β-strand 13/tethered agonist linkage, interactions between the GAIN or other ECR elements and 7TM domains are not well-characterized and have only been observed indirectly in a few isolated studies. In HEK293T cells co-expressing Myc-tagged ADGRB1 NTF and a CTF ADGRB1 variant (ΔNTF), anti-Myc was used to co-immunoprecipitate the ADGRB1 CTF (
      • Stephenson J.R.
      • Paavola K.J.
      • Schaefer S.A.
      • Kaur B.
      • Van Meir E.G.
      • Hall R.A.
      Brain-specific angiogenesis inhibitor-1 signaling, regulation, and enrichment in the postsynaptic density.
      ). ADGRG1 harbors two disease-causing mutations, R565W and L640R, in extracellular loops 2 and 3 (ECL2 and -3) of the 7TM, respectively (
      • Jin Z.
      • Tietjen I.
      • Bu L.
      • Liu-Yesucevitz L.
      • Gaur S.K.
      • Walsh C.A.
      • Piao X.
      Disease-associated mutations affect GPR56 protein trafficking and cell surface expression.
      ). Relative cell surface abundance of the mutant receptors was impaired, but it was not when the mutants were expressed as CTF-only variants (
      • Kishore A.
      • Hall R.A.
      Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.
      ). The differential effect of the NTF on WT and mutant receptor trafficking suggests that the mutant residues may alter critical interactions between the ECLs and the NTF that are important for trafficking and possibly receptor activity. Additional evidence comes from a study that reported that NTFs and CTFs of chimeric AGPCRs had the ability to exchange or to swap via a “split personality” model (
      • Silva J.P.
      • Lelianova V.
      • Hopkins C.
      • Volynski K.E.
      • Ushkaryov Y.
      Functional cross-interaction of the fragments produced by the cleavage of distinct adhesion G-protein-coupled receptors.
      ). Given the intricacies of how the tethered agonist is embedded within the GAIN domain, it is unlikely that AGPCRs are capable of NTF exchange that includes re-embedding of the tethered agonist, especially considering that GAIN domains are destabilized following NTF/CTF dissociation or when constructed recombinantly to lack β-strand 13. These observations of NTF swapping may, however, be evidence of additional, conserved GAIN-CTF contact points that are distinct from β-strand 13.
      Whereas additional GAIN or NTF direct interactions with the CTF remain undefined, evidence for them would help explain how AGPCRs are allosterically activated by ligands to produce more modest signaling strength outputs than orthosteric agonism (Fig. 2E). Several endogenous and engineered soluble or diffusible binding partners of AGPCRs have been identified that bind to AGPCR NTFs and induce signaling changes. One example is the docosahexaenoic acid metabolite synaptamide, which was identified as an ADGRF1 ligand and proposed to activate cAMP signaling to promote synaptogenesis and anti-neuroinflammatory responses (
      • Kim H.-Y.
      • Spector A.A.
      N-Docosahexaenoylethanolamine: a neurotrophic and neuroprotective metabolite of docosahexaenoic acid.
      ,
      • Huang B.X.
      • Hu X.
      • Kwon H.S.
      • Fu C.
      • Lee J.W.
      • Southall N.
      • Marugan J.
      • Kim H.Y.
      Synaptamide activates the adhesion GPCR GPR110 (ADGRF1) through GAIN domain binding.
      ,
      • Lee J.W.
      • Huang B.X.
      • Kwon H.
      • Rashid M.A.
      • Kharebava G.
      • Desai A.
      • Patnaik S.
      • Marugan J.
      • Kim H.Y.
      Orphan GPR110 (ADGRF1) targeted by N-docosahexaenoylethanolamine in development of neurons and cognitive function.
      ). Synaptamide binds directly to the GAIN domain and results in a modest increase in cAMP levels, as shown by gene reporter assays (
      • Huang B.X.
      • Hu X.
      • Kwon H.S.
      • Fu C.
      • Lee J.W.
      • Southall N.
      • Marugan J.
      • Kim H.Y.
      Synaptamide activates the adhesion GPCR GPR110 (ADGRF1) through GAIN domain binding.
      ). Functional studies of ADGRG1 in neural progenitor cells demonstrated that ADGRG1 NTF-targeting antibodies could stimulate G12/13 signaling (
      • Iguchi T.
      • Sakata K.
      • Yoshizaki K.
      • Tago K.
      • Mizuno N.
      • Itoh H.
      Orphan G protein-coupled receptor GPR56 regulates neural progenitor cell migration via a Gα12/13 and Rho pathway.
      ). Recent structural studies of the ADGRG1 NTF corroborated this, showing that NTF-targeted antibodies could induce small, antibody-specific decreases or increases in G protein signaling (
      • Salzman G.S.
      • Zhang S.
      • Gupta A.
      • Koide A.
      • Koide S.
      • Araç D.
      Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region.
      ,
      • Salzman G.S.
      • Ackerman S.D.
      • Ding C.
      • Koide A.
      • Leon K.
      • Luo R.
      • Stoveken H.M.
      • Fernandez C.G.
      • Tall G.G.
      • Piao X.
      • Monk K.R.
      • Koide S.
      • Araç D.
      Structural basis for regulation of GPR56/ADGRG1 by its alternatively spliced extracellular domains.
      ). The changes in signaling were modest and within 0.5-fold of basal levels as opposed to tethered agonist– or synthetic peptide agonist–activated receptors that exhibit manyfold increases in signaling over holoreceptors (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ,
      • Liebscher I.
      • Schön J.
      • Petersen S.C.
      • Fischer L.
      • Auerbach N.
      • Demberg L.M.
      • Mogha A.
      • Cöster M.
      • Simon K.-U.
      • Rothemund S.
      • Monk K.R.
      • Schöneberg T.
      A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133.
      ,
      • Paavola K.J.
      • Stephenson J.R.
      • Ritter S.L.
      • Alter S.P.
      • Hall R.A.
      The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity.
      ,
      • Kishore A.
      • Purcell R.H.
      • Nassiri-Toosi Z.
      • Hall R.A.
      Stalk-dependent and stalk-independent signaling by the adhesion G protein-coupled receptors GPR56 (ADGRG1) and BAI1 (ADGRB1).
      ,
      • Wilde C.
      • Fischer L.
      • Lede V.
      • Kirchberger J.
      • Rothemund S.
      • Schöneberg T.
      • Liebscher I.
      The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist.
      ,
      • Demberg L.M.
      • Winkler J.
      • Wilde C.
      • Simon K.U.
      • Schön J.
      • Rothemund S.
      • Schöneberg T.
      • Prömel S.
      • Liebscher I.
      Activation of adhesion G protein-coupled receptors: agonist specificity of Stachel sequence-derived peptides.
      ). It is difficult to envision how soluble antibody ligands alone could support the anchoring and force requirements that are proposed to be needed for NTF dissociation, and there was no observation of NTF dissociation following the ADGRG1 antibody treatments. Therefore, AGPCR NTF-directed antibodies may be useful probes for understanding allosteric regulation of AGPCR activation that is independent of receptor fragment dissociation.
      Allosteric modulation of AGPCRs also occurs through NTF interactions with anchored protein ligands in cis (on the same cell) or in trans (from an adjacent cell). Distinct from those mentioned above, these ligands are anchored binding partners, often receptors themselves, and may convey conformational change to the 7TM via the NTF/GAIN. ADGRA2 interacts in cis with the protein RECK to regulate Wnt7/Frizzled receptor activation for modulation of angiogenesis in the central nervous system (
      • Cho C.
      • Smallwood P.M.
      • Nathans J.
      Reck and Gpr124 are essential receptor cofactors for Wnt7a/Wnt7b-specific signaling in mammalian CNS angiogenesis and blood-brain barrier regulation.
      ,
      • Vallon M.
      • Yuki K.
      • Nguyen T.D.
      • Chang J.
      • Yuan J.
      • Siepe D.
      • Miao Y.
      • Essler M.
      • Noda M.
      • Garcia K.C.
      • Kuo C.J.
      A RECK-WNT7 receptor-ligand interaction enables isoform-specific regulation of Wnt bioavailability.
      ). ADGRA2 is predicted to be noncleaved due to its atypical GPS (Table 1), so it makes sense for the receptor to mediate signaling allosterically in a large “signalosome” complex rather than through tethered agonism. Additional in cis interactions with AGPCRs have been observed, directly or indirectly, for ADGRB3 and stabilin-2, ADGRC1 and Vangl-2 or Frizzled-6, and ADGRL1 and contactin-6 (
      • Hamoud N.
      • Tran V.
      • Aimi T.
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      • Pelletier A.
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      • Kim I.S.
      • Kania A.
      • Yuzaki M.
      • Bouvier M.
      • Côté J.F.
      Spatiotemporal regulation of the GPCR activity of BAI3 by C1qL4 and stabilin-2 controls myoblast fusion.
      ,
      • Devenport D.
      • Oristian D.
      • Heller E.
      • Fuchs E.
      Mitotic internalization of planar cell polarity proteins preserves tissue polarity.
      ,
      • Zuko A.
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      • Post H.
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      • Shimoda Y.
      • Pasterkamp R.J.
      • Burbach J.P.
      Association of cell adhesion molecules contactin-6 and latrophilin-1 regulates neuronal apoptosis.
      ). Most ADGRL (latrophilin) receptors interact in trans with teneurin and fibronectin leucine-rich repeat transmembrane protein (FLRT) ligands to form trans-synaptic signaling complexes that in some contexts are implied to be stable due to their role in maintaining the architecture of the synapse (
      • Scholz N.
      • Guan C.
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      • Grotemeyer A.
      • Maiellaro I.
      • Gao S.
      • Beck S.
      • Pawlak M.
      • Sauer M.
      • Asan E.
      • Rothemund S.
      • Winkler J.
      • Prömel S.
      • Nagel G.
      • Langenhan T.
      • et al.
      Mechano-dependent signaling by latrophilin/CIRL quenches cAMP in proprioceptive neurons.
      ,
      • Scholz N.
      • Monk K.R.
      • Kittel R.J.
      • Langenhan T.
      Adhesion GPCRs as a putative class of metabotropic mechanosensors.
      ,
      • Scholz N.
      • Gehring J.
      • Guan C.
      • Ljaschenko D.
      • Fischer R.
      • Lakshmanan V.
      • Kittel R.J.
      • Langenhan T.
      The adhesion GPCR latrophilin/CIRL shapes mechanosensation.
      ,
      • O'Sullivan M.L.
      • de Wit J.
      • Savas J.N.
      • Comoletti D.
      • Otto-Hitt S.
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      • Ghosh A.
      FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development.
      ,
      • Ranaivoson F.M.
      • Liu Q.
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      • von Daake S.
      • Li S.
      • Lee D.
      • Demeler B.
      • Hendrickson W.A.
      • Comoletti D.
      Structural and mechanistic insights into the latrophilin3-FLRT3 complex that mediates glutamatergic synapse development.
      ,
      • Li J.
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      • Sando R.
      • Jiang X.
      • Kibrom A.
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      • Leon K.
      • Katanski C.
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      • Lu Y.C.
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      • Skiniotis G.
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      Structural basis for teneurin function in circuit-wiring: a toxin motif at the synapse.
      ,
      • Boucard A.A.
      • Maxeiner S.
      • Südhof T.C.
      Latrophilins function as heterophilic cell-adhesion molecules by binding to teneurins: regulation by alternative splicing.
      ,
      • Sando R.
      • Jiang X.
      • Südhof T.C.
      Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins.
      ). The stabilities of these cis or trans ligand/NTF complexes provide additional evidence for the idea that AGPCRs function in adhesion capacities and in doing so may allosterically regulate signaling in the absence of fragment dissociation.
      NTF allosteric regulation of the 7TM is also supported by evidence that the GAIN domain may act as an autoinhibitor of the 7TM and that this inhibition can be relieved or enhanced by ligand binding to NTF sites (Fig. 2D) (
      • Kishore A.
      • Hall R.A.
      Versatile signaling activity of adhesion GPCRs.
      ,
      • Paavola K.J.
      • Hall R.A.
      Adhesion G protein-coupled receptors: signaling, pharmacology, and mechanisms of activation.
      ). Autoinhibition experiments can be difficult to interpret while excluding instances of NTF shedding and resultant tethered agonism. NTF autoinhibition was inferred from observed increases in signaling following NTF deletion for multiple receptors (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ,
      • Kishore A.
      • Hall R.A.
      Versatile signaling activity of adhesion GPCRs.
      ,
      • Paavola K.J.
      • Hall R.A.
      Adhesion G protein-coupled receptors: signaling, pharmacology, and mechanisms of activation.
      ,
      • Paavola K.J.
      • Stephenson J.R.
      • Ritter S.L.
      • Alter S.P.
      • Hall R.A.
      The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity.
      ,
      • Okajima D.
      • Kudo G.
      • Yokota H.
      Brain-specific angiogenesis inhibitor 2 (BAI2) may be activated by proteolytic processing.
      ,
      • Ward Y.
      • Lake R.
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      LPA receptor heterodimerizes with CD97 to amplify LPA-initiated RHO-dependent signaling and invasion in prostate cancer cells.
      ,
      • Paavola K.J.
      • Sidik H.
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      • Eckart M.
      • Talbot W.S.
      Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126.
      ). As aforementioned, some antibodies directed toward the ADGRG1 NTF imparted subtle signaling decreases (
      • Salzman G.S.
      • Zhang S.
      • Gupta A.
      • Koide A.
      • Koide S.
      • Araç D.
      Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region.
      ). Although not direct evidence, the inhibitory effects of these antibodies supports the idea that in specific contexts, the NTF may repress activity of the CTF (red star in Fig. 2D). An ADGRG1 construct with an intact GAIN domain but deleted N-terminal pentraxin/laminin/neurexin/sex hormone–binding globulin-like (PLL) adhesive domain had enhanced signaling over full-length ADGRG1 (
      • Salzman G.S.
      • Zhang S.
      • Gupta A.
      • Koide A.
      • Koide S.
      • Araç D.
      Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region.
      ,
      • Salzman G.S.
      • Ackerman S.D.
      • Ding C.
      • Koide A.
      • Leon K.
      • Luo R.
      • Stoveken H.M.
      • Fernandez C.G.
      • Tall G.G.
      • Piao X.
      • Monk K.R.
      • Koide S.
      • Araç D.
      Structural basis for regulation of GPR56/ADGRG1 by its alternatively spliced extracellular domains.
      ). This implies that the PLL may enforce GAIN autoinhibition of the CTF or inhibit the CTF directly. However, exposure of recombinant ADGRG1 NTF to a ΔNTF ADGRG1 receptor resulted in no change in activated receptor signaling, indicating that the NTF may not provide inhibition unless it is noncovalently bound through the TM1 stalk (
      • Stoveken H.M.
      • Hajduczok A.G.
      • Xu L.
      • Tall G.G.
      Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.
      ).
      Whereas many details remain unknown for understanding orthosteric agonism and allosteric modulation, it is clear that the AGPCR NTFs are critical for regulating 7TM signaling. The extracellular domains (ECDs) of Class B1 and Class C GPCRs are also critical regulators of 7TM activation. Consequently, drawing parallels to these receptors may aid in understanding of how adhesion GPCRs are capable of transducing distal extracellular signals across the receptor to impart G protein activation.

      Class B1 GPCR-activating transitions and peptide agonism: Parallels for AGPCR activation?

      The Class B1 or secretin-like GPCR family is most closely related to adhesion GPCRs. There have been substantial structural determination efforts and insight into the transitions that occur between the inactive and active states of family B1 members in recent years (Fig. 3A) (
      • de Graaf C.
      • Song G.
      • Cao C.
      • Zhao Q.
      • Wang M.-W.
      • Wu B.
      • Stevens R.C.
      Extending the structural view of class B GPCRs.
      ,
      • Hollenstein K.
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      • Wang M.W.
      • Marshall F.H.
      • Stevens R.C.
      Insights into the structure of class B GPCRs.
      ,
      • Krumm B.
      • Roth B.L.
      A structural understanding of class B GPCR selectivity and activation revealed.
      ,
      • Willard F.S.
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      Discovery and pharmacology of the covalent GLP-1 receptor (GLP-1R) allosteric modulator BETP: a novel tool to probe GLP-1R pharmacology.
      ,
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      Molecular mechanisms of class B GPCR activation: insights from adrenomedullin receptors.
      ,
      • Wu F.
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      • Wu L.
      • Han G.W.
      • Ren Q.
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      • Reedtz-Runge S.
      • Song G.
      • Stevens R.C.
      Full-length human GLP-1 receptor structure without orthosteric ligands.
      ,
      • Ma S.
      • Shen Q.
      • Zhao L.H.
      • Mao C.
      • Zhou X.E.
      • Shen D.D.
      • de Waal P.W.
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      • Sexton P.M.
      • Wootten D.
      • Melcher K.
      • Zhang Y.
      • et al.
      Molecular basis for hormone recognition and activation of corticotropin-releasing factor receptors.
      ). As opposed to tethered-peptide agonists, family B1 members are activated by soluble peptide hormone agonists. Prominent examples include secretin, parathyroid hormone, glucagon, and glucagon-like peptides, among others (
      • Donnelly D.
      The structure and function of the glucagon-like peptide-1 receptor and its ligands.
      ,
      • Miller L.J.
      • Dong M.
      • Harikumar K.G.
      Ligand binding and activation of the secretin receptor, a prototypic family B G protein-coupled receptor.
      ,
      • Ehrenmann J.
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      • Rappas M.
      • Kummer L.
      • Doré A.S.
      • Plückthun A.
      High-resolution crystal structure of parathyroid hormone 1 receptor in complex with a peptide agonist.
      ). Family B1 members have ∼120–160 amino acid N-terminal ECDs that extend from stalks emanating from TM1, akin, although not homologous, to the GAIN domains of AGPCRs. Family B1 ECDs do not undergo autoproteolysis. In the unliganded state, family B1 ECDs were proposed to contact the three extracellular loops and are thought to constrain movements of the 7TM helical bundle, thereby stabilizing receptor low-activity states (Fig. 3A, left) (
      • Koth C.M.
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      Molecular basis for negative regulation of the glucagon receptor.
      ,
      • Yang L.
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      • Potter C.S.
      • Zhou H.
      • Griffin P.R.
      • et al.
      Conformational states of the full-length glucagon receptor.
      ). Family B1 ECDs serve as the initial docking site for the C termini of the peptide hormone agonists (
      • 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.
      ,
      • Castro M.
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      • Palm D.
      • Lohse M.J.
      • Vilardaga J.P.
      Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.
      ). Upon peptide binding, the orientation of the ECD is thought to change such that it is released from its strong contacts with the ECLs (Fig. 3A) (
      • Koth C.M.
      • Murray J.M.
      • Mukund S.
      • Madjidi A.
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      Molecular basis for negative regulation of the glucagon receptor.
      ). This opens the extracellular face of the 7TM bundle to allow entry of the N terminus of the agonist peptide hormone and its binding to the interior core. Thus, the orthosteric site of family B1 peptide agonists consist of two components, the ECD/C-terminal binding site and the 7TM bundle/N-terminal binding site.
      Figure thumbnail gr3
      Figure 3Parallels of adhesion GPCR allosteric activation to Class B1 and Class C GPCRs. A, family B1 GPCRs are receptors for soluble peptide agonists and are closely related to adhesion GPCRs. B1 receptors possess ECDs that inhibit signaling of the 7TM domain in the absence of a ligand via interactions with ECLs. In the active state of the receptor, the N terminus of the peptide agonist (orange) binds within the 7TM bundle, whereas the C terminus binds to the ECD. Shown is the structure of active glucagon-like peptide-1 receptor (GLP1R) (gray and blue, ribbon) bound to glucagon-like peptide-1 (GLP1) peptide (orange, space-filled, PDB entry 5VAI). B, Class C GPCRs are distantly related to adhesion GPCRs but also possess large ECDs. Class C receptors are obligate dimers that possess an extracellular VFT domain (blue) and a CRD (yellow) N-terminal to the 7TM bundle. Binding of ligands to the VFT domain results in activation of the 7TM. Long-range transmission of the bound ligand signal from the VFT involves conformational changes of the CRD that impart changes to the transmembrane helices of the 7TM bundle to favor an active-state conformation. This is aided, in part, by a critical interaction with ECL2 of the 7TM domain that acts as a rigid linker for a subtle rotational shift in the 7TMs. C, it is not certain how AGPCR ligands allosterically modulate adhesion GPCR signaling in the absence of NTF/CTF dissociation. In the absence of ligand, the GAIN domain may interact with the ECLs of the 7TM bundle to repress active conformations (left), similarly to the ECDs of family B1 receptors. In the presence of an allosteric ligand, the activation signal may be transmitted through the GAIN domain to the 7TM (right). This could activate the receptor by relieving repression conferred by the NTF or by utilizing the CTF stalk as a fulcrum in a manner akin to class C receptors that use a rigid body to impart activating conformational changes to the 7TM.
      Current structural information of adhesion GPCRs is limited to views of extracellular portions of the receptor, including GAIN domains, the GAIN plus adjacent NTF subdomains, or, for ADGRG1, the entire NTF (
      • Araç D.
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      Structural basis for adhesion G protein-coupled receptor Gpr126 function.
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      Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism.
      ). If the ECRs or NTFs of adhesion GPCRs parallel the function of family B1 ECDs, then they may bind the ECLs and act to constrain the 7TM bundle in a low-activity state (Fig. 3C). Multiple lines of evidence were discussed above demonstrating that distal ligand binding events can allosterically induce receptor activation in the absence of AGPCR NTF/CTF dissociation (
      • Wilde C.
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      The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist.
      ,
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      • et al.
      Mechano-dependent signaling by latrophilin/CIRL quenches cAMP in proprioceptive neurons.
      ,
      • Scholz N.
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      Adhesion GPCRs as a putative class of metabotropic mechanosensors.
      ,
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      Synaptamide activates the adhesion GPCR GPR110 (ADGRF1) through GAIN domain binding.
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      • Aust G.
      Mechano-dependent phosphorylation of the PDZ-binding motif of CD97/ADGRE5 modulates cellular detachment.
      ). The combined actions of ligand binding and force may alter the conformation of the CTF toward higher-activity states. The only known point of contact between the NTF and CTF is the stalk region that is contiguous with TM1. Conformational changes to TM1 that are induced allosterically through the stalk may account for instances of 7TM helical bundle activation, but the evidence for interactions between the NTF or GAIN domain and the AGPCR ECLs must also be considered (Fig. 3C). Ligand engagement events could alter these putative interactions and release constraints on receptor activation, paralleling the first step of family B1 receptor activation (derepression; Fig. 3A).
      AGPCR orthosteric agonism may also share parallels to family B1 soluble peptide hormone agonism. If AGPCR NTFs/GAINs serve to constrain the 7TM bundle, then upon force-mediated dissociation of the NTF from the CTF, this inhibition is relieved while tethered-peptide agonists are released from the interior core of the GAIN domain. The partially activated state of the receptor (after NTF disinhibition) would favor the fully active form and drive first-order binding of the tethered-peptide agonist to its orthosteric site. Akin to family B1 receptors, the N termini of AGPCR tethered-peptide agonists may be expected to bind within the interior of the 7TM bundle, and the C-terminal elements of the tethered-peptide agonist/stalk region might bind to the ECLs. The N-terminal ∼7 amino acids of adhesion GPCR tethered-peptide agonists are much more hydrophobic than family B1 peptide agonist N termini, suggesting an orthosteric site position that lies deeper within the interior of the 7TM bundle. Family B1 peptide agonists are highly conserved at their N termini, with receptor-specific variations occurring at the C termini (
      • 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.
      ). This feature is shared with adhesion GPCR tethered-peptide agonists that also have highly conserved N termini and divergent C termini.

      Class C GPCRs as a parallel for transmission of distal activation signals?

      Class C GPCRs are distantly related to AGPCRs and possess completely distinct ECR architectures. Class C receptors may nonetheless provide clues for understanding how AGPCRs allosterically regulate signaling without NTF/CTF dissociation (Fig. 3B). Class C receptors are dimers that possess large ECRs comprising a Venus flytrap (VFT) domain and an intervening rigid structure, such as a cysteine-rich domain (CRD), that are linked N-terminally to the 7TM bundle (Fig. 3B) (
      • Pin J.P.
      • Galvez T.
      • Prézeau L.
      Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors.
      ,
      • Romano C.
      • Yang W.L.
      • O'Malley K.L.
      Metabotropic glutamate receptor 5 is a disulfide-linked dimer.
      ). Class C GPCR ligand binding occurs at the distal VFT domains (
      • Urwyler S.
      Allosteric modulation of family C G-protein-coupled receptors: from molecular insights to therapeutic perspectives.
      ). In the ligand-free state, a prototypical Class C receptor dimer possesses two VFT domains in an open conformation, and ligand binding causes domain closure. This precedes 7TM bundle structural shifts that facilitate G protein activation (
      • Kniazeff J.
      • Prézeau L.
      • Rondard P.
      • Pin J.P.
      • Goudet C.
      Dimers and beyond: the functional puzzles of class C GPCRs.
      ). The exact mechanisms of how the activation signal is transduced from the ligand-bound VFT to the 7TM bundle are emerging, and it is thought that ligand binding induces inter- and intraprotomer conformational changes that result in 7TM activation (Fig. 3B, right) (
      • Lei T.
      • Hu Z.
      • Ding R.
      • Chen J.
      • Li S.
      • Zhang F.
      • Pu X.
      • Zhao N.
      Exploring the activation mechanism of a metabotropic glutamate receptor homodimer via molecular dynamics simulation.
      ,
      • Monnier C.
      • Tu H.
      • Bourrier E.
      • Vol C.
      • Lamarque L.
      • Trinquet E.
      • Pin J.P.
      • Rondard P.
      Trans-activation between 7TM domains: implication in heterodimeric GABAB receptor activation.
      ). Ligand binding draws the two VFT domains in close proximity, which alters the conformation of the intervening rigid body to impose rotational shifts of the 7TM bundles that stabilize an active state (
      • Tateyama M.
      • Abe H.
      • Nakata H.
      • Saito O.
      • Kubo Y.
      Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1α.
      ,
      • Tateyama M.
      • Kubo Y.
      Dual signaling is differentially activated by different active states of the metabotropic glutamate receptor 1α.
      ,
      • 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.
      ). Recent cryo-EM structural work with mGlu5 has helped to form this hypothesis while also revealing a critical interaction between the CRD and ECL2, where the CRD-ECL2 interaction is proposed to serve as a rigid turning point to convey structural shifts between the CRD and 7TM domain (
      • 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.
      ). The importance of the rigid linker-ECL2 interaction was affirmed in a recent structural determination of the heterodimeric GABAB receptor that lacks typical CRDs (
      • Papasergi-Scott M.M.
      • Robertson M.J.
      • Seven A.B.
      • Panova O.
      • Mathiesen J.M.
      • Skiniotis G.
      Structures of metabotropic GABAB receptor.
      ). An ECL2 truncation of the ligand-bound protomer, GABAB1, destabilized this interaction and resulted in increased basal signaling, whereas ECL truncation of the other protomer, GABAB2, only increased GABAB efficacy, indicating that the rigid linker-ECL2 interaction mediates both inter- and intraprotomer interactions.
      In instances where AGPCR ligands bind to the N-terminal adhesive domains and allosterically promote signaling without NTF/CTF dissociation, the information would also need to be transmitted distally across the receptor to the 7TM bundle. The most obvious domain to act as the transmitter of these intramolecular signals is the GAIN domain, which might function analogously to the Class C rigid body. The Class C rigid link between the VFT and 7TM generally possesses a cysteine-rich β-sheet architecture that confers ligand-mediated contortions to the 7TM, which may parallel the dense β-sheet architecture of the GAINB domain of AGPCRs. In line with this, the ADGRG1 GAIN domain forms a critical disulfide link to the PLL adhesive domain (Fig. 1D) (
      • Salzman G.S.
      • Ackerman S.D.
      • Ding C.
      • Koide A.
      • Leon K.
      • Luo R.
      • Stoveken H.M.
      • Fernandez C.G.
      • Tall G.G.
      • Piao X.
      • Monk K.R.
      • Koide S.
      • Araç D.
      Structural basis for regulation of GPR56/ADGRG1 by its alternatively spliced extracellular domains.
      ). Disruption of this disulfide bond via C-to-S mutations surprisingly increased basal signaling ∼2-fold in a gene reporter readout (
      • Salzman G.S.
      • Ackerman S.D.
      • Ding C.
      • Koide A.
      • Leon K.
      • Luo R.
      • Stoveken H.M.
      • Fernandez C.G.
      • Tall G.G.
      • Piao X.
      • Monk K.R.
      • Koide S.
      • Araç D.
      Structural basis for regulation of GPR56/ADGRG1 by its alternatively spliced extracellular domains.
      ). Additionally, an ADGRG6 splice variant that adopted more diverse ECR conformations as judged by negative-stain EM images also had greater basal signaling (
      • Leon K.
      • Cunningham R.L.
      • Riback J.A.
      • Feldman E.
      • Li J.
      • Sosnick T.R.
      • Zhao M.
      • Monk K.R.
      • Araç D.
      Structural basis for adhesion G protein-coupled receptor Gpr126 function.
      ). These studies indicate that disruption of AGPCR NTF rigidity may result in a more dynamic GAIN domain that influences 7TM activation. Future studies may explore this putative function of the GAIN domain as conferring rigidity and stabilization of inactive states. Binding of allosteric ligands may impart flexibility, allowing for activating structural changes to be conferred to the 7TM bundle (Fig. 3C).
      Given the lack of hard evidence for NTF/ECL interactions, it is plausible that the tethered agonist, which links the two domains together in the holoreceptor conformation, may also be key in regulating allosteric modulation via 7TM conformational shifts. The tethered agonist stalk (beyond the core seven residues embedded within the GAIN) may serve as a pivot point to convey 7TM contortions via TM1. This would allow for receptor activation if NTF/ECL interactions do not exist, but it is also possible that both types of interactions are relevant and may work together (Fig. 3C). Supporting this role for the stalk, reduced basal signaling of an ADGRG5 SNP was attributed to its missing glutamine residue within the middle of the stalk apart from the core tethered agonist. A synthetic peptide agonist derived from this stalk enhanced signaling independent of the glutamine (
      • Wilde C.
      • Fischer L.
      • Lede V.
      • Kirchberger J.
      • Rothemund S.
      • Schöneberg T.
      • Liebscher I.
      The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist.
      ). This implies that the stalk of the ADGRG5 SNP that contains the glutamine positively impacts holoreceptor basal activity independent of the tethered agonist, possibly by conveying structural information to the 7TM. In sum, there is much to learn about the nature of AGPCR ECR interactions with the 7TM domain, and the potential parallels to Class B1 and Class C mechanisms may help guide research design.

      How do AGPCR ligands modulate receptor activity?

      Insights into AGPCR activation mechanisms may be gleaned from the endogenous ligands that target them, some of which were introduced previously. Wide-ranging deorphanization studies have uncovered several endogenous ligands that bind to individual AGPCRs (reviewed in Refs.
      • Purcell R.H.
      • Hall R.A.
      Adhesion G protein–coupled receptors as drug targets.
      ,
      • Langenhan T.
      Adhesion G protein-coupled receptors—candidate metabotropic mechanosensors and novel drug targets.
      ,
      • Kishore A.
      • Hall R.A.
      Versatile signaling activity of adhesion GPCRs.
      ,
      • Folts C.J.
      • Giera S.
      • Li T.
      • Piao X.
      Adhesion G protein-coupled receptors as drug targets for neurological diseases.
      ,
      • Langenhan T.
      • Aust G.
      • Hamann J.
      Sticky signaling–adhesion class G protein-coupled receptors take the stage.
      ,
      • Wacker D.
      • Stevens R.C.
      • Roth B.L.
      How ligands illuminate GPCR molecular pharmacology.
      , and
      • Moreno-Salinas A.L.
      • Avila-Zozaya M.
      • Ugalde-Silva P.
      • Hernandez-Guzman D.A.
      • Missirlis F.
      • Boucard A.A.
      Latrophilins: a neuro-centric view of an evolutionary conserved adhesion G protein-coupled receptor subfamily.
      ). The identities of the ligands provide useful clues for understanding receptor activation mechanisms in physiological contexts. About half of all AGPCRs remain orphans, and of those with identified ligands, it is unanimously unclear how the ligands impart receptor activation. Table 2 and Fig. S1 detail the currently known endogenous AGPCR ligands and proposes a means to classify adhesion GPCRs based on the type(s) of ligand that each receptor binds. Some AGPCRs have multiple ligands and span multiple categories of the classification system: 1) trans-cell–presented proteins, 2) extracellular matrix components, and 3) soluble proteins, peptides, lipids, and small molecules. Cis-cell–presented protein ligands represent an emerging category with only a select few examples.
      Table 2Endogenous adhesion GPCR ligands
      ClassReceptorECM componentsLipids/soluble proteins/small moleculesTrans-presented proteinsReferences
      AADGRA1Orphan
      ADGRA2Integrin-αVβ3, glycosaminoglycans, syndecan-1,2
      • Vallon M.
      • Essler M.
      Proteolytically processed soluble tumor endothelial marker (TEM) 5 mediates endothelial cell survival during angiogenesis by linking integrin αvβ3 to glycosaminoglycans.
      ,
      • Chong Z.-S.
      • Ohnishi S.
      • Yusa K.
      • Wright G.J.
      Pooled extracellular receptor-ligand interaction screening using CRISPR activation.
      ADGRA3Orphan
      BADGRB1αVβ5 integrinPhosphatidylserine, lipopolysaccharideRTN4R, CD36
      • Park D.
      • Tosello-Trampont A.-C.
      • Elliott M.R.
      • Lu M.
      • Haney L.B.
      • Ma Z.
      • Klibanov A.L.
      • Mandell J.W.
      • Ravichandran K.S.
      BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module.
      ,
      • Chong Z.-S.
      • Ohnishi S.
      • Yusa K.
      • Wright G.J.
      Pooled extracellular receptor-ligand interaction screening using CRISPR activation.
      ,
      • Koh J.T.
      • Kook H.
      • Kee H.J.
      • Seo Y.W.
      • Jeong B.C.
      • Lee J.H.
      • Kim M.Y.
      • Yoon K.C.
      • Jung S.
      • Kim K.K.
      Extracellular fragment of brain-specific angiogenesis inhibitor 1 suppresses endothelial cell proliferation by blocking αvβ5 integrin.
      ,
      • Das S.
      • Owen K.A.
      • Ly K.T.
      • Park D.
      • Black S.G.
      • Wilson J.M.
      • Sifri C.D.
      • Ravichandran K.S.
      • Ernst P.B.
      • Casanova J.E.
      Brain angiogenesis inhibitor 1 (BAI1) is a pattern recognition receptor that mediates macrophage binding and engulfment of Gram-negative bacteria.
      ,
      • Kaur B.
      • Cork S.M.
      • Sandberg E.M.
      • Devi N.S.
      • Zhang Z.
      • Klenotic P.A.
      • Febbraio M.
      • Shim H.
      • Mao H.
      • Tucker-Burden C.
      • Silverstein R.L.
      • Brat D.J.
      • Olson J.J.
      • Van Meir E.G.
      Vasculostatin inhibits intracranial glioma growth and negatively regulates in vivo angiogenesis through a CD36-dependent mechanism.
      ADGRB2Glutaminase-interacting protein (GIP)
      • Zencir S.
      • Ovee M.
      • Dobson M.J.
      • Banerjee M.
      • Topcu Z.
      • Mohanty S.
      Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein.
      ADGRB3C1ql1-C1ql4
      • Bolliger M.F.
      • Martinelli D.C.
      • Südhof T.C.
      The cell-adhesion G protein-coupled receptor BAI3 is a high-affinity receptor for C1q-like proteins.
      ,
      • Sigoillot S.M.
      • Iyer K.
      • Binda F.
      • González-Calvo I.
      • Talleur M.
      • Vodjdani G.
      • Isope P.
      • Selimi F.
      The secreted protein C1QL1 and its receptor BAI3 control the synaptic connectivity of excitatory inputs converging on cerebellar Purkinje cells.
      CADGRC1Orphan
      ADGRC2Orphan
      ADGRC3Dystroglycan
      • Lindenmaier L.B.
      • Parmentier N.
      • Guo C.
      • Tissir F.
      • Wright K.M.
      Dystroglycan is a scaffold for extracellular axon guidance decisions.
      DADGRD1Orphan
      ADGRD2Orphan
      EADGRE1Unknown NK cell receptor
      • Warschkau H.
      • Kiderlen A.F.
      A monoclonal antibody directed against the murine macrophage surface molecule F4/80 modulates natural immune response to Listeria monocytogenes.
      ADGRE2Chondroitin sulfate, Integrins-αVβ3
      Predicted based on structural similarities.
      , α5β1
      Predicted based on structural similarities.
      CD90
      Predicted based on structural similarities.
      • Lin H.-H.
      • Stacey M.
      • Hamann J.
      • Gordon S.
      • McKnight A.J.
      Human EMR2, a novel EGF-TM7 molecule on chromosome 19p13.1, is closely related to CD97.
      ,
      • Stacey M.
      • Chang G.-W.
      • Davies J.Q.
      • Kwakkenbos M.J.
      • Sanderson R.D.
      • Hamann J.
      • Gordon S.
      • Lin H.-H.
      The epidermal growth factor-like domains of the human EMR2 receptor mediate cell attachment through chondroitin sulfate glycosaminoglycans.
      ADGRE3Unknown ligand on macrophages and neutrophils
      • Stacey M.
      • Lin H.-H.
      • Hilyard K.L.
      • Gordon S.
      • McKnight A.J.
      Human epidermal growth factor (EGF) module-containing mucin-like hormone receptor 3 is a new member of the EGF-TM7 family that recognizes a ligand on human macrophages and activated neutrophils.
      ADGRE4Unknown B cell ligand
      • Stacey M.
      • Chang G.-W.
      • Sanos S.L.
      • Chittenden L.R.
      • Stubbs L.
      • Gordon S.
      • Lin H.-H.
      EMR4, a novel epidermal growth factor (EGF)-TM7 molecule up-regulated in activated mouse macrophages, binds to a putative cellular ligand on B lymphoma cell line A20.
      ADGRE5Chondroitin sulfate, integrins αVβ3, α5β1CD55, CD90, lysophosphatidic acid receptor
      • Ward Y.
      • Lake R.
      • Yin J.J.
      • Heger C.D.
      • Raffeld M.
      • Goldsmith P.K.
      • Merino M.
      • Kelly K.
      LPA receptor heterodimerizes with CD97 to amplify LPA-initiated RHO-dependent signaling and invasion in prostate cancer cells.
      ,
      • Stacey M.
      • Chang G.-W.
      • Davies J.Q.
      • Kwakkenbos M.J.
      • Sanderson R.D.
      • Hamann J.
      • Gordon S.
      • Lin H.-H.
      The epidermal growth factor-like domains of the human EMR2 receptor mediate cell attachment through chondroitin sulfate glycosaminoglycans.
      ,
      • Wang T.
      • Ward Y.
      • Tian L.
      • Lake R.
      • Guedez L.
      • Stetler-Stevenson W.G.
      • Kelly K.
      CD97, an adhesion receptor on inflammatory cells, stimulates angiogenesis through binding integrin counterreceptors on endothelial cells.
      • Wandel E.
      • Saalbach A.
      • Sittig D.
      • Gebhardt C.
      • Aust G.
      Thy-1 (CD90) is an interacting partner for CD97 on activated endothelial cells.
      • Hamann J.
      • Vogel B.
      • van Schijndel G.M.
      • van Lier R.A.
      The seven-span transmembrane receptor CD97 has a cellular ligand (CD55, DAF).
      FADGRF1Synaptamide
      • Kim H.-Y.
      • Spector A.A.
      N-Docosahexaenoylethanolamine: a neurotrophic and neuroprotective metabolite of docosahexaenoic acid.
      ,
      • Huang B.X.
      • Hu X.
      • Kwon H.S.
      • Fu C.
      • Lee J.W.
      • Southall N.
      • Marugan J.
      • Kim H.Y.
      Synaptamide activates the adhesion GPCR GPR110 (ADGRF1) through GAIN domain binding.
      ADGRF2Orphan
      ADGRF3Orphan
      ADGRF4Orphan
      ADGRF5Surfactant protein-D
      • Fukuzawa T.
      • Ishida J.
      • Kato A.
      • Ichinose T.
      • Ariestanti D.M.
      • Takahashi T.
      • Ito K.
      • Abe J.
      • Suzuki T.
      • Wakana S.
      • Fukamizu A.
      • Nakamura N.
      • Hirose S.
      Lung surfactant levels are regulated by Ig-Hepta/GPR116 by monitoring surfactant protein D.
      ,
      • Schneberger D.
      • DeVasure J.M.
      • Kirychuk S.A.
      • Wyatt T.A.
      Organic barn dust inhibits surfactant protein D production through protein kinase-Cα dependent increase of GPR116.
      GADGRG1Collagen III, heparin, transglutaminase-2, lamininProgastrin
      • Stoveken H.M.
      • Larsen S.D.
      • Smrcka A.V.
      • Tall G.G.
      Gedunin- and Khivorin-derivatives are small-molecule partial agonists for adhesion G protein-coupled receptors GPR56/ADGRG1 and GPR114/ADGRG5.
      ,
      • Zhu B.
      • Luo R.
      • Jin P.
      • Li T.
      • Oak H.C.
      • Giera S.
      • Monk K.R.
      • Lak P.
      • Shoichet B.K.
      • Piao X.
      GAIN domain-mediated cleavage is required for activation of G protein-coupled receptor 56 (GPR56) by its natural ligands and a small-molecule agonist.
      ,
      • Luo R.
      • Jeong S.-J.
      • Jin Z.
      • Strokes N.
      • Li S.
      • Piao X.
      G protein-coupled receptor 56 and collagen III, a receptor-ligand pair, regulates cortical development and lamination.
      ,
      • Giera S.
      • Luo R.
      • Ying Y.
      • Ackerman S.D.
      • Jeong S.-J.
      • Stoveken H.M.
      • Folts C.J.
      • Welsh C.A.
      • Tall G.G.
      • Stevens B.
      • Monk K.R.
      • Piao X.
      Microglial transglutaminase-2 drives myelination and myelin repair via GPR56/ADGRG1 in oligodendrocyte precursor cells.
      ,
      • Xu L.
      • Begum S.
      • Hearn J.D.
      • Hynes R.O.
      GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis.
      ,
      • Stoveken H.M.
      • Bahr L.L.
      • Anders M.W.
      • Wojtovich A.P.
      • Smrcka A.V.
      • Tall G.G.
      Dihydromunduletone is a small-molecule selective adhesion G protein-coupled receptor antagonist.
      ,
      • Jin G.
      • Sakitani K.
      • Wang H.
      • Jin Y.
      • Dubeykovskiy A.
      • Worthley D.L.
      • Tailor Y.
      • Wang T.C.
      The G-protein coupled receptor 56, expressed in colonic stem and cancer cells, binds progastrin to promote proliferation and carcinogenesis.
      • Ackerman S.D.
      • Luo R.
      • Poitelon Y.
      • Mogha A.
      • Harty B.L.
      • D'Rozario M.
      • Sanchez N.E.
      • Lakkaraju A.K.K.
      • Gamble P.
      • Li J.
      • Qu J.
      • MacEwan M.R.
      • Ray W.Z.
      • Aguzzi A.
      • Feltri M.L.
      • et al.
      GPR56/ADGRG1 regulates development and maintenance of peripheral myelin.
      • Huang K.Y.
      • Lin H.H.
      The activation and signaling mechanisms of GPR56/ADGRG1 in melanoma cell.
      ADGRG2Orphan
      ADGRG3Orphan
      ADGRG4Orphan
      ADGRG5Orphan
      ADGRG6Collagen IV, laminin-211Cellular prion protein
      • Paavola K.J.
      • Sidik H.
      • Zuchero J.B.
      • Eckart M.
      • Talbot W.S.
      Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126.
      ,
      • Petersen S.C.
      • Luo R.
      • Liebscher I.
      • Giera S.
      • Jeong S.-J.
      • Mogha A.
      • Ghidinelli M.
      • Feltri M.L.
      • Schöneberg T.
      • Piao X.
      • Monk K.R.
      The adhesion GPCR GPR126 has distinct, domain-dependent functions in Schwann cell development mediated by interaction with laminin-211.
      ,
      • Küffer A.
      • Lakkaraju A.K.
      • Mogha A.
      • Petersen S.C.
      • Airich K.
      • Doucerain C.
      • Marpakwar R.
      • Bakirci P.
      • Senatore A.
      • Monnard A.
      • Schiavi C.
      • Nuvolone M.
      • Grosshans B.
      • Hornemann S.
      • Bassilana F.
      • et al.
      The prion protein is an agonistic ligand of the G protein-coupled receptor Adgrg6.
      ADGRG7Orphan
      LADGRL1Teneurin-2/4, neurexin-1α, -1β, -2β, -3β, FLRT1/3
      • O'Sullivan M.L.
      • de Wit J.
      • Savas J.N.
      • Comoletti D.
      • Otto-Hitt S.
      • Yates J.R.
      • Ghosh A.
      FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development.
      ,
      • Silva J.P.
      • Lelianova V.G.
      • Ermolyuk Y.S.
      • Vysokov N.
      • Hitchen P.G.
      • Berninghausen O.
      • Rahman M.A.
      • Zangrandi A.
      • Fidalgo S.
      • Tonevitsky A.G.
      • Dell A.
      • Volynski K.E.
      • Ushkaryov Y.A.
      Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities.
      ,
      • Boucard A.A.
      • Ko J.
      • Südhof T.C.
      High affinity neurexin binding to cell adhesion G-protein-coupled receptor CIRL1/latrophilin-1 produces an intercellular adhesion complex.
      ,
      • Krasnoperov V.G.
      • Bittner M.A.
      • Beavis R.
      • Kuang Y.
      • Salnikow K.V.
      • Chepurny O.G.
      • Little A.R.
      • Plotnikov A.N.
      • Wu D.
      • Holz R.W.
      • Petrenko A.G.
      α-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor.
      ADGRL2Teneurin-2, FLRT3
      • O'Sullivan M.L.
      • de Wit J.
      • Savas J.N.
      • Comoletti D.
      • Otto-Hitt S.
      • Yates J.R.
      • Ghosh A.
      FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development.
      ,
      • Jackson V.A.
      • Meijer D.H.
      • Carrasquero M.
      • van Bezouwen L.S.
      • Lowe E.D.
      • Kleanthous C.
      • Janssen B.J.C.
      • Seiradake E.
      Structures of Teneurin adhesion receptors reveal an ancient fold for cell-cell interaction.
      ADGRL3Teneurin-3, FLRT1/3, UNC5A
      • O'Sullivan M.L.
      • de Wit J.
      • Savas J.N.
      • Comoletti D.
      • Otto-Hitt S.
      • Yates J.R.
      • Ghosh A.
      FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development.
      ,
      • Boucard A.A.
      • Maxeiner S.
      • Südhof T.C.
      Latrophilins function as heterophilic cell-adhesion molecules by binding to teneurins: regulation by alternative splicing.
      ,
      • Jackson V.A.
      • Mehmood S.
      • Chavent M.
      • Roversi P.
      • Carrasquero M.
      • Del Toro D.
      • Seyit-Bremer G.
      • Ranaivoson F.M.
      • Comoletti D.
      • Sansom M.S.
      • Robinson C.V.
      • Klein R.
      • Seiradake E.
      Super-complexes of adhesion GPCRs and neural guidance receptors.
      ADGRL4Orphan
      VADGRV1Orphan
      * Predicted based on structural similarities.

      Trans-cell–presented proteins

      Trans-signaling complexes are formed through interactions of the extracellular extensions of protein ligands presented by neighboring cells and the N-terminal adhesive modules of AGPCRs to provide modes of cell-to-cell adhesion and communication. As discussed previously, stable trans-cell AGPCR and protein ligand complexes may signal through allosteric activation modes and/or tethered agonism. Tethered agonism imparted by trans-cell–adhesive ligands is thought to occur by ligand binding to its AGPCR binding site(s) in a manner that is tighter than the strength of the noncovalent contacts that embed β-strand 13 within the GAIN domain. Consequently, shear force created by the two cells moving in relation to each other serves to dissociate the ligand-anchored NTF from the CTF to promote signaling. Oftentimes multiple trans-cell ligands bind simultaneously to AGPCR N-terminal adhesive modules, which is thought to provide strong multivalent binding that is sufficient to anchor the NTF. Prominent examples of AGPCRs that utilize trans-cell ligands are those present at synaptic junctions that bind to ligands spanning the synapse. ADGRL (latrophilin) and ADGRB (BAI) receptors are both enriched in synaptic junctions and serve as models of trans-cell synaptic AGPCR signaling.
      Most ADGRL receptors (ADGRL1–4), excluding ADGRL4, are localized on axons, axonal growth cones, and nerve terminals (
      • O'Sullivan M.L.
      • de Wit J.
      • Savas J.N.
      • Comoletti D.
      • Otto-Hitt S.
      • Yates J.R.
      • Ghosh A.
      FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development.
      ,
      • Moreno-Salinas A.L.
      • Avila-Zozaya M.
      • Ugalde-Silva P.
      • Hernandez-Guzman D.A.
      • Missirlis F.
      • Boucard A.A.
      Latrophilins: a neuro-centric view of an evolutionary conserved adhesion G protein-coupled receptor subfamily.
      ,
      • Silva J.P.
      • Lelianova V.G.
      • Ermolyuk Y.S.
      • Vysokov N.
      • Hitchen P.G.
      • Berninghausen O.
      • Rahman M.A.
      • Zangrandi A.
      • Fidalgo S.
      • Tonevitsky A.G.
      • Dell A.
      • Volynski K.E.
      • Ushkaryov Y.A.
      Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities.
      ,
      • Boucard A.A.
      • Ko J.
      • Südhof T.C.
      High affinity neurexin binding to cell adhesion G-protein-coupled receptor CIRL1/latrophilin-1 produces an intercellular adhesion complex.
      ). They are structurally distinguished from other AGPCRs by the presence of an N-terminal olfactomedin (Olf) domain and a lectin-like (Lec) domain. ADGRL receptors regulate interneuron adhesion and the migration of growth cones (actin-rich neuronal extensions) while promoting synapse formation and remodeling through control of cytoskeletal rearrangements (
      • O'Sullivan M.L.
      • de Wit J.
      • Savas J.N.
      • Comoletti D.
      • Otto-Hitt S.
      • Yates J.R.
      • Ghosh A.
      FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development.
      ,
      • Ranaivoson F.M.
      • Liu Q.
      • Martini F.
      • Bergami F.
      • von Daake S.
      • Li S.
      • Lee D.
      • Demeler B.
      • Hendrickson W.A.
      • Comoletti D.
      Structural and mechanistic insights into the latrophilin3-FLRT3 complex that mediates glutamatergic synapse development.
      ,
      • Boucard A.A.
      • Maxeiner S.
      • Südhof T.C.
      Latrophilins function as heterophilic cell-adhesion molecules by binding to teneurins: regulation by alternative splicing.
      ,
      • Sando R.
      • Jiang X.
      • Südhof T.C.
      Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins.
      ,
      • Silva J.P.
      • Lelianova V.G.
      • Ermolyuk Y.S.
      • Vysokov N.
      • Hitchen P.G.
      • Berninghausen O.
      • Rahman M.A.
      • Zangrandi A.
      • Fidalgo S.
      • Tonevitsky A.G.
      • Dell A.
      • Volynski K.E.
      • Ushkaryov Y.A.
      Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities.
      ). Defects or variants in ADGRL genes are associated with neuronal disorders including attention deficit and hyperactivity disorder, autism spectrum disorder, schizophrenia, rhombencephalosynapsis, and microcephaly (
      • Arcos-Burgos M.
      • Jain M.
      • Acosta M.T.
      • Shively S.
      • Stanescu H.
      • Wallis D.
      • Domené S.
      • Vélez J.I.
      • Karkera J.D.
      • Balog J.
      • Berg K.
      • Kleta R.
      • Gahl W.A.
      • Roessler E.
      • Long R.
      • et al.
      A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication.
      ,
      • Ribasés M.
      • Ramos-Quiroga J.A.
      • Sánchez-Mora C.
      • Bosch R.
      • Richarte V.
      • Palomar G.
      • Gastaminza X.
      • Bielsa A.
      • Arcos-Burgos M.
      • Muenke M.
      • Castellanos F.X.
      • Cormand B.
      • Bayés M.
      • Casas M.
      Contribution of LPHN3 to the genetic susceptibility to ADHD in adulthood: a replication study.
      ,
      • Domené S.
      • Stanescu H.
      • Wallis D.
      • Tinloy B.
      • Pineda D.E.
      • Kleta R.
      • Arcos-Burgos M.
      • Roessler E.
      • Muenke M.
      Screening of human LPHN3 for variants with a potential impact on ADHD susceptibility.
      ,
      • Chen C.H.
      • Chen H.I.
      • Chien W.H.
      • Li L.H.
      • Wu Y.Y.
      • Chiu Y.N.
      • Tsai W.C.
      • Gau S.S.
      High resolution analysis of rare copy number variants in patients with autism spectrum disorder from Taiwan.
      ,
      • Legge S.E.
      • Hamshere M.L.
      • Ripke S.
      • Pardinas A.F.
      • Goldstein J.I.
      • Rees E.
      • Richards A.L.
      • Leonenko G.
      • Jorskog L.F.
      • Clozapine-Induced Agranulocytosis C.
      • Chambert K.D.
      • Collier D.A.
      • Genovese G.
      • Giegling I.
      • Holmans P.
      • et al.
      Genome-wide common and rare variant analysis provides novel insights into clozapine-associated neutropenia.
      ,
      • Vezain M.
      • Lecuyer M.
      • Rubio M.
      • Dupé V.
      • Ratié L.
      • David V.
      • Pasquier L.
      • Odent S.
      • Coutant S.
      • Tournier I.
      • Trestard L.
      • Adle-Biassette H.
      • Vivien D.
      • Frebourg T.
      • Gonzalez B.J.
      • et al.
      A de novo variant in ADGRL2 suggests a novel mechanism underlying the previously undescribed association of extreme microcephaly with severely reduced sulcation and rhombencephalosynapsis.
      ). ADGRL receptors bind to three classes of single-membrane pass, trans-presented protein ligands: teneurins, neurexins, and FLRTs. Teneurins bind to the ADGRL Lec domain, whereas neurexins and FLRTs interact with the ADGRL Olf domain (
      • Sando R.
      • Jiang X.
      • Südhof T.C.
      Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins.
      ,
      • Li J.
      • Xie Y.
      • Cornelius S.
      • Jiang X.
      • Sando R.
      • Kordon S.P.
      • Pan M.
      • Leon K.
      • Südhof T.C.
      • Zhao M.
      • Araç D.
      Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism.
      ,
      • Boucard A.A.
      • Ko J.
      • Südhof T.C.
      High affinity neurexin binding to cell adhesion G-protein-coupled receptor CIRL1/latrophilin-1 produces an intercellular adhesion complex.
      ,
      • Del Toro D.
      • Carrasquero-Ordaz M.A.
      • Chu A.
      • Ruff T.
      • Shahin M.
      • Jackson V.A.
      • Chavent M.
      • Berbeira-Santana M.
      • Seyit-Bremer G.
      • Brignani S.
      • Kaufmann R.
      • Lowe E.
      • Klein R.
      • Seiradake E.
      Structural basis of teneurin-latrophilin interaction in repulsive guidance of migrating neurons.
      ). These proteins comprise trans-synaptic signaling complexes and provide models for understanding how AGPCRs can be activated by trans-presented ligands. For instance, ADGRL1 interacts with both teneurin-2 (also known as Lasso) and FLRT simultaneously to regulate dendritic arborization, axonal extension, and synaptogenesis (
      • Sando R.
      • Jiang X.
      • Südhof T.C.
      Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins.
      ,
      • Li J.
      • Xie Y.
      • Cornelius S.
      • Jiang X.
      • Sando R.
      • Kordon S.P.
      • Pan M.
      • Leon K.
      • Südhof T.C.
      • Zhao M.
      • Araç D.
      Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism.
      ,
      • Del Toro D.
      • Carrasquero-Ordaz M.A.
      • Chu A.
      • Ruff T.
      • Shahin M.
      • Jackson V.A.
      • Chavent M.
      • Berbeira-Santana M.
      • Seyit-Bremer G.
      • Brignani S.
      • Kaufmann R.
      • Lowe E.
      • Klein R.
      • Seiradake E.
      Structural basis of teneurin-latrophilin interaction in repulsive guidance of migrating neurons.
      ). These neuronal reshaping processes are thought to result from Rho/Rac-mediated actin cytoskeletal changes downstream of G12/13 signaling, through which ADGRL3 was recently shown to signal (
      • Moers A.
      • Nürnberg A.
      • Goebbels S.
      • Wettschureck N.
      • Offermanns S.
      Gα12/Gα13 deficiency causes localized overmigration of neurons in the developing cerebral and cerebellar cortices.
      ,
      • Kranenburg O.
      • Poland M.
      • van Horck F.P.
      • Drechsel D.
      • Hall A.
      • Moolenaar W.H.
      Activation of RhoA by lysophosphatidic acid and Gα12/13 subunits in neuronal cells: induction of neurite retraction.
      ,
      • Katoh H.
      • Aoki J.
      • Yamaguchi Y.
      • Kitano Y.
      • Ichikawa A.
      • Negishi M.
      Constitutively active Gα12, Gα13, and Gαq induce Rho-dependent neurite retraction through different signaling pathways.
      ,
      • Barrett K.
      • Leptin M.
      • Settleman J.
      The Rho GTPase and a putative RhoGEF mediate a signaling pathway for the cell shape changes in Drosophila gastrulation.
      ,
      • Suzuki N.
      • Hajicek N.
      • Kozasa T.
      Regulation and physiological functions of G12/13-mediated signaling pathways.
      ,
      • Cruz-Ortega J.S.
      • Boucard A.A.
      Actin cytoskeleton remodeling defines a distinct cellular function for adhesion G protein-coupled receptors ADGRL/latrophilins 1, 2 and 3.
      ,
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • Okashah N.
      • Langenhan T.
      • Inoue A.
      • Lambert N.A.
      • Tall G.G.
      • Javitch J.A.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ). ADGRLs also couple to Gi/o and Gq, which may also influence neuronal migration and synaptogenesis, as the asymmetric production of cAMP or Ca2+ within the cell influences growth cone guidance (
      • Moreno-Salinas A.L.
      • Avila-Zozaya M.
      • Ugalde-Silva P.
      • Hernandez-Guzman D.A.
      • Missirlis F.
      • Boucard A.A.
      Latrophilins: a neuro-centric view of an evolutionary conserved adhesion G protein-coupled receptor subfamily.
      ,
      • Müller A.
      • Winkler J.
      • Fiedler F.
      • Sastradihardja T.
      • Binder C.
      • Schnabel R.
      • Kungel J.
      • Rothemund S.
      • Hennig C.
      • Schöneberg T.
      • Prömel S.
      Oriented cell division in the C. elegans embryo is coordinated by G-protein signaling dependent on the adhesion GPCR LAT-1.
      ,
      • Lelianova V.G.
      • Davletov B.A.
      • Sterling A.
      • Rahman M.A.
      • Grishin E.V.
      • Totty N.F.
      • Ushkaryov Y.A.
      α-Latrotoxin receptor, latrophilin, is a novel member of the secretin family of G protein-coupled receptors.
      ,
      • Rahman M.A.
      • Ashton A.C.
      • Meunier F.A.
      • Davletov B.A.
      • Dolly J.O.
      • Ushkaryov Y.A.
      Norepinephrine exocytosis stimulated by alpha-latrotoxin requires both external and stored Ca2+ and is mediated by latrophilin, G proteins and phospholipase C.
      ,
      • Tojima T.
      • Hines J.H.
      • Henley J.R.
      • Kamiguchi H.
      Second messengers and membrane trafficking direct and organize growth cone steering.
      ).
      As with many AGPCRs, ADGRLs are self-cleaved receptors (Table 1) and will activate signaling following NTF/CTF dissociation via tethered-peptide agonism (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • Brunger A.T.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ,
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • Okashah N.
      • Langenhan T.
      • Inoue A.
      • Lambert N.A.
      • Tall G.G.
      • Javitch J.A.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ,