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Membrane receptor activation mechanisms and transmembrane peptide tools to elucidate them

Open AccessPublished:December 25, 2019DOI:https://doi.org/10.1074/jbc.REV119.009457
      Single-pass membrane receptors contain extracellular domains that respond to external stimuli and transmit information to intracellular domains through a single transmembrane (TM) α-helix. Because membrane receptors have various roles in homeostasis, signaling malfunctions of these receptors can cause disease. Despite their importance, there is still much to be understood mechanistically about how single-pass receptors are activated. In general, single-pass receptors respond to extracellular stimuli via alterations in their oligomeric state. The details of this process are still the focus of intense study, and several lines of evidence indicate that the TM domain (TMD) of the receptor plays a central role. We discuss three major mechanistic hypotheses for receptor activation: ligand-induced dimerization, ligand-induced rotation, and receptor clustering. Recent observations suggest that receptors can use a combination of these activation mechanisms and that technical limitations can bias interpretation. Short peptides derived from receptor TMDs, which can be identified by screening or rationally developed on the basis of the structure or sequence of their targets, have provided critical insights into receptor function. Here, we explore recent evidence that, depending on the target receptor, TMD peptides cannot only inhibit but also activate target receptors and can accommodate novel, bifunctional designs. Furthermore, we call for more sharing of negative results to inform the TMD peptide field, which is rapidly transforming into a suite of unique tools with the potential for future therapeutics.

      Introduction

      Membrane receptors transmit information about extracellular stimuli into the cytoplasm. This is achieved through a combination of changes in conformation and oligomeric state by membrane proteins with a single transmembrane domain (TMD).
      The abbreviations used are: TMD
      TM domain
      TM
      transmembrane
      LIR
      ligand-induced rotation
      VEGFR
      vascular endothelial growth factor receptor
      RTK
      receptor tyrosine kinase
      TCR
      T-cell receptor
      LID
      ligand-induced dimerization
      EGF
      epidermal growth factor
      EGFR
      epidermal growth factor receptor
      GHR
      growth hormone receptor
      HGH
      human growth hormone
      EpoR
      erythropoietin receptor
      GpA
      glycophorin A
      MHC
      major histocompatibility complex
      pMHC
      peptide-major histocompatibility complex
      PTPRJ
      protein-tyrosine phosphatase receptor J
      CHAMPS
      computed helical anti-membrane proteins
      βcR
      β common subunit
      InsR
      insulin receptor
      PDB
      Protein Data Bank
      CAT
      chloramphenicol acetyltransferase
      IL-3
      interleukin 3
      CP
      core peptide
      MTP
      membrane-targeting peptide
      FDA
      Food and Drug Administration
      InsR
      insulin receptor
      PIP2
      phosphatidylinositol 4,5-bisphosphate.
      The factors that govern receptor oligomerization remain poorly understood yet are critical for obtaining a complete description of receptor function. The existence of TMD dimers shows that self-assembly information is encoded by the TMD. Recently, studies using short peptides composed of the TMDs of receptors have provided unique insights into receptor function. TMD peptides have generally been thought to interact with the TMD of the target receptor and inhibit its dimerization. However, in recent years, TMD peptides against some receptor targets have been found to promote receptor oligomerization. This suggests that interactions between TMD peptides and their targets may provide insights on receptor-activation mechanisms. This insight and the recent development of the first bi-functional TMD peptide, TYPE7, prompt our review of the subject.
      The proteins discussed in this review are single-pass receptors, which contain one α-helical TMD and an extracellular ligand-binding domain. We have chosen to omit multispanning receptors from this review, as they may also undergo large changes in their transmembrane tertiary structure, which is beyond the scope of this review. A large superfamily of single-pass receptors—the receptor tyrosine kinases (RTKs)—possesses an intracellular kinase domain. Other non-RTK receptors discussed include the T-cell receptor (TCR), which contains multiple separate subunits with one TMD each. A large number of single-pass receptors have been implicated in disease due to their roles in cell proliferation and differentiation. Therefore, gaining a better understanding of these receptors is of critical importance. Here, we review how TMD peptides can be used as tools to better understand TMD-mediated self-assembly. We direct the reader elsewhere for complementary reviews focused on progress toward therapeutic TMD peptides (
      • Sigalov A.B.
      New therapeutic strategies targeting transmembrane signal transduction in the immune system.
      ,
      • Stone T.A.
      • Deber C.M.
      Therapeutic design of peptide modulators of protein–protein interactions in membranes.
      ,
      • Yin H.
      • Flynn A.D.
      Drugging membrane protein interactions.
      ,
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      Small molecule and peptide recognition of protein transmembrane domains.
      ,
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      Development of agents that modulate protein–protein interactions in membranes.
      ), design of TMD peptides (
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      • DeGrado W.F.
      ,
      • Cymer F.
      • Schneider D.
      Lessons from viruses: controlling the function of transmembrane proteins by interfering transmembrane helices.
      ), or prior reviews of similar focus (
      • Slivka P.F.
      • Wong J.
      • Caputo G.A.
      • Yin H.
      Peptide probes for protein transmembrane domains.
      ,
      • Yin H.
      Exogenous agents that target transmembrane domains of proteins.
      ).

      Role of the transmembrane domain in activation of single-pass membrane proteins

      The plasma membrane offers both challenges and opportunities for membrane proteins. Unlike soluble proteins, which can diffuse and rotate in three dimensions, membrane proteins are anchored to the membrane by their TMD. Because single-pass receptors are constrained to the bilayer, they must function while their TMD is restricted to four principal motions: translation, piston, pivot, and rotation (Fig. 1A) (
      • Matthews E.E.
      • Zoonens M.
      • Engelman D.M.
      Dynamic helix interactions in transmembrane signaling.
      ). Furthermore, their secondary structure is severely constrained, as the energetic cost of unfolding a peptide backbone in the membrane is a staggering +4 kcal mol−1 per amino acid (
      • Cymer F.
      • von Heijne G.
      • White S.H.
      Mechanisms of integral membrane protein insertion and folding.
      ). For perspective, this means that inserting just two unfolded amino acids into the bilayer would have an energetic cost similar to phosphorylating one molecule of ADP into ATP. With such constraints, how could a signal be effectively transmitted through a membrane with minimal secondary and tertiary structural change? For many proteins, the answer is assembly. Receptor homo- or hetero-association allows a given receptor to activate phosphorylation cascades only when an extracellular stimulus is present. Receptor associations rely at least in part on the interactions of receptor TMDs. Thus, the same lipid bilayer that hosts them and restricts their conformation, also ensures that they are in an optimal position to encounter another receptor. In this section, we will explore TMD interactions that facilitate receptor activation.
      Figure thumbnail gr1
      Figure 1Common structural motifs of TMD dimers. A, isolated transmembrane α-helices are constrained to four principal motions: piston movement, translation, tilt, and rotation. B, common characteristics of interhelical interaction motifs. The left-handed TMD dimer is typically found at an approximate −20° crossing angle. Similar to soluble coiled coils, the interaction interface can be described in a heptad repeat nomenclature, abcdefg. Interhelical hydrogen bonding (cyan) is commonly found between a–a′ and d–e′. The average interhelical distance is 11 Å (
      • Zhang S.Q.
      • Kulp D.W.
      • Schramm C.A.
      • Mravic M.
      • Samish I.
      • DeGrado W.F.
      The membrane- and soluble-protein helix-helix interactome: similar geometry via different interactions.
      ). The right-handed TMD dimer typically forms at an approximate +40° crossing angle. Right-handed dimers are best described by a tetrad repeat nomenclature, abcd. At the a and d positions, small residues such as glycine are enriched, which create an angled groove where helices may pack tightly together. Interhelical hydrogen bonding is commonly found between residues at positions a and d′. The average interhelical distance is ∼9 Å (
      • Zhang S.Q.
      • Kulp D.W.
      • Schramm C.A.
      • Mravic M.
      • Samish I.
      • DeGrado W.F.
      The membrane- and soluble-protein helix-helix interactome: similar geometry via different interactions.
      ). Representative structures shown are as follows: left-handed is EphA2 (PDB code 2K9Y), and right-handed is glycophorin A (PDB code 5EH4).
      Not surprisingly, studies on diverse single-pass receptors show that the TMD can strongly affect receptor functionality. This is demonstrated by substitution studies in which the TMD of a receptor is replaced with that of a constitutively active receptor. For example, substituting the TMD of the muscle-specific kinase, MuSK, for that of Neu (V664E) (an oncogenic mutant of the Neu/ErbB2 receptor) created a receptor that constitutively self-assembles into signaling-active receptor clusters (
      • Jones G.
      • Moore C.
      • Hashemolhosseini S.
      • Brenner H.R.
      Constitutively active MuSK is clustered in the absence of agrin and induces ectopic postsynaptic-like membranes in skeletal muscle fibers.
      ).
      Mutations in the TMD that alter dimerization can also lead to disease. TMD mutations cause aberrant activation in many receptor families. Half of the 20 classes of RTKs have members with such mutations. There are documented examples of activating or inactivating transmembrane mutants for epidermal growth factor receptor (
      • Bargmann C.I.
      • Hung M.C.
      • Weinberg R.A.
      Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185.
      ), ErbB2 (
      • Bargmann C.I.
      • Hung M.C.
      • Weinberg R.A.
      Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185.
      ), insulin receptor (
      • Longo N.
      • Shuster R.C.
      • Griffin L.D.
      • Langley S.D.
      • Elsas L.J.
      Activation of insulin receptor signaling by a single amino acid substitution in the transmembrane domain.
      ), platelet-derived growth factor receptor (
      • Velghe A.I.
      • Van Cauwenberghe S.
      • Polyansky A.A.
      • Chand D.
      • Montano-Almendras C.P.
      • Charni S.
      • Hallberg B.
      • Essaghir A.
      • Demoulin J.-B.
      PDGFRA alterations in cancer: characterization of a gain-of-function V536E transmembrane mutant as well as loss-of-function and passenger mutations.
      ), vascular endothelial growth factor receptor (VEGFR) (
      • Sarabipour S.
      • Ballmer-Hofer K.
      • Hristova K.
      VEGFR-2 conformational switch in response to ligand binding.
      ), fibroblast growth factor receptor (
      • Li E.
      • You M.
      • Hristova K.
      FGFR3 dimer stabilization due to a single amino acid pathogenic mutation.
      ,
      • You M.
      • Spangler J.
      • Li E.
      • Han X.
      • Ghosh P.
      • Hristova K.
      Effect of pathogenic cysteine mutations on FGFR3 transmembrane domain dimerization in detergents and lipid bilayers.
      ,
      • Webster M.K.
      • Donoghue D.J.
      Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia.
      ,
      • Shiang R.
      • Thompson L.M.
      • Zhu Y.-Z.
      • Church D.M.
      • Fielder T.J.
      • Bocian M.
      • Winokur S.T.
      • Wasmuth J.J.
      Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia.
      ), nerve growth factor receptor (
      • Monshipouri M.
      • Jiang H.
      • Lazarovici P.
      NGF Stimulation of ERK phosphorylation is impaired by a point mutation in the transmembrane domain of TrkA receptor.
      ), Eph receptor (
      • Sharonov G.V.
      • Bocharov E.V.
      • Kolosov P.M.
      • Astapova M.V.
      • Arseniev A.S.
      • Feofanov A.V.
      Point mutations in dimerization motifs of the transmembrane domain stabilize active or inactive state of the EphA2 receptor tyrosine kinase.
      ), discoidin domain receptor (
      • Noordeen N.A.
      • Carafoli F.
      • Hohenester E.
      • Horton M.A.
      • Leitinger B.
      A transmembrane leucine zipper is required for activation of the dimeric receptor tyrosine kinase DDR1.
      ), RET (
      • Benej M.
      • Fekecsova S.
      • Poturnajova M.
      Assessing the effect of RET transmembrane domain mutations in receptor self-association capability using the in vivo TOXCAT system.
      ), and ROS (
      • Jong S.M.
      • Wang L.H.
      Two point mutations in the transmembrane domain of P68gag-ros inactive its transforming activity and cause a delay in membrane association.
      ). Other non-RTK single-pass membrane proteins suffer these activating mutations as well. For example the T-cell receptor ζ chain TMD contains a central aspartic acid which, when mutated to valine, abrogates dimerization resulting in inactivation of the TCR complex (
      • Rutledge T.
      • Cosson P.
      • Manolios N.
      • Bonifacino J.S.
      • Klausner R.D.
      Transmembrane helical interactions: ζ chain dimerization and functional association with the T cell antigen receptor.
      ). Yet there are yet other receptors, such as multispanning and nonsignaling membrane proteins, that are aberrantly activated by TMD mutations. For example, mutating an aspartic acid in the TMD of the nine-pass protein presenilin (1 or 2) reduces heterodimerization and promotes β-amyloid production (
      • Kimberly W.T.
      • Xia W.
      • Rahmati T.
      • Wolfe M.S.
      • Selkoe D.J.
      The transmembrane aspartates in presenilin 1 and 2 are obligatory for γ-secretase activity and amyloid β-protein generation.
      ,
      • Wolfe M.S.
      • Xia W.
      • Ostaszewski B.L.
      • Diehl T.S.
      • Kimberly W.T.
      • Selkoe D.J.
      Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and γ-secretase activity.
      ). Note that not all of the mutations discussed above are naturally occurring, but nevertheless they demonstrate that TMDs are critical for the proper function of membrane proteins in a variety of contexts.
      Although TMDs play a role in receptor activation, we are still learning the structural and sequence requirements that account for the observed specific TMD interactions. It is also unclear how membrane proteins regulate these interactions to avoid spurious activation in the absence of ligand stimulation. In the following sections, we will discuss specificity of TMD interactions and how TMD interactions contribute to receptor activation mechanisms.

      Specificity of transmembrane helical interactions

      In a way, the discovery of specific TMD interactions resembles the discovery of specific soluble receptor–ligand interactions in the early 1900s (
      • Maehle A.-H.
      A binding question: the evolution of the receptor concept.
      ). In the latter case, it was thought that with the great diversity of proteins in the body, it would be extremely unlikely that a ligand could specifically bind to just one receptor type. Similarly, specific TMD interactions were widely thought to be unlikely, given the similar hydrophobicity of TMDs and the expected frequency of stochastic membrane protein collisions. In both cases, controversy and indirect evidence abounded, but key evidence was lacking until experiments were designed to systematically address the questions. A major breakthrough came with the first high-resolution crystal structure of an integral membrane protein complex, the photosynthetic reaction center. Although suggested before, this structure unambiguously showed tight transmembrane contacts, indicating that independent helices specifically associate in the membrane (
      • Deisenhofer J.
      • Epp O.
      • Miki K.
      • Huber R.
      • Michel H.
      Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3 Å resolution.
      ).
      This was further directly tested by Popot et al. (
      • Popot J.-L.
      • Gerchman S.-E.
      • Engelman D.M.
      Refolding of bacteriorhodopsin in lipid bilayers.
      ) when they cleaved bacteriorhodopsin into two inactive fragments and successfully reconstituted them into vesicles, where bacteriorhodopsin regained its fold and function. This was taken a step further by cleaving bacteriorhodopsin into its seven constitutive helices and studying them independently, a study that was complicated by some of the peptides being too soluble or aggregation-prone to readily fold into the membrane (
      • Hunt J.F.
      • Earnest T.N.
      • Bousché O.
      • Kalghatgi K.
      • Reilly K.
      • Horváth C.
      • Rothschild K.J.
      • Engelman D.M.
      A biophysical study of integral membrane protein folding.
      ). This research led to the two-stage model, which posits that transmembrane helices are independently folding units that can then further assemble with one another (
      • Popot J.L.
      • Engelman D.M.
      Membrane protein folding and oligomerization: the two-stage model.
      ).
      Pioneering work on glycophorin A (GpA) provided the first structural details of a specific TMD interaction. Previously, it was thought that interhelical interactions would only be driven by the hydrophobic effect. Polar residues would aggregate together within the bilayer as nonpolar residues do in solution. In other words, nonpolar residues were thought to contribute only weakly, if at all. Eventually, alanine-scanning mutagenesis of the GpA TMD showed that certain residues, including hydrophobic ones, contribute more to dimer formation than others and revealed a key concept with deep ramifications: TMDs have preferred interaction interfaces (
      • Lemmon M.A.
      • Flanagan J.M.
      • Treutlein H.R.
      • Zhang J.
      • Engelman D.M.
      Sequence specificity in the dimerization of transmembrane α-helices.
      ,
      • Treutlein H.R.
      • Lemmon M.A.
      • Engelman D.M.
      • Brünger A.T.
      The glycophorin–a transmembrane domain dimer–sequence-specific propensity for a right-handed supercoil of helices.
      ). Furthermore, the residues that contribute the most to the dimerization of GpA, when put into a polyleucine sequence, also confer dimerization propensity (
      • Lemmon M.A.
      • Treutlein H.R.
      • Adams P.D.
      • Brünger A.T.
      • Engelman D.M.
      A dimerization motif for transmembrane α-helices.
      ). This was the first discovery of a sequence motif that could confer dimerization specificity to a transmembrane α-helix. Contrary to the hydrophobic effect model, the critical region of the interface is composed of glycines and valines (LIXXGVXXGVXXT). Even a single methyl introduction on one of the interfacial glycines (e.g. Gly to Ala) is enough to disrupt the dimer (
      • Lemmon M.A.
      • Flanagan J.M.
      • Treutlein H.R.
      • Zhang J.
      • Engelman D.M.
      Sequence specificity in the dimerization of transmembrane α-helices.
      ). The authors reasoned that valine residues from one helix formed a ridge that packed into a groove created by glycine residues on the other (i.e. knobs-into-holes). This suggests that it is not only hydrophobicity but side-chain packing that determines specific interhelical association.
      When the structure of the GpA TMD was solved, it corroborated earlier conclusions. Specifically, van der Waals–induced TMD interaction is indeed possible (
      • MacKenzie K.R.
      • Prestegard J.H.
      • Engelman D.M.
      A transmembrane helix dimer: structure and implications.
      ), and the dimer is strong. GpA dimerizes with a free energy of dissociation of 9.1 kcal mol−1 (
      • Fleming K.G.
      • Ackerman A.L.
      • Engelman D.M.
      The effect of point mutations on the free energy of transmembrane α-helix dimerization.
      ). Additionally, it was found that noncanonical hydrogen bonding between the α-hydrogen and carbonyl oxygen strongly contributes to the dimerization (
      • Senes A.
      • Ubarretxena-Belandia I.
      • Engelman D.M.
      The Cα–H···O hydrogen bond: a determinant of stability and specificity in transmembrane helix interactions.
      ). Still, GpA shows us that transmembrane sequence motifs can result in specific interactions. In the next section, we will discuss such sequence motifs. For a more thorough historical review of the specificity of GpA interactions, we suggest Ref.
      • MacKenzie K.R.
      Folding and stability of α-helical integral membrane proteins.
      .

      Common transmembrane interaction motifs

      The structure of GpA shows that, as predicted (
      • Lemmon M.A.
      • Flanagan J.M.
      • Treutlein H.R.
      • Zhang J.
      • Engelman D.M.
      Sequence specificity in the dimerization of transmembrane α-helices.
      ), the transmembrane interface contains two critical glycine residues separated by three amino acids (
      • MacKenzie K.R.
      • Prestegard J.H.
      • Engelman D.M.
      A transmembrane helix dimer: structure and implications.
      ). This so-called GXXXG sequence motif is highly over-represented in transmembrane α-helices compared with soluble helices (
      • Russ W.P.
      • Engelman D.M.
      The GxxxG motif: a framework for transmembrane helix–helix association.
      ,
      • Senes A.
      • Gerstein M.
      • Engelman D.M.
      Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with β-branched residues at neighboring positions.
      ). Later, meta-analyses of the solved structures of helical dimers revealed additional interaction motifs. Just four different structural motifs account for two-thirds of all the helical pairs (
      • Gimpelev M.
      • Forrest L.R.
      • Murray D.
      • Honig B.
      Helical packing patterns in membrane and soluble proteins.
      ,
      • Walters R.F.
      • DeGrado W.F.
      Helix-packing motifs in membrane proteins.
      ). Indeed, most RTKs contain at least one predicted dimerization motif in the TMD (
      • Sternberg M.J.
      • Gullick W.J.
      A sequence motif in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization.
      ). It should be noted that any such analysis is subject to the limited number of solved membrane protein structures and that there are likely more motifs that will be discovered as more TMD interactions are studied. In general, single-pass receptor TMDs form parallel interactions (i.e. the N termini of the TMDs are on the same side of the membrane) rather than anti-parallel interactions (
      • Moore D.T.
      • Berger B.W.
      • DeGrado W.F.
      Protein–protein interactions in the membrane: sequence, structural, and biological motifs.
      ). For this reason, we will only discuss the two parallel motifs in this review. For a thorough review of transmembrane helical interaction motifs, including the two antiparallel motifs not discussed here, we suggest Ref.
      • Rath A.
      • Johnson R.M.
      • Deber C.M.
      Peptides as transmembrane segments: decrypting the determinants for helix–helix interactions in membrane proteins.
      .
      The GXXXG motif first discovered in GpA belongs to the largest class of motifs, the GASright motif (
      • Gimpelev M.
      • Forrest L.R.
      • Murray D.
      • Honig B.
      Helical packing patterns in membrane and soluble proteins.
      ,
      • Walters R.F.
      • DeGrado W.F.
      Helix-packing motifs in membrane proteins.
      ). The “right” in GASright refers to the crossing of the two helices, where the top of the helix closest to the viewer is pointing right. They cross approximately at a right-handed 40° angle (Fig. 1B). The “GAS” of this motif refers to the small amino acids glycine, alanine, and serine, which, when placed four residues apart, form an angled groove down the helix. When opposed, two helices with the same groove will pack tightly, and the association will be stabilized by van der Waals forces as well as Cα-H—O=C noncanonical backbone hydrogen bonding, which contributes 0.88 kcal mol−1 per bond (
      • Senes A.
      • Ubarretxena-Belandia I.
      • Engelman D.M.
      The Cα–H···O hydrogen bond: a determinant of stability and specificity in transmembrane helix interactions.
      ,
      • Russ W.P.
      • Engelman D.M.
      The GxxxG motif: a framework for transmembrane helix–helix association.
      ,
      • Anderson S.M.
      • Mueller B.K.
      • Lange E.J.
      • Senes A.
      Combination of Cα-H hydrogen bonds and van der Waals packing modulates the stability of GxxxG-mediated dimers in membranes.
      ,
      • Arbely E.
      • Arkin I.T.
      Experimental measurement of the strength of a Cα-H···O bond in a lipid bilayer.
      ). Further analysis shows that this motif is precisely optimized for stabilization of these noncanonical hydrogen bonds (
      • Mueller B.K.
      • Subramaniam S.
      • Senes A.
      A frequent, GxxxG-mediated, transmembrane association motif is optimized for the formation of interhelical Cα–H hydrogen bonds.
      ). This suggests a causal link between the prevalence of GXXXG motifs and strong noncanonical hydrogen bonds.
      Another major class of parallel motifs is the left-handed dimer (Fig. 1B). These typically have a smaller crossing angle, around 15–20° (
      • Walters R.F.
      • DeGrado W.F.
      Helix-packing motifs in membrane proteins.
      ). These left-handed interactions are often stabilized by heptad repeats, repeating units of seven amino acids (abcdefg) (
      • Chambers P.
      • Pringle C.R.
      • Easton A.J.
      Heptad repeat sequences are located adjacent to hydrophobic regions in several types of virus fusion glycoproteins.
      ) where the interfacial residues are often at the a, d, e, or g positions (
      • Langosch D.
      • Heringa J.
      Interaction of transmembrane helices by a knobs-into-holes packing characteristic of soluble coiled coils.
      ). This motif is based on the remarkably accurate 1953 prediction by Francis Crick that two soluble, symmetric α-helices could tightly coil around each other with their residues packing in a knobs-into-holes manner, but only if they were offset by 20° and their a and d positions were polar (
      • Crick F.H.
      The packing of α-helices: simple coiled-coils.
      ). Later, similar periodic repeats were found in many soluble proteins (
      • McLachlan A.D.
      • Karn J.
      Periodic features in the amino acid sequence of nematode myosin rod.
      ,
      • McLachlan A.D.
      • Stewart M.
      Tropomyosin coiled-coil interactions: evidence for an unstaggered structure.
      ,
      • Cohen C.
      • Parry D.A.D.
      α-Helical coiled coils — a widespread motif in proteins.
      ), and finally, a crystal structure confirmed the first knobs-into-holes–mediated soluble coiled coil (
      • O'Shea E.K.
      • Klemm J.D.
      • Kim P.S.
      • Alber T.
      X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil.
      ). As mentioned above, the same periodicity that allows knobs-into-holes packing can be found in transmembrane α-helices, which tend to have two to three heptad repeating units. Meta-analysis confirmed a preference for a–a′ hydrogen bonding in TMD interactions (
      • Zhang S.Q.
      • Kulp D.W.
      • Schramm C.A.
      • Mravic M.
      • Samish I.
      • DeGrado W.F.
      The membrane- and soluble-protein helix-helix interactome: similar geometry via different interactions.
      ). However, multiple studies have failed to find site-specific preference for amino acid type in these motifs (
      • Walters R.F.
      • DeGrado W.F.
      Helix-packing motifs in membrane proteins.
      ,
      • Langosch D.
      • Heringa J.
      Interaction of transmembrane helices by a knobs-into-holes packing characteristic of soluble coiled coils.
      ), and they have led to speculation that the apparent motif may simply be a consequence of helix packing (i.e. the interacting residues will appear periodic) and that left-handed sequence motifs may be a red herring (
      • Li E.
      • Wimley W.C.
      • Hristova K.
      Transmembrane helix dimerization: beyond the search for sequence motifs.
      ).

      Exceptions and unanswered questions

      Despite earlier evidence that motifs drive association, TMD dimers have been found that contain no discernable sequence motif (
      • Li E.
      • Wimley W.C.
      • Hristova K.
      Transmembrane helix dimerization: beyond the search for sequence motifs.
      ). Furthermore, sequence motifs do not always confer dimerization propensity. For example, only very weak dimerization of the TMD of protein tyrosine kinase 7 occurs, despite the presence of a GXXXG motif (
      • Kobus F.J.
      • Fleming K.G.
      The GxxxG-containing transmembrane domain of the CCK4 oncogene does not encode preferential self-interactions.
      ). Similarly, the RTKs EphA1 (
      • Artemenko E.O.
      • Egorova N.S.
      • Arseniev A.S.
      • Feofanov A.V.
      Transmembrane domain of EphA1 receptor forms dimers in membrane-like environment.
      ) and EphA2 (
      • Bocharov E.V.
      • Mayzel M.L.
      • Volynsky P.E.
      • Mineev K.S.
      • Tkach E.N.
      • Ermolyuk Y.S.
      • Schulga A.A.
      • Efremov R.G.
      • Arseniev A.S.
      Left-handed dimer of EphA2 transmembrane domain: helix packing diversity among receptor tyrosine kinases.
      ) form dimers in bicelles, but apparently prefer a left-handed interface over their GXXXG motifs. Furthermore, there are many favorable geometries that have no apparent sequence motif, lending more support to the idea of structural classes of interactions (e.g. left-handed parallel), rather than sequence motifs (
      • Zhang S.Q.
      • Kulp D.W.
      • Schramm C.A.
      • Mravic M.
      • Samish I.
      • DeGrado W.F.
      The membrane- and soluble-protein helix-helix interactome: similar geometry via different interactions.
      ). In fact, the presence of a GXXXG motif is not a strong predictor of dimerization (
      • Teese M.G.
      • Langosch D.
      Role of GxxxG motifs in transmembrane domain interactions.
      ), and predicting interaction with GXXXG motifs often involves “idealizing” the helices into straight rods, ignoring helical kinks (
      • Anderson S.M.
      • Mueller B.K.
      • Lange E.J.
      • Senes A.
      Combination of Cα-H hydrogen bonds and van der Waals packing modulates the stability of GxxxG-mediated dimers in membranes.
      ). This point was demonstrated by a double-mutant screen of GpA, which found that GXXXG is neither necessary nor sufficient for dimerization in the GpA TMD (
      • Doura A.K.
      • Fleming K.G.
      Complex interactions at the helix–helix interface stabilize the glycophorin A transmembrane dimer.
      ). Although in some contexts sequence and structural motifs are correlated, it seems that targeting a desired structure is more likely to yield predictable structural outcomes than relying on sequence motifs, as we will see under “Structure-based design.”

      Structural contribution of transmembrane domains to activation mechanisms

      Understanding the activation mechanisms of single-pass receptors has remained tantalizingly out of reach despite decades of intense research. Small victories only serve to pose more questions, and a full description of receptor-mediated signaling remains elusive. Regardless, several clues have been unearthed, and as our understanding grows, so does the complexity of the models required to describe the mechanisms. Some receptors require simple descriptions, whereas others require more complex models.
      As we study the TMDs of single-pass receptors, a pattern is emerging. Most receptor TMDs, including all of the RTKs, and toll-like receptors, have a tendency to self-associate (
      • Finger C.
      • Escher C.
      • Schneider D.
      The single transmembrane domains of human receptor tyrosine kinases encode self-Interactions.
      ,
      • Godfroy 3rd, J.I.
      • Roostan M.
      • Moroz Y.S.
      • Korendovych I.V.
      • Yin H.
      Isolated Toll-like receptor transmembrane domains are capable of oligomerization.
      ). This associative property contributes to the overall dimerization of the protein and is typically balanced with the associative and dissociative properties of soluble domains. For example, the extracellular ligand-binding domain of EphA2 promotes dimerization (
      • Himanen J.P.
      • Goldgur Y.
      • Miao H.
      • Myshkin E.
      • Guo H.
      • Buck M.
      • Nguyen M.
      • Rajashankar K.R.
      • Wang B.
      • Nikolov D.B.
      Ligand recognition by A-class Eph receptors: crystal structures of the EphA2 ligand-binding domain and the EphA2/ephrin-A1 complex.
      ), whereas the intracellular sterile α-motif domain inhibits it (
      • Singh D.R.
      • Ahmed F.
      • Paul M.D.
      • Gedam M.
      • Pasquale E.B.
      • Hristova K.
      The SAM domain inhibits EphA2 interactions in the plasma membrane.
      ,
      • Shi X.
      • Hapiak V.
      • Zheng J.
      • Muller-Greven J.
      • Bowman D.
      • Lingerak R.
      • Buck M.
      • Wang B.-C.
      • Smith A.W.
      A role of the SAM domain in EphA2 receptor activation.
      ). Each receptor likely maintains a unique balance of forces such that only the right conditions will tip the scales in favor of receptor oligomerization and activation.
      In this section, we will discuss three mechanistic hypotheses for receptor activation in order of increasing complexity. We propose that there is no universal model for receptor activation and that receptors may use more than one mechanism to achieve full activation.

      Ligand-induced dimerization (LID)

      Early work on the epidermal growth factor receptor (EGFR) suggested that the receptor is more active in the dimeric form than in the monomeric form (
      • Yarden Y.
      • Schlessinger J.
      Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor.
      ,
      • Yarden Y.
      • Schlessinger J.
      Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation.
      ). This led to the LID hypothesis, which posits that ligand binding induces dimerization, which is sufficient for activation (Fig. 2) (
      • Heldin C.-H.
      Dimerization of cell surface receptors in signal transduction.
      ,
      • Schlessinger J.
      Ligand-induced, receptor-mediated dimerization and activation of EGF receptor.
      ,
      • Lemmon M.A.
      Ligand-induced ErbB receptor dimerization.
      ). Most of the early arguments for LID were based on observations that bivalent ligands may “cross-link” receptor monomers together (
      • Burgess A.W.
      • Cho H.-S.
      • Eigenbrot C.
      • Ferguson K.M.
      • Garrett T.P.J.
      • Leahy D.J.
      • Lemmon M.A.
      • Sliwkowski M.X.
      • Ward C.W.
      • Yokoyama S.
      An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors.
      ).
      Figure thumbnail gr2
      Figure 2Role of TMD in receptor activation mechanisms. The LID hypothesis posits that ligand binding to the extracellular domains of the receptor brings receptor monomers together into a dimer that is signaling-competent. The LIR hypothesis assumes that an inactive dimer exists and that ligand binding induces a rotation of the receptor to bring the intracellular kinase domains into the active configuration for signaling. Clustering occurs when receptors are stabilized as large higher-order oligomeric signaling complexes. These mechanisms are not necessarily mutually exclusive, and some receptors may use a combination of them. Blue and orange represent different TMD interfaces. Domains are not to scale, and the ligand is not shown for clarity.
      After the initial observation of EGFR dimerization, a study on the human growth hormone receptor (GHR) provided the first strong evidence for LID. In this thorough analysis, Cunningham et al. (
      • Cunningham B.
      • Ultsch M.
      • De Vos A.M.
      • Mulkerrin M.G.
      • Clauser K.R.
      • Wells J.A.
      Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule.
      ) show that human growth hormone (HGH) forms a 1:2 complex with the extracellular domain of GHR; two GHR receptors bind to a single HGH molecule. This provided a mechanistic model for how receptor “cross-linking” might occur. The major determinants for activation would thus be in the extracellular ligand-binding domain. Another study on the erythropoietin receptor (EpoR) provided evidence to support this finding, showing that a cysteine mutation in the extracellular region of EpoR caused it to become a constitutively-active dimer by allowing the formation of intermolecular disulfide bonds (
      • Watowich S.S.
      • Yoshimura A.
      • Longmore G.D.
      • Hilton D.J.
      • Yoshimura Y.
      • Lodish H.F.
      Homodimerization and constitutive activation of the erythropoietin receptor.
      ).
      In the simple LID hypothesis, the TMD could either be passive or active. Early experimental evidence seemed to support the passivity of the TMD (
      • Kashles O.
      • Szapary D.
      • Bellot F.
      • Ullrich A.
      • Schlessinger J.
      • Schmidt A.
      Ligand-induced stimulation of epidermal growth factor receptor mutants with altered transmembrane regions.
      ,
      • Carpenter C.D.
      • Ingraham H.A.
      • Cochet C.
      • Walton G.M.
      • Lazar C.S.
      • Sowadski J.M.
      • Rosenfeld M.G.
      • Gill G.N.
      Structural analysis of the transmembrane domain of the epidermal growth factor receptor.
      ). Thus, it was thought that the extracellular domain would drive the receptors together, and the TMD would transmit that proximity information through the membrane to the intracellular domains. However, as discussed later, the TMDs of RTKs form dimers by themselves, which suggests an active role for the TMD. These studies collectively support the LID hypothesis, where a ligand stabilizes the dimeric form of the receptor, which is signaling-active. However, this hypothesis has difficulty explaining some observations, as we will see in the next section.

      Ligand-induced rotation (LIR)

      Since the birth of the LID hypothesis, evidence began to appear against it. Receptors can be found as unliganded dimers particularly at high protein densities in the membrane. This list includes RTKs such as EGFR (
      • Moriki T.
      • Maruyama H.
      • Maruyama I.N.
      Activation of preformed EGF receptor dimers by ligand-induced rotation of the transmembrane domain.
      ), EphA2 (
      • Seiradake E.
      • Harlos K.
      • Sutton G.
      • Aricescu A.R.
      • Jones E.Y.
      An extracellular steric seeding mechanism for Eph-ephrin signaling platform assembly.
      ,
      • Himanen J.P.
      • Yermekbayeva L.
      • Janes P.W.
      • Walker J.R.
      • Xu K.
      • Atapattu L.
      • Rajashankar K.R.
      • Mensinga A.
      • Lackmann M.
      • Nikolov D.B.
      • Dhe-Paganon S.
      Architecture of Eph receptor clusters.
      ), and the insulin receptor (
      • Massague J.
      • Pilch P.F.
      • Czech M.P.
      Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries.
      ), as well as cytokine receptors such as the EpoR (
      • Livnah O.
      • Stura E.A.
      • Middleton S.A.
      • Johnson D.L.
      • Jolliffe L.K.
      • Wilson I.A.
      Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation.
      ). A full list can be found in Ref.
      • Maruyama I.N.
      Activation of transmembrane cell-surface receptors via a common mechanism? The “Rotation Model,”.
      .
      Several studies have further shown that dimerization alone is not sufficient for receptor activation. For example, some cysteine mutations in the membrane-proximal region of ErbB2 induce dimerization, but not transforming activity (
      • Cao H.
      • Bangalore L.
      • Dompé C.
      • Bormann B.J.
      • Stern D.F.
      An extra cysteine proximal to the transmembrane domain induces differential cross-linking of p185neu and p185neu.
      ), nor is dimerization sufficient for activation of TCR (
      • Ratcliffe M.J.
      • Coggeshall K.M.
      • Newell M.K.
      • Julius M.H.
      T cell receptor aggregation, but not dimerization, induces increased cytosolic calcium concentrations and reveals a lack of stable association between CD4 and the T cell receptor.
      ). The confusion was further compounded by the apparent observation of an inactive dimer structure of the EGFR intracellular kinase domain (
      • Jura N.
      • Endres N.F.
      • Engel K.
      • Deindl S.
      • Das R.
      • Lamers M.H.
      • Wemmer D.E.
      • Zhang X.
      • Kuriyan J.
      Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment.
      ). Furthermore, varying degrees of self-association have been observed for all of the RTK TMDs, which indicates that some regulatory mechanism must be in place to prevent spontaneous activation (
      • Finger C.
      • Escher C.
      • Schneider D.
      The single transmembrane domains of human receptor tyrosine kinases encode self-Interactions.
      ). This evidence points to allosteric, rather than oligomeric, regulation of receptor activity. It should be noted that this was observed via the bacteria-based TOXCAT assay (
      • Russ W.P.
      • Engelman D.M.
      TOXCAT: a measure of transmembrane helix association in a biological membrane.
      ). In this assay, TMDs are tagged with ToxR, a transcription factor that induces expression of chloramphenicol acetyltransferase (CAT) upon dimerization. The levels of CAT are determined by an enzymatic assay and compared with a control condition. TOXCAT can detect very weak interactions but measures only relative, not thermodynamic, strength of association.
      Allostery is an obvious alternative to dimerization (
      • Ishizaka K.
      • Campbell D.H.
      Biologic activity of soluble antigen-antibody complexes: IV. The inhibition of the skin reactivity of soluble complexes and the PCA reaction by heterologous complexes.
      ). However, classical allosteric models rely on globular domain conformational changes. How could changes of the dimeric extracellular domains of receptors lead to rearrangement of the intracellular domains into an active state? This would be a constrained structural change, as the TMD would have to remain an α-helix as discussed above. A possible solution to this conundrum was proposed in Ref.
      • Moriki T.
      • Maruyama H.
      • Maruyama I.N.
      Activation of preformed EGF receptor dimers by ligand-induced rotation of the transmembrane domain.
      . If the dimeric extracellular domains were to rotate upon ligand binding, then the transmembrane and intracellular domains would also rotate without sacrificing their dimeric state. This hypothesis was dubbed the LIR mechanism (Fig. 2).
      A strong test of the LIR hypothesis was performed on the Neu and platelet-derived growth factor β-receptors (
      • Bell C.A.
      • Tynan J.A.
      • Hart K.C.
      • Meyer A.N.
      • Robertson S.C.
      • Donoghue D.J.
      Rotational coupling of the transmembrane and kinase domains of the Neu receptor tyrosine kinase.
      ). In this study, the TMD of the target receptors was replaced with a polyvaline sequence that is unable form a dimer on its own. The receptors only dimerized upon addition of two glutamic acids at positions a and e in the TMD sequence. Although dimerization occurred when the glutamic acids were in any position, activation only occurred periodically—when the glutamic acids were on one “face” of the helix. A similar observation was made on ErbB2; Burke and Stern (
      • Burke C.L.
      • Stern D.F.
      Activation of Neu (ErbB-2) Mediated by disulfide bond-induced dimerization reveals a receptor tyrosine kinase dimer interface.
      ) added cysteines at several positions in the juxtamembrane domain and determined that this region contains an additional dimerization interface, which may explain the rotational coupling of the TMD for activation. Both interfaces must line up face-to-face. For example, GHR forms a constitutive dimer yet is inactive in the absence of ligand. By blocking the “inactive” transmembrane interface with alanine mutations, the receptor can be activated in the absence of ligand (
      • Brown R.J.
      • Adams J.J.
      • Pelekanos R.A.
      • Wan Y.
      • McKinstry W.J.
      • Palethorpe K.
      • Seeber R.M.
      • Monks T.A.
      • Eidne K.A.
      • Parker M.W.
      • Waters M.J.
      Model for growth hormone receptor activation based on subunit rotation within a receptor dimer.
      ).
      Another approach for testing the coupling of TMD rotational conformation to receptor activation consists of using the Put3 domain (
      • Mattoon D.
      • Gupta K.
      • Doyon J.
      • Loll P.J.
      • DiMaio D.
      Identification of the transmembrane dimer interface of the bovine papillomavirus E5 protein.
      ). The soluble coiled-coil Put3, when shortened to varying degrees, can be used as a scaffold for rotating the dimerization interface. Using this approach, Matsushita et al. (
      • Matsushita C.
      • Tamagaki H.
      • Miyazawa Y.
      • Aimoto S.
      • Smith S.O.
      • Sato T.
      Transmembrane helix orientation influences membrane binding of the intracellular juxtamembrane domain in Neu receptor peptides.
      ) found that the juxtamembrane domain of ErbB2 associates with the membrane more favorably in certain orientations of the transmembrane dimer. Similarly, Mohan et al. (
      • Mohan K.
      • Ueda G.
      • Kim A.R.
      • Jude K.M.
      • Fallas J.A.
      • Guo Y.
      • Hafer M.
      • Miao Y.
      • Saxton R.A.
      • Piehler J.
      • Sankaran V.G.
      • Baker D.
      • Garcia K.C.
      Topological control of cytokine receptor signaling induces differential effects in hematopoiesis.
      ) found that synthetic ligands activate EpoR depending on the distance and angle that they induce between the extracellular domains. This might provide a mechanism for how LIR results in activation: certain orientations may favor dissociation of the juxtamembrane domain from the membrane interface, relieving autoinhibition of the kinase domain.
      Structural studies suggest that motif switching might be a critical aspect of the LIR hypothesis. The difference between what is considered a strong transmembrane dimer (GpA) and a weak one (GpA destabilized by the G83I mutation) is only on the order of <2 kcal mol−1 (
      • Nash A.
      • Notman R.
      • Dixon A.M.
      De novo design of transmembrane helix-helix interactions and measurement of stability in a biological membrane.
      ). Therefore, weak or nonideal TMD interfaces may still be able to mediate dimerization in an active state. This may be the case for VEGFR. When two glutamic acids are introduced into the transmembrane domain, the interaction interface is rotated 180°, resulting in constitutive activation (
      • Manni S.
      • Mineev K.S.
      • Usmanova D.
      • Lyukmanova E.N.
      • Shulepko M.A.
      • Kirpichnikov M.P.
      • Winter J.
      • Matkovic M.
      • Deupi X.
      • Arseniev A.S.
      • Ballmer-Hofer K.
      Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation.
      ). Therefore, a weak TMD dimer in the active state may actually be preferred in order to prevent spontaneous signaling (
      • Weiner D.B.
      • Liu J.
      • Cohen J.A.
      • Williams W.V.
      • Greene M.I.
      A point mutation in the Neu oncogene mimics ligand induction of receptor aggregation.
      ). TMDs that have so far been observed “switching” conformation are EGFR (
      • Bocharov E.V.
      • Lesovoy D.M.
      • Pavlov K.V.
      • Pustovalova Y.E.
      • Bocharova O.V.
      • Arseniev A.S.
      Alternative packing of EGFR transmembrane domain suggests that protein–lipid interactions underlie signal conduction across membrane.
      ) and GHR (
      • Bocharov E.V.
      • Lesovoy D.M.
      • Bocharova O.V.
      • Urban A.S.
      • Pavlov K.V.
      • Volynsky P.E.
      • Efremov R.G.
      • Arseniev A.S.
      Structural basis of the signal transduction via transmembrane domain of the human growth hormone receptor.
      ). The effect has also been predicted for others such as EphA2 (
      • Sharonov G.V.
      • Bocharov E.V.
      • Kolosov P.M.
      • Astapova M.V.
      • Arseniev A.S.
      • Feofanov A.V.
      Point mutations in dimerization motifs of the transmembrane domain stabilize active or inactive state of the EphA2 receptor tyrosine kinase.
      ,
      • Bocharov E.V.
      • Mayzel M.L.
      • Volynsky P.E.
      • Mineev K.S.
      • Tkach E.N.
      • Ermolyuk Y.S.
      • Schulga A.A.
      • Efremov R.G.
      • Arseniev A.S.
      Left-handed dimer of EphA2 transmembrane domain: helix packing diversity among receptor tyrosine kinases.
      ).
      The evidence above strongly suggests that for some receptors (a) there is a preferred TMD dimer orientation for activation, (b) dimerization is not sufficient for activation, (c) the TMD plays a critical role in the LIR mechanism, and (d) the juxtamembrane domain may play a regulatory role in dimerization.
      Receptor clustering
      “We now know that the receptor for insulin is only one of a much larger family of structurally-related cell-surface receptors, and there are persuasive data to demonstrate that this whole family of growth factor and related receptors uses ligand-induced aggregation as a primary control mechanism” (
      • Metzger H.
      Transmembrane signaling: the joy of aggregation.
      ).
      The LIR and LID hypotheses for receptor activation invoke a type of allosteric regulation of signaling. In both mechanisms, the receptor is considered inactive until stimulation by a ligand induces a conformational change that stabilizes the signaling-competent active conformation (Fig. 3). This requires only dimerization. However, some receptors form tetramers, and even large-scale oligomers (i.e. clusters) after stimulation. The LID and LIR hypotheses ignore this common phenomenon, perhaps because it is a problem of a different scale. Receptors that cluster become only fully active when they are at high local densities in the membrane. This makes kinases more likely to encounter their substrates, especially when the substrate is another receptor. Here, we will discuss how clustering activates and regulates receptors, and the role that the transmembrane domain may play in this phenomenon (Fig. 2).
      Figure thumbnail gr3
      Figure 3Receptor activation can rely on different processes. LID requires that receptors come into close contact upon ligand binding. The left panel shows that in the presence of ligand, a free energy minimum appears when receptors are in contact (10 Å is the approximate distance from helix centers (
      • Zhang S.Q.
      • Kulp D.W.
      • Schramm C.A.
      • Mravic M.
      • Samish I.
      • DeGrado W.F.
      The membrane- and soluble-protein helix-helix interactome: similar geometry via different interactions.
      )). In the absence of ligand, receptors will be separated by some context-dependent average distance between monomers. LIR requires reorientation of TMDs. The central panel shows that receptor TMDs may have more than one permitted crossing angle, whereas only one is favored in the presence of ligand. This example shows a ligand-induced shift toward the right-handed (R) conformation. Clustering requires that ligand binding induces formation of larger oligomers. The right panel shows that in the absence of ligand, high-order oligomers are energetically unfavorable, whereas the presence of ligand stabilizes clusters. These processes are not mutually exclusive and may all occur in the same receptor.
      Many receptors cluster upon ligand stimulation. Examples include the following: immune receptors such as the B-cell (
      • Casten L.A.
      • Pierce S.K.
      Receptor-mediated B cell antigen processing. Increased antigenicity of a globular protein covalently coupled to antibodies specific for B cell surface structures.
      ,
      • Cheng P.C.
      • Dykstra M.L.
      • Mitchell R.N.
      • Pierce S.K.
      A role for lipid rafts in B cell antigen receptor signaling and antigen targeting.
      ,
      • Kläsener K.
      • Maity P.C.
      • Hobeika E.
      • Yang J.
      • Reth M.
      B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk.
      ,
      • Maity P.C.
      • Blount A.
      • Jumaa H.
      • Ronneberger O.
      • Lillemeier B.F.
      • Reth M.
      B cell antigen receptors of the IgM and IgD classes are clustered in different protein islands that are altered during B cell activation.
      ,
      • Stone M.B.
      • Shelby S.A.
      • Núñez M.F.
      • Wisser K.
      • Veatch S.L.
      Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.
      ) and T-cell receptors (
      • Taylor M.J.
      • Husain K.
      • Gartner Z.J.
      • Mayor S.
      • Vale R.D.
      A DNA-based T cell receptor reveals a role for receptor clustering in ligand discrimination.
      ); RTKs such as EGFR (
      • Schechter Y.
      • Hernaez L.
      • Schlessinger J.
      • Cuatrecasas P.
      Local aggregation of hormone–receptor complexes is required for activation by epidermal growth factor.
      ); the platelet-derived growth factor β-receptor (PDGFβR) (
      • Hammacher A.
      • Mellström K.
      • Heldin C.H.
      • Westermark B.
      Isoform-specific induction of actin reorganization by platelet-derived growth factor suggests that the functionally active receptor is a dimer.
      ,
      • Heldin C.-H.
      • Ernlund A.
      • Rorsman C.
      • Rönnstrand L.
      Dimerization of B-type platelet-derived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation.
      ,
      • Seifert R.A.
      • Hart C.E.
      • Phillips P.E.
      • Forstrom J.W.
      • Ross R.
      • Murray M.J.
      • Bowen-Pope D.F.
      Two different subunits associate to create isoform-specific platelet-derived growth factor receptors.
      ); GHR (
      • Cunningham B.
      • Ultsch M.
      • De Vos A.M.
      • Mulkerrin M.G.
      • Clauser K.R.
      • Wells J.A.
      Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule.
      ); and Eph receptors (
      • Wimmer-Kleikamp S.H.
      • Janes P.W.
      • Squire A.
      • Bastiaens P.I.
      • Lackmann M.
      Recruitment of Eph receptors into signaling clusters does not require ephrin contact.
      ). Clustering may also modulate the activation of integrins (
      • Miyamoto S.
      • Akiyama S.K.
      • Yamada K.M.
      Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function.
      ). With the exception of GHR, each of these receptors will be discussed in detail below. This is not meant to be a comprehensive list, as there are yet other receptors not discussed here that form clusters. Rather, it is meant to highlight that clustering is a common feature of many receptor activation mechanisms.
      A key difference in the examples mentioned above is the effect of ligand stimulation on how receptors associate into a cluster. For example, EGFR forms, on average, tetrameric complexes upon stimulation (
      • Kozer N.
      • Barua D.
      • Orchard S.
      • Nice E.C.
      • Burgess A.W.
      • Hlavacek W.S.
      • Clayton A.H.
      Exploring higher-order EGFR oligomerisation and phosphorylation—a combined experimental and theoretical approach.
      ,
      • Huang Y.
      • Bharill S.
      • Karandur D.
      • Peterson S.M.
      • Marita M.
      • Shi X.
      • Kaliszewski M.J.
      • Smith A.W.
      • Isacoff E.Y.
      • Kuriyan J.
      Molecular basis for multimerization in the activation of the epidermal growth factor receptor.
      ,
      • Clayton A.H.
      • Walker F.
      • Orchard S.G.
      • Henderson C.
      • Fuchs D.
      • Rothacker J.
      • Nice E.C.
      • Burgess A.W.
      Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor-A multidimensional microscopy analysis.
      ,
      • Clayton A.H.
      • Orchard S.G.
      • Nice E.C.
      • Posner R.G.
      • Burgess A.W.
      Predominance of activated EGFR higher-order oligomers on the cell surface.
      ), whereas Eph receptors form much larger micron-sized clusters (
      • Davis S.
      • Gale N.W.
      • Aldrich T.H.
      • Maisonpierre P.C.
      • Lhotak V.
      • Pawson T.
      • Goldfarb M.
      • Yancopoulos G.D.
      Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity.
      ,
      • Egea J.
      • Nissen U.V.
      • Dufour A.
      • Sahin M.
      • Greer P.
      • Kullander K.
      • Mrsic-Flogel T.D.
      • Greenberg M.E.
      • Kiehn O.
      • Vanderhaeghen P.
      • Klein R.
      Regulation of EphA4 kinase activity is required for a subset of axon guidance decisions suggesting a key role for receptor clustering in Eph function.
      ,
      • Schaupp A.
      • Sabet O.
      • Dudanova I.
      • Ponserre M.
      • Bastiaens P.
      • Klein R.
      The composition of EphB2 clusters determines the strength in the cellular repulsion response.
      ,
      • Singh D.R.
      • Kanvinde P.
      • King C.
      • Pasquale E.B.
      • Hristova K.
      The EphA2 receptor is activated through induction of distinct, ligand-dependent oligomeric structures.
      ). Quantitatively assessing cluster stoichiometry in cells is difficult; therefore, there are still only a few studies that report it. However, it is clear that clustering plays an important role in signaling and deserves further investigation. Special emphasis should be placed on testing a wide range of receptors in native conditions.
      The clustering model may explain why overexpression of many receptors leads to malignancy. Simply concentrating the receptors is enough to induce clustering and activate signaling, as modeled in Ref.
      • Stone M.B.
      • Shelby S.A.
      • Núñez M.F.
      • Wisser K.
      • Veatch S.L.
      Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.
      . As examples, malignancy is induced by overexpression of EphA2 (
      • Miyazaki T.
      • Kato H.
      • Fukuchi M.
      • Nakajima M.
      • Kuwano H.
      EphA2 overexpression correlates with poor prognosis in esophageal squamous cell carcinoma.
      ,
      • Saito T.
      • Masuda N.
      • Miyazaki T.
      • Kanoh K.
      • Suzuki H.
      • Shimura T.
      • Asao T.
      • Kuwano H.
      Expression of EphA2 and E-cadherin in colorectal cancer: correlation with cancer metastasis.
      ), EGFR (
      • Barker 2nd, F.G.
      • Simmons M.L.
      • Chang S.M.
      • Prados M.D.
      • Larson D.A.
      • Sneed P.K.
      • Wara W.M.
      • Berger M.S.
      • Chen P.
      • Israel M.A.
      • Aldape K.D.
      EGFR overexpression and radiation response in glioblastoma multiforme.
      ), Axl (an RTK) (
      • Song X.
      • Wang H.
      • Logsdon C.D.
      • Rashid A.
      • Fleming J.B.
      • Abbruzzese J.L.
      • Gomez H.F.
      • Evans D.B.
      • Wang H.
      Overexpression of receptor tyrosine kinase Axl promotes tumor cell invasion and survival in pancreatic ductal adenocarcinoma.
      ), hepatocyte growth factor receptor (
      • Gumustekin M.
      • Kargi A.
      • Bulut G.
      • Gozukizil A.
      • Ulukus C.
      • Oztop I.
      • Atabey N.
      HGF/c-Met overexpressions, but not met mutation, correlates with progression of non-small cell lung cancer.
      ), and many others (
      • Blume-Jensen P.
      • Hunter T.
      Oncogenic kinase signalling.
      ). The concentration effect can also be studied by presenting cells with artificial substrates coated with ligands at fixed intervals (
      • Shaw A.
      • Lundin V.
      • Petrova E.
      • Förds F.
      • Benson E.
      • Al-Amin A.
      • Herland A.
      • Blokzijl A.
      • Högberg B.
      • Teixeira A.I.
      Spatial control of membrane receptor function using ligand nanocalipers.
      ). Receptors that rely on clustering will only be activated by these substrates when the density of ligands is high enough. Eph receptors, such as EphA2 (
      • Möser C.
      • Lorenz J.
      • Sajfutdinow M.
      • Smith D.
      Pinpointed stimulation of EphA2 receptors via DNA-templated oligovalence.
      ) and EphB2 (
      • Hortigüela V.
      • Larrañaga E.
      • Cutrale F.
      • Seriola A.
      • García-Díaz M.
      • Lagunas A.
      • Andilla J.
      • Loza-Alvarez P.
      • Samitier J.
      • Ojosnegros S.
      • Martínez E.
      Nanopatterns of surface-bound ephrinB1 produce multivalent ligand–receptor interactions that tune EphB2 receptor clustering.
      ), have been activated in this way, suggesting that Eph receptors rely on clustering for activity. TCR also responds more efficiently to dense clusters of peptide–major histocompatibility complex (pMHC) ligands than sparse ones (
      • Anikeeva N.
      • Fischer N.O.
      • Blanchette C.D.
      • Sykulev Y.
      Extent of MHC clustering regulates selectivity and effectiveness of T cell responses.
      ). Various applications and findings of ligand nanopatterning studies are reviewed in Ref.
      • Zhang K.
      • Gao H.
      • Deng R.
      • Li J.
      Emerging applications of nanotechnology for controlling cell-surface receptor clustering.
      .
      In the LID and LIR hypotheses, dimerization in the correct conformation is theoretically sufficient for full-signaling activation (Fig. 2). However, some studies show that there are receptors for which dimerization does not cause maximal signaling. For example, almost a century ago, Karl Landsteiner (
      • Landsteiner K.
      Experiments on anaphylaxis to azoproteins.
      ) could generate an allergic response with polyvalent but not monovalent antigens against what was later found to be the immunoglobin E receptor. If dimerization were sufficient, some concentration of ligand should be able to produce a full response, even if the receptor/ligand stoichiometry differs from 2:1. The effect was also observed in the unrelated insulin receptor, where polyvalent but not monovalent antibodies could elicit cellular responses (
      • Kahn C.R.
      • Baird K.L.
      • Jarrett D.B.
      • Flier J.S.
      Direct demonstration that receptor crosslinking or aggregation is important in insulin action.
      ). As expected, clustering of ligands is also especially important for Eph receptor activation: only ligands “pre-clustered” with an antibody fragment can fully induce signaling, whereas monomeric ligands are only partial agonists (
      • Davis S.
      • Gale N.W.
      • Aldrich T.H.
      • Maisonpierre P.C.
      • Lhotak V.
      • Pawson T.
      • Goldfarb M.
      • Yancopoulos G.D.
      Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity.
      ,
      • Egea J.
      • Nissen U.V.
      • Dufour A.
      • Sahin M.
      • Greer P.
      • Kullander K.
      • Mrsic-Flogel T.D.
      • Greenberg M.E.
      • Kiehn O.
      • Vanderhaeghen P.
      • Klein R.
      Regulation of EphA4 kinase activity is required for a subset of axon guidance decisions suggesting a key role for receptor clustering in Eph function.
      ,
      • Salaita K.
      • Nair P.M.
      • Petit R.S.
      • Neve R.M.
      • Das D.
      • Gray J.W.
      • Groves J.T.
      Restriction of receptor movement alters cellular response: physical force sensing by EphA2.
      ). This effect can also be replicated by allowing monovalent ligands to diffuse laterally in a bilayer. The T-cell receptor forms clusters upon activation with a diffusive pMHC but not with an immobilized pMHC (
      • Ma Y.
      • Pandzic E.
      • Nicovich P.R.
      • Yamamoto Y.
      • Kwiatek J.
      • Pageon S.V.
      • Benda A.
      • Rossy J.
      • Gaus K.
      An intermolecular FRET sensor detects the dynamics of T cell receptor clustering.
      ). Not surprisingly, mobile bilayer-bound ligands also activate EphA2 (
      • Salaita K.
      • Nair P.M.
      • Petit R.S.
      • Neve R.M.
      • Das D.
      • Gray J.W.
      • Groves J.T.
      Restriction of receptor movement alters cellular response: physical force sensing by EphA2.
      ,
      • Chen Z.
      • Oh D.
      • Biswas K.H.
      • Yu C.
      • Zaidel-Bar R.
      • Groves J.T.
      • Pozen D.E.
      Spatially modulated ephrinA1:EphA2 signaling increases local contractility and global focal adhesion dynamics to promote cell motility.
      ,
      • Greene A.C.
      • Lord S.J.
      • Tian A.
      • Rhodes C.
      • Kai H.
      • Groves J.T.
      Spatial organization of EphA2 at the cell-cell interface modulates trans-endocytosis of ephrinA1.
      ,
      • Lohmüller T.
      • Xu Q.
      • Groves J.T.
      Nanoscale obstacle arrays frustrate transport of EphA2–Ephrin-A1 clusters in cancer cell lines.
      ). Various receptors cluster upon activation, and there is evidence that the TMD is important for this process.

      Transmembrane domain-induced clustering

      Clustering induced by TMDs does not require a sophisticated model. According to the fluid mosaic model, proteins and lipids diffuse randomly in the lipid bilayer (
      • Singer S.J.
      • Nicolson G.L.
      The fluid mosaic model of the structure of cell membranes.
      ). As discussed above, this is clearly an oversimplification. However, even without including suborganization, the fluid mosaic model already goes far enough as to suggest how proteins may be prone to clustering. Confinement of diffusion to two dimensions greatly increases the effective concentration. Thus, the membrane enhances interactions simply by restricting the diffusive space (
      • Grasberger B.
      • Minton A.P.
      • DeLisi C.
      • Metzger H.
      Interaction between proteins localized in membranes.
      ). Aggregation is promoted by the concentration of proteins on the membrane by the “volume exclusion effect” (
      • Metzger H.
      Transmembrane signaling: the joy of aggregation.
      ,
      • Minton A.P.
      Excluded volume as a determinant of macromolecular structure and reactivity.
      ). Furthermore, membrane properties can directly affect TMD dimerization. For example, oligomerization is enhanced by the ordering of the bilayer caused by cholesterol (
      • Mall S.
      • Broadbridge R.
      • Sharma R.P.
      • East J.M.
      • Lee A.G.
      Self-association of model transmembrane α-helices is modulated by lipid structure.
      ,
      • Cristian L.
      • Lear J.D.
      • DeGrado W.F.
      Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers.
      ) or by a transition to the gel phase (
      • Anbazhagan V.
      • Schneider D.
      The membrane environment modulates self-association of the human GpA TM domain-implications for membrane protein folding and transmembrane signaling.
      ). Oligomerization also relies on matching of the hydrophobic thickness of the TMD and the surrounding lipid. If the lipid bilayer is too thin or too thick, oligomerization can be reduced in model membranes (
      • Cristian L.
      • Lear J.D.
      • DeGrado W.F.
      Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers.
      ,
      • Anbazhagan V.
      • Schneider D.
      The membrane environment modulates self-association of the human GpA TM domain-implications for membrane protein folding and transmembrane signaling.
      ). For a more thorough review of the role of the lipid bilayer on single-pass membrane protein oligomerization, we suggest Ref.
      • Bocharov E.V.
      • Mineev K.S.
      • Pavlov K.V.
      • Akimov S.A.
      • Kuznetsov A.S.
      • Efremov R.G.
      • Arseniev A.S.
      Helix-helix interactions in membrane domains of bitopic proteins: specificity and role of lipid environment.
      .
      Receptor clustering has profound effects on signaling efficiency and sensitivity (
      • Bray D.
      • Levin M.D.
      • Morton-Firth C.J.
      Receptor clustering as a cellular mechanism to control sensitivity.
      ). For example, the B-cell receptor co-clusters with its downstream effector Lyn, while excluding the phosphatase CD45 (
      • Stone M.B.
      • Shelby S.A.
      • Núñez M.F.
      • Wisser K.
      • Veatch S.L.
      Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.
      ). Likewise, TCR complex clustering results in exclusion of CD45, prolonging the phosphorylation and thus signaling of receptors in the phospho-protected cluster (
      • Su X.
      • Ditlev J.A.
      • Hui E.
      • Xing W.
      • Banjade S.
      • Okrut J.
      • King D.S.
      • Taunton J.
      • Rosen M.K.
      • Vale R.D.
      Phase separation of signaling molecules promotes T cell receptor signal transduction.
      ). In another recent example, a TMD peptide targeting the single-pass protein-tyrosine phosphatase receptor J (PTPRJ) disrupts receptor dimerization and enhances its activation, reducing EGFR phosphorylation even though no direct PTPRJ–EGFR interaction has been observed (
      • Bloch E.
      • Sikorski E.L.
      • Pontoriero D.
      • Day E.K.
      • Berger B.W.
      • Lazzara M.J.
      • Thévenin D.
      Disrupting the transmembrane domain–mediated oligomerization of protein tyrosine phosphatase receptor J inhibits EGFR-driven cancer cell phenotypes.
      ). Sub-localization of molecules in clusters may be explained by interactions with the cytoskeleton, where proteins that interact more strongly with actin migrate more effectively toward the center of the cluster (
      • Hartman N.C.
      • Nye J.A.
      • Groves J.T.
      Cluster size regulates protein sorting in the immunological synapse.
      ). This effect may be further enhanced by the suborganization of the membrane.
      There is mounting evidence that the plasma membrane can be segregated laterally into subdomains, sometimes referred to as lipid rafts, although this topic is still debated. Lipid mixtures can phase-separate into liquid ordered and disordered domains (
      • Ahmed S.N.
      • Brown D.A.
      • London E.
      On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes.
      ,
      • Thompson T.E.
      • Tillack T.W.
      Organization of glycosphingolipids in bilayers and plasma membranes of mammalian cells.
      ). Some membrane proteins have a preference for different membrane subdomains (
      • Toulmay A.
      • Prinz W.A.
      Direct imaging reveals stable, micrometer-scale lipid domains that segregate proteins in live cells.
      ). It has been hypothesized that the thickness of the bilayer is a key determinant for protein sorting to lipid domains (
      • Lin Q.
      • London E.
      Altering hydrophobic sequence lengths shows that hydrophobic mismatch controls affinity for ordered lipid domains (Rafts) in the multitrans membrane strand protein perfringolysin O.
      ). However, transmembrane length does not appear to affect the clustering of receptors (
      • Kashles O.
      • Szapary D.
      • Bellot F.
      • Ullrich A.
      • Schlessinger J.
      • Schmidt A.
      Ligand-induced stimulation of epidermal growth factor receptor mutants with altered transmembrane regions.
      ,
      • Grau B.
      • Javanainen M.
      • García-Murria M.J.
      • Kulig W.
      • Vattulainen I.
      • Mingarro I.
      • Martínez-Gil L.
      The role of hydrophobic matching on transmembrane helix packing in cells.
      ). It has been recently demonstrated that, in cell membranes, protein TMDs encode preferential partitioning information based on their lipid-accessible surface area, not in their hydrophobic length (
      • Lorent J.H.
      • Diaz-Rohrer B.
      • Lin X.
      • Spring K.
      • Gorfe A.A.
      • Levental K.R.
      • Levental I.
      Structural determinants and functional consequences of protein affinity for membrane rafts.
      ). This disagrees with studies in model membranes, suggesting compensatory forces that will be discussed below. Lipid subdomain partitioning further increases the effective concentration of receptors to such a point that transient clustering and activation might be expected. However, full activation requires more than transient clustering. A cluster must be sustained for a set period of time before a signal is produced (
      • DeLisi C.
      The biophysics of ligand–receptor interactions.
      ). Despite this fact, multiple factors acting in concert may be able to induce receptor activation (e.g. strong localization to these domains and interaction with other receptors). These can be brought about by seemingly minor changes in some cases. For example, a single TMD mutation induces constitutive clustering and activation of the Neu receptor (
      • Weiner D.B.
      • Liu J.
      • Cohen J.A.
      • Williams W.V.
      • Greene M.I.
      A point mutation in the Neu oncogene mimics ligand induction of receptor aggregation.
      ). The membrane is thus a critical factor in understanding receptor activation (
      • Bocharov E.V.
      • Mineev K.S.
      • Pavlov K.V.
      • Akimov S.A.
      • Kuznetsov A.S.
      • Efremov R.G.
      • Arseniev A.S.
      Helix-helix interactions in membrane domains of bitopic proteins: specificity and role of lipid environment.
      ).

      Implications for understanding receptor activation

      If there is a single, unified picture of single-pass receptor activation, it is a blurry one. Receptors bind ligands, and this triggers a signal. This much is clear. But what happens in-between is still poorly understood. Over the years, different hypotheses have been proposed. The LID hypothesis assumes that receptors are monomers and only signal upon dimerization and that close contact is the only requirement (Fig. 2). The LIR hypothesis assumes that receptors can form inactive dimers and only activate upon allosteric rotation to the correct intracellular conformation. So far, the transmembrane domain interface (i.e. crossing angle) has been implicated as a possible mechanism. Using only these two hypotheses, Paul and Hristova (
      • Paul M.D.
      • Hristova K.
      The transition model of RTK activation: a quantitative framework for understanding RTK signaling and RTK modulator activity.
      ) recently proposed a general model of RTK activation. Although it is currently unclear how broadly applicable this model is, these studies help to synthesize common elements of receptor activation. The clustering hypothesis assumes that receptors somehow prevent maximal signaling until forming large clusters. It is important to keep in mind that these hypotheses are not necessarily mutually exclusive.
      Each receptor likely has a unique set of factors that regulate activity. Receptors may in principle use all three of the activation mechanisms discussed above (Fig. 3). One can imagine a monomeric receptor that transiently forms inactive dimers, but only upon ligand binding does it dimerize in the correct configuration. Then, factors such as the cytoskeleton and membrane suborganization limit its diffusion until receptor dimers are recruited into a large signaling cluster and an optimal signal is generated. In fact, all of the above phenomena have been observed for a single receptor, EGFR (
      • Yarden Y.
      • Schlessinger J.
      Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor.
      ,
      • Bocharov E.V.
      • Lesovoy D.M.
      • Pavlov K.V.
      • Pustovalova Y.E.
      • Bocharova O.V.
      • Arseniev A.S.
      Alternative packing of EGFR transmembrane domain suggests that protein–lipid interactions underlie signal conduction across membrane.
      ,
      • Schechter Y.
      • Hernaez L.
      • Schlessinger J.
      • Cuatrecasas P.
      Local aggregation of hormone–receptor complexes is required for activation by epidermal growth factor.
      ,
      • Saffarian S.
      • Li Y.
      • Elson E.L.
      • Pike L.J.
      Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis.
      ).
      Other regulating factors may provide receptor specificity as well. Let us briefly review the influences discussed. There are extracellular influences, such as the distribution of ligands on opposing cells or surfaces (
      • Salaita K.
      • Nair P.M.
      • Petit R.S.
      • Neve R.M.
      • Das D.
      • Gray J.W.
      • Groves J.T.
      Restriction of receptor movement alters cellular response: physical force sensing by EphA2.
      ). Similarly, extracellular matrix components such as fibronectin can bind to specific receptors to concentrate them around features such as focal adhesions (
      • Boudreau N.
      • Bissell M.J.
      Extracellular matrix signaling: integration of form and function in normal and malignant cells.
      ). As mentioned before, lipid order favors oligomerization (
      • Mall S.
      • Broadbridge R.
      • Sharma R.P.
      • East J.M.
      • Lee A.G.
      Self-association of model transmembrane α-helices is modulated by lipid structure.
      ,
      • Cristian L.
      • Lear J.D.
      • DeGrado W.F.
      Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers.
      ). Hydrophobic matching of the TMD and lipid bilayer also favors oligomerization (
      • Cristian L.
      • Lear J.D.
      • DeGrado W.F.
      Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers.
      ,
      • Anbazhagan V.
      • Schneider D.
      The membrane environment modulates self-association of the human GpA TM domain-implications for membrane protein folding and transmembrane signaling.
      ). Specific helix–lipid interactions, in contrast, may hinder oligomerization (
      • Hasan M.
      • Patel D.
      • Ellis N.
      • Brown S.P.
      • Lewandowski J.R.
      • Dixon A.M.
      Modulation of transmembrane domain interactions in Neu receptor tyrosine kinase by membrane fluidity and cholesterol.
      ). Lipid lateral organization may also play a role. As an example, the juxtamembrane domain of EGFR binds to PIP2 with a free energy of −0.9 kcal mol−1 (
      • Hedger G.
      • Sansom M.S.P.
      Lipid interaction sites on channels, transporters and receptors: recent insights from molecular dynamics simulations.
      ) and could thus be expected to localize to regions enriched with PIP2. Of course, regulation of protein levels also affects oligomerization; simply increasing the levels of receptors in the membrane will make transient collisions between proteins more likely (
      • Stone M.B.
      • Shelby S.A.
      • Núñez M.F.
      • Wisser K.
      • Veatch S.L.
      Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.
      ). Finally, intracellular factors may promote specific protein oligomerization. For example, the actin cytoskeleton limits diffusion (
      • Hartman N.C.
      • Nye J.A.
      • Groves J.T.
      Cluster size regulates protein sorting in the immunological synapse.
      ), and scaffolding proteins provide substrates on which receptors can specifically associate (
      • Pawson T.
      • Scott J.D.
      Signaling through scaffold, anchoring, and adaptor proteins.
      ).
      As it is becoming clear that different receptors use different activation mechanisms, high-resolution descriptions of individual receptors would be useful. The relative contributions of dimerization, rotation, and clustering for individual receptors should be the focus of future studies.

      Transmembrane peptides to modulate receptor oligomerization

      Single-pass receptor activation mechanisms rely on TMDs, at least as an anchor and at most as a participant in stabilizing oligomeric states. The role of the TMD can be studied directly with peptides that target the TMD (i.e. TMD peptides) (Table S1). TMD peptides may either be synthesized or expressed in cells, a point that may affect their efficacy and will be discussed later. Interactions between receptors and TMD peptides can inform on the receptor activation mechanism. For example, if a receptor requires TMD-mediated dimerization to function, then a TMD peptide targeting that receptor should be able to competitively inhibit receptor dimerization. For some receptors, this simple case is observed. Others require more complex models (Fig. 4). The various effects of TMD peptides have much to teach us about the diversities and similarities between single-pass receptor activation mechanisms. In this section, we will discuss the different approaches to designing TMD peptides to modulate receptor function and how the resulting peptides can be used to study activation.
      Figure thumbnail gr4
      Figure 4TMD peptide functional consequences. TMD peptides can have different functional effects based on the receptor activation mechanism. TMD peptides (green) may competitively inhibit receptor dimerization, leading to reduced signaling or, alternatively, increased signaling in the case of integrins. Receptor complexes may also be stabilized by TMD peptides such as the traptamers and TYPE7, leading to activation, although how this occurs structurally is still unclear. TMD peptides interact preferentially with a specific interface of the TMD shown in blue.

      Structure-based design

      An obvious, yet technically challenging approach to designing TMD peptides is to design a TMD peptide based on the structure of the target. The challenge of this approach lies in being able to accurately predict the forces that drive TMD association. TMD interactions can be stabilized by electrostatic interactions, polar side-chain hydrogen bonds (
      • Choma C.
      • Gratkowski H.
      • Lear J.D.
      • DeGrado W.F.
      Asparagine-mediated self-association of a model transmembrane helix.
      ,
      • Zhou F.X.
      • Cocco M.J.
      • Russ W.P.
      • Brunger A.T.
      • Engelman D.M.
      Interhelical hydrogen bonding drives strong interactions in membrane proteins.
      ), backbone hydrogen bonds (
      • Senes A.
      • Ubarretxena-Belandia I.
      • Engelman D.M.
      The Cα–H···O hydrogen bond: a determinant of stability and specificity in transmembrane helix interactions.
      ), van der Waals interactions (
      • Mravic M.
      • Thomaston J.L.
      • Tucker M.
      • Solomon P.E.
      • Liu L.
      • DeGrado W.F.
      Packing of apolar side chains enables accurate design of highly stable membrane proteins.
      ), or a combination of these forces. As backbone hydrogen bonding and van der Waals interactions rely on packing of helices and side chains, respectively, structural complementarity is, in principle, sufficient for helix association.
      The process of designing TMD peptides based on structure was developed by Yin et al. (
      • Yin H.
      • Slusky J.S.
      • Berger B.W.
      • Walters R.S.
      • Vilaire G.
      • Litvinov R.I.
      • Lear J.D.
      • Caputo G.A.
      • Bennett J.S.
      • DeGrado W.F.
      Computational design of peptides that target transmembrane helices.
      ). Briefly, a “template” dimer structure is populated with the amino acids of the target sequence on one helix and a random sequence on the other. Next, a Monte Carlo repacking algorithm varies the side chains on the random helix in order to generate nonclashing combinations. Finally, an energy function determines optimal residues for each position. The resulting peptides are called computed helical anti-membrane proteins (CHAMPs).
      Integrin receptors detect extracellular matrix components to control cell adhesion. Unlike many receptors, the TMDs of α- and β-integrin heterodimers dissociate when activated by agonist-induced extracellular domain rearrangements (
      • Takagi J.
      • Springer T.A.
      Integrin activation and structural rearrangement.
      ). For integrin heterodimers containing a β3-subunit, this results in platelet aggregation. The simple dimer–monomer activation mechanism of integrins offered a convenient platform to test whether TMD peptides could be designed against them.
      Before designing peptides based on structure, Yin et al. (
      • Yin H.
      • Slusky J.S.
      • Berger B.W.
      • Walters R.S.
      • Vilaire G.
      • Litvinov R.I.
      • Lear J.D.
      • Caputo G.A.
      • Bennett J.S.
      • DeGrado W.F.
      Computational design of peptides that target transmembrane helices.
      ) first designed a TMD peptide based on the sequence. This rational design approach allowed them to first test the feasibility of targeting the integrin transmembrane domain. Site-directed mutagenesis had already suggested that disruption of the αIIbβ3 helical dimer would induce signaling (
      • Partridge A.W.
      • Liu S.
      • Kim S.
      • Bowie J.U.
      • Ginsberg M.H.
      Transmembrane domain helix packing stabilizes integrin αIIbβ3 in the low affinity state.
      ). However, it was unclear whether a synthetic TMD peptide could produce the same effect. Thus, the authors synthesized a peptide composed of the αIIb TMD (αIIb-TM) (Table S1) with hopes that it would bind to the β3-subunit and disrupt the αIIbβ3 TMD interaction (
      • Yin H.
      • Litvinov R.I.
      • Vilaire G.
      • Zhu H.
      • Li W.
      • Caputo G.A.
      • Moore D.T.
      • Lear J.D.
      • Weisel J.W.
      • Degrado W.F.
      • Bennett J.S.
      Activation of platelet αIIbβ3 by an exogenous peptide corresponding to the transmembrane domain of αIIb.
      ). Indeed, αIIb-TM binds to and activates the αIIbβ3 integrin, demonstrating that a TMD peptide can disrupt TMD dimer formation and lead to integrin activation.
      CHAMP peptides, unlike αIIb-TM, are not required to have homology with any native sequence. CHAMPs were first designed to disrupt the αIIbβ3 and αvβ3 TMD interactions (
      • Yin H.
      • Slusky J.S.
      • Berger B.W.
      • Walters R.S.
      • Vilaire G.
      • Litvinov R.I.
      • Lear J.D.
      • Caputo G.A.
      • Bennett J.S.
      • DeGrado W.F.
      Computational design of peptides that target transmembrane helices.
      ). Using the side-chain repacking method described above, the authors designed sequences that were predicted to structurally complement the α-subunit TMD. The resulting peptides (i.e. anti-αv and anti-αIIb) (Table S1) activate their target receptors by competing with the integrin transmembrane domain heterodimer (
      • Caputo G.A.
      • Litvinov R.I.
      • Li W.
      • Bennett J.S.
      • Degrado W.F.
      • Yin H.
      Computationally designed peptide inhibitors of protein–protein interactions in membranes.
      ). Anti-αIIb induces platelet aggregation, whereas anti-αv induces platelet adhesion to osteopontin-coated plates (a feature of αvβ3 activation) (
      • Yin H.
      • Slusky J.S.
      • Berger B.W.
      • Walters R.S.
      • Vilaire G.
      • Litvinov R.I.
      • Lear J.D.
      • Caputo G.A.
      • Bennett J.S.
      • DeGrado W.F.
      Computational design of peptides that target transmembrane helices.
      ), but neither induces the other effect. This demonstrates not only the validity of the CHAMP approach, but that TMD peptides can be specifically targeted.
      A key advantage of the CHAMP design approach is that the target sequence is the only input. This means that studying the effects of disrupting heterodimeric interactions is possible, even if a native interacting partner is unknown. It also means that peptides can be composed of noncanonical amino acids (
      • Shandler S.J.
      • Korendovych I.V.
      • Moore D.T.
      • Smith-Dupont K.B.
      • Streu C.N.
      • Litvinov R.I.
      • Billings P.C.
      • Gai F.
      • Bennett J.S.
      • DeGrado W.F.
      Computational design of a β-peptide that targets transmembrane helices.
      ). Another advantage is that CHAMP peptides do not need to replicate complex native interactions. For example, the structure of the αIIbβ3 heterodimer contains an arginine–aspartate salt bridge that is not possible with anti-αIIb, yet anti-αIIb activates αIIbβ3 regardless (
      • Lau T.-L.
      • Kim C.
      • Ginsberg M.H.
      • Ulmer T.S.
      The structure of the integrin αIIbβ3 transmembrane complex explains integrin transmembrane signalling.
      ).
      The CHAMP design method assumes that tight helical packing will lead to interhelical associations. However, site-directed mutagenesis of the αIIbβ3 heterodimer shows that mutation of β3 (i.e. I693A) results in underpacking of the dimer interface, yet it has no effect on integrin activity (
      • Lau T.-L.
      • Kim C.
      • Ginsberg M.H.
      • Ulmer T.S.
      The structure of the integrin αIIbβ3 transmembrane complex explains integrin transmembrane signalling.
      ). This is likely due to the presence of strong polar interactions (e.g. the arginine–aspartate salt bridge) acting on the same dimer. van der Waals-only interaction models have dramatically improved lately due to incorporation of more sophisticated prediction programs such as Rosetta membrane (
      • Alford R.F.
      • Koehler Leman J.K.
      • Weitzner B.D.
      • Duran A.M.
      • Tilley D.C.
      • Elazar A.
      • Gray J.J.
      An integrated framework advancing membrane protein modeling and design.
      ,
      • Mravic M.
      • Hu H.
      • Lu Z.
      • Bennett J.S.
      • Sanders C.R.
      • Orr A.W.
      • DeGrado W.F.
      De novo designed transmembrane peptides activating the α5β1 integrin.
      ). This concept has been used to accurately design a transmembrane helical pentamer using only van der Waals interactions (
      • Mravic M.
      • Thomaston J.L.
      • Tucker M.
      • Solomon P.E.
      • Liu L.
      • DeGrado W.F.
      Packing of apolar side chains enables accurate design of highly stable membrane proteins.
      ). Future prediction models will benefit from incorporating polar and nonpolar interactions as well as protein–lipid and protein–solvent (e.g. side-chain snorkeling) interactions.

      Sequence-based design

      Screening

      Genetic-based selections for functional TMD interactions is perhaps the most efficient way to identify new sequences that activate receptors via their TMDs. In recent years, the DiMaio and co-workers have used this strategy to discover a large number of artificial transmembrane peptides with divergent sequences that activate PDGFβR (
      • Freeman-Cook L.L.
      • Dixon A.M.
      • Frank J.B.
      • Xia Y.
      • Ely L.
      • Gerstein M.
      • Engelman D.M.
      • DiMaio D.
      Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor β receptor.
      ) or EpoR (
      • Cammett T.J.
      • Jun S.J.
      • Cohen E.B.
      • Barrera F.N.
      • Engelman D.M.
      • Dimaio D.
      Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor.
      ) or even reduce levels of the C–C chemokine receptor type 5, potentially by binding to its TMD and destabilizing the protein (
      • Scheideman E.H.
      • Marlatt S.A.
      • Xie Y.
      • Hu Y.
      • Sutton R.E.
      • DiMaio D.
      Transmembrane protein aptamers that inhibit CCR5 expression and HIV coreceptor function.
      ). These peptides are unusual in that they induce rather than inhibit receptor oligomerization.

      Platelet-derived growth factor β receptor

      Platelet-derived growth factor receptors are a small subfamily of RTKs. Both PDGFRs, α and β, have a single TMD connecting an extracellular ligand-binding domain to an intracellular kinase domain. Both receptors are also required for PDGF-induced activation (
      • Seifert R.A.
      • Hart C.E.
      • Phillips P.E.
      • Forstrom J.W.
      • Ross R.
      • Murray M.J.
      • Bowen-Pope D.F.
      Two different subunits associate to create isoform-specific platelet-derived growth factor receptors.
      ). PDGFβR activity can also be induced by bovine papillomavirus infection. This virus oncogenically transforms fibroblasts via a short oncoprotein called E5 (
      • Bergman P.
      • Ustav M.
      • Sedman J.
      • Moreno-Lopéz J.
      • Vennström B.
      • Pettersson U.
      The E5 gene of bovine papillomavirus type 1 is sufficient for complete oncogenic transformation of mouse fibroblasts.
      ), and the transforming ability of E5 largely depends on the presence of PDGFβR (
      • Petti L.
      • Nilson L.A.
      • DiMaio D.
      Activation of the platelet-derived growth factor receptor by the bovine papillomavirus E5 transforming protein.
      ,
      • Petti L.
      • DiMaio D.
      Specific interaction between the bovine papillomavirus E5 transforming protein and the β receptor for platelet-derived growth factor in stably transformed and acutely transfected cells.
      ). E5 is only 44 amino acids long and is largely composed of a single TMD (
      • Yang Y.-C.
      • Spalholz B.A.
      • Rabson M.S.
      • Howley P.M.
      Dissociation of transforming and trans-activation functions for bovine papillomavirus type 1.
      ,
      • Schlegel R.
      • Wade-Glass M.
      • Rabson M.S.
      • Yang Y.C.
      The E5 transforming gene of bovine papillomavirus encodes a small, hydrophobic polypeptide.
      ) that specifically interacts with the PDGFβR TMD and activates PDGFβR signaling (
      • Cohen B.D.
      • Goldstein D.J.
      • Rutledge L.
      • Vass W.C.
      • Lowy D.R.
      • Schlegel R.
      • Schiller J.T.
      Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and the epidermal growth factor receptor cytoplasmic domain.
      ). E5 must be a dimer to interact with PDGFβR and transform cells, and dimerization is mediated by a central transmembrane glutamine and two C-terminal cysteines in E5 (
      • Horwitz B.H.
      • Burkhardt A.L.
      • Schlegel R.
      • DiMaio D.
      44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids.
      ,
      • Meyer A.N.
      • Xu Y.-F.
      • Webster M.K.
      • Smith A.E.
      • Donoghue D.J.
      Cellular transformation by a transmembrane peptide: structural requirements for the bovine papillomavirus E5 oncoprotein.
      ,
      • Nilson L.A.
      • Gottlieb R.L.
      • Polack G.W.
      • DiMaio D.
      Mutational analysis of the interaction between the bovine papillomavirus E5 transforming protein and the endogenous β receptor for platelet-derived growth factor in mouse C127 cells.
      ). For a comprehensive review of the E5–PDGFβR interaction, see Ref.
      • Talbert-Slagle K.
      • DiMaio D.
      The bovine papillomavirus E5 protein and the PDGF β receptor: it takes two to tango.
      .
      Armed with the knowledge that a short transmembrane oncoprotein could activate PDGFβR, Freeman-Cook and co-workers sought to elucidate the sequence requirements for activation of PDGFβR (
      • Freeman-Cook L.L.
      • Dixon A.M.
      • Frank J.B.
      • Xia Y.
      • Ely L.
      • Gerstein M.
      • Engelman D.M.
      • DiMaio D.
      Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor β receptor.
      ). To do this, they set up a genetic screening strategy to identify novel transmembrane sequences that could activate PDGFβR. By randomizing nucleotides at certain positions, the authors generated libraries of millions of short hydrophobic peptide sequences. When constructs containing these sequences were introduced into murine fibroblasts, peptides that activate the endogenous PDGFβR caused the appearance of transformed foci. Similarly, in murine hematopoietic BaF3 cells, only cells expressing a peptide that activates PDGFβR were able to proliferate in the absence of interleukin 3 (IL-3). Thus, isolating transformed cells or cells grown in the absence of IL-3 and amplifying their DNA revealed which sequences activate PDGFβR. These peptides were named transmembrane protein aptamers (traptamers) (Table S1).
      The genetic screens started conservatively. First, they only randomized the hydrophobic amino acids in the TMD, leaving the critical central glutamine intact (
      • Freeman-Cook L.L.
      • Dixon A.M.
      • Frank J.B.
      • Xia Y.
      • Ely L.
      • Gerstein M.
      • Engelman D.M.
      • DiMaio D.
      Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor β receptor.
      ). Next, they expanded the screen to include the glutamine and lengthened the peptides to include the positions of two critical cysteines. They also allowed hydrophilic amino acids to be inserted sparsely (
      • Freeman-Cook L.L.
      • Dimaio D.
      Modulation of cell function by small transmembrane proteins modeled on the bovine papillomavirus E5 protein.
      ). When they further expanded the screen to the 20 most central TMD residues allowing only hydrophobic residues to be inserted, they found that van der Waals forces alone could generate remarkably specific interactions (
      • Ptacek J.B.
      • Edwards A.P.
      • Freeman-Cook L.L.
      • DiMaio D.
      Packing contacts can mediate highly specific interactions between artificial transmembrane proteins and the PDGFβ receptor.
      ). Some interactions were even more specific for PDGFβR than is E5. Thus, each expansion of the screen led to new sequences that could activate PDGFβR, even when supposedly critical residues were mutated. This demonstrates the main advantage of screening for transmembrane sequences that activate receptors: it allows for high-throughput, side-by-side comparisons of activating ability among potential TMD peptides (
      • Marlatt S.A.
      • Kong Y.
      • Cammett T.J.
      • Korbel G.
      • Noonan J.P.
      • Dimaio D.
      Construction and maintenance of randomized retroviral expression libraries for transmembrane protein engineering.
      ).
      Screening has resulted in traptamers with extremely simple sequences. For example, some traptamers contain only two different residues, leucine and isoleucine, and are affectionately named LIL traptamers (Table S1). In contrast, the per-residue probability of finding a leucine or isoleucine at a given position in a TMD is only 26% (analysis of Table S4 from Ref.
      • Sharpe H.J.
      • Stevens T.J.
      • Munro S.
      A comprehensive comparison of transmembrane domains reveals organelle-specific properties.
      ). The specificity of LIL traptamers can be altered by mutations that change the position of a single side-chain methyl group (
      • He L.
      • Steinocher H.
      • Shelar A.
      • Cohen E.B.
      • Heim E.N.
      • Kragelund B.B.
      • Grigoryan G.
      • DiMaio D.
      Single methyl groups can act as toggle switches to specify transmembrane Protein–protein interactions.
      ). Remarkably, one active traptamer was isolated that contains only a single isoleucine in a polyleucine sequence (
      • Heim E.N.
      • Marston J.L.
      • Federman R.S.
      • Edwards A.P.
      • Karabadzhak A.G.
      • Petti L.M.
      • Engelman D.M.
      • DiMaio D.
      Biologically active LIL proteins built with minimal chemical diversity.
      ).
      What can these discoveries teach us about PDGFβR activation? A recent study may provide insights. Using Rosetta, the authors generated a compelling mechanism for E5-induced PDGFβR dimerization (
      • Karabadzhak A.G.
      • Petti L.M.
      • Barrera F.N.
      • Edwards A.P.B.
      • Moya-Rodríguez A.
      • Polikanov Y.S.
      • Freites J.A.
      • Tobias D.J.
      • Engelman D.M.
      • DiMaio D.
      Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF β receptor in an active dimeric conformation.
      ). Their model predicts that the PDGFβR dimer is stabilized between two E5 dimers. Much like E5, LIL traptamers activate PDGFβR and thus may also stabilize the PDGFβR dimer. Unlike E5, however, LIL traptamers do not have polar residues with which to form hydrogen bonds. This hints at an entirely van der Waals–stabilized complex, which may look very different from the E5–PDGFβR complex. Regardless, the simplest explanation for PDGFβR appears to be that ligand-induced dimerization is sufficient for activation. However, it is still unclear whether the PDGFβR dimer must be stabilized in a specific conformation. It would also be interesting to determine whether all LIL traptamer–PDGFβR complexes share the same hexameric stoichiometry as the E5–PDGFβR complex, which would suggest a common mechanism for dimer stabilization.
      Traptamers that target other receptors have been isolated. For example, DiMaio and co-workers (
      • He L.
      • Steinocher H.
      • Shelar A.
      • Cohen E.B.
      • Heim E.N.
      • Kragelund B.B.
      • Grigoryan G.
      • DiMaio D.
      Single methyl groups can act as toggle switches to specify transmembrane Protein–protein interactions.
      ,
      • He L.
      • Cohen E.B.
      • Edwards A.P.B.
      • Xavier-Ferrucio J.
      • Bugge K.
      • Federman R.S.
      • Absher D.
      • Myers R.M.
      • Kragelund B.B.
      • Krause D.S.
      • DiMaio D.
      Transmembrane protein aptamer induces cooperative signaling by the EPO receptor and the cytokine receptor β-common subunit.
      ) set up a screen for transmembrane sequences that induce IL-3–independent proliferation of BaF3 cells engineered to express EpoR, but not parental BaF3 cells lacking EpoR expression. This led to the discovery of traptamers that activate EpoR but not PDGFβR. Case studies on select EpoR traptamers demonstrated that they may use fundamentally different mechanisms to activate the receptor. One traptamer, ELI-3, does not require EpoR cytoplasmic tyrosines, but requires activation of a secondary receptor, the cytokine receptor β common subunit (βcR) (
      • He L.
      • Cohen E.B.
      • Edwards A.P.B.
      • Xavier-Ferrucio J.
      • Bugge K.
      • Federman R.S.
      • Absher D.
      • Myers R.M.
      • Kragelund B.B.
      • Krause D.S.
      • DiMaio D.
      Transmembrane protein aptamer induces cooperative signaling by the EPO receptor and the cytokine receptor β-common subunit.
      ), whereas another traptamer, EBC5-16, requires EpoR tyrosines, but not βcR.
      Screening to discover TMD peptides has several advantages. In theory, the approach outlined above can be applied to find TMD peptide activators of any receptor that induces cell proliferation or some other selectable phenotype. Because traptamers are not based on pre-existing, evolved sequences, they may display activities that have not evolved in nature. Also, these TMD peptides may use novel mechanisms, such as the ELI-3–induced heteroactivation of EpoR and βcR. However, this may also be a disadvantage of screening. Without secondary screens, the mechanism of activation cannot be selected. For example, selecting TMD peptides that activate PDGFβR, but only by a different mechanism, would require extra screening steps. Another disadvantage is that the screening approach described above is survival-based, and thus only selects for activating TMD peptides. Discovering deactivating TMD peptides would require a different approach. For example, He et al. (
      • He L.
      • Hoffmann A.R.
      • Serrano C.
      • Hristova K.
      • Wimley W.C.
      High-throughput selection of transmembrane sequences that enhance receptor tyrosine kinase activation.
      ) set up a SDS-PAGE–based screen to find ErbB2 TMD sequences that dimerize more readily than the WT sequence. One can imagine adding a second step to this screen in which peptides are exogenously added or expressed and then tested for their ability to inhibit ErbB2 activation. This approach might result in TMD peptides that deactivate their targets. In general, however, the success of screening is directly tied to the ability of the screen to detect the desired TMD peptides.

      Rational design

      Sequence-based rational design of TMD peptides permits testing specific hypotheses. The simplest and most common approach is to isolate the TMD of the target receptor and present it as a peptide to cells expressing the full-length receptor. To our knowledge, the first time this was achieved was with GpA. The GpA TMD peptide (
      • Tomita M.
      • Marchesi V.T.
      Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin.
      ) shifts the full-length GpA from a dimer to an apparent monomer through hetero-association (
      • Furthmayr H.
      • Marchesi V.T.
      Subunit structure of human erythrocyte glycophorin A.
      ). This line of experiments represents the first demonstration that a TMD peptide can compete with a full-length membrane protein to disrupt dimerization (
      • Bormann B.J.
      • Knowles W.J.
      • Marchesi V.T.
      Synthetic peptides mimic the assembly of transmembrane glycoproteins.
      ,
      • Sternberg M.J.
      • Gullick W.J.
      Neu receptor dimerization.
      ).
      Rational design of TMD peptides has been used on diverse protein targets. For example, there are TMD peptides that disrupt hetero-association between toll-like receptors 2 and 6 (Table S1) (
      • Shmuel-Galia L.
      • Aychek T.
      • Fink A.
      • Porat Z.
      • Zarmi B.
      • Bernshtein B.
      • Brenner O.
      • Jung S.
      • Shai Y.
      Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis.
      ,
      • Fink A.
      • Reuven E.M.
      • Arnusch C.J.
      • Shmuel-Galia L.
      • Antonovsky N.
      • Shai Y.
      Assembly of the TLR2/6 transmembrane domains is essential for activation and is a target for prevention of sepsis.
      ). Similarly, a peptide derived from the TMD of the p75 neutrophin receptor inhibits p75-tyrosine kinase receptor B association (Table S1) (
      • Saadipour K.
      • MacLean M.
      • Pirkle S.
      • Ali S.
      • Lopez-Redondo M.-L.
      • Stokes D.L.
      • Chao M.V.
      The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.
      ). The same principle that applies to single-pass receptors also works for G-protein–coupled receptors such as the CXC motif chemokine receptor 4 (
      • Tarasova N.I.
      • Rice W.G.
      • Michejda C.J.
      Inhibition of G-protein–coupled receptor function by disruption of transmembrane domain interactions.
      ), dopamine D2 receptor (
      • Ng G.Y.
      • O'Dowd B.F.
      • Lee S.P.
      • Chung H.T.
      • Brann M.R.
      • Seeman P.
      • George S.R.
      Dopamine D2 receptor dimers and receptor-blocking peptides.
      ), and β2-adrenergic receptor (Table S1) (
      • Hebert T.E.
      • Moffett S.
      • Morello J.-P.
      • Loisel T.P.
      • Bichet D.G.
      • Barret C.
      • Bouvier M.
      A peptide derived from a β2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation.
      ). As we will see in the forthcoming sections, this approach has proven successful for several other receptors, and, when coupled with studies on receptor activity, can shed light on receptor activation mechanisms.

      T-cell receptor

      The T-cell receptor (TCR) is found on T lymphocytes and is required for cell-mediated adaptive immune response (
      • Alberts B.
      • Johnson A.
      • Lewis J.
      • Raff M.
      • Roberts K.
      • Walter P.
      The Adaptive Immune System.
      ). TCR activates upon binding to the MHC complex on antigen-presenting cells. This prompts a signaling cascade that results in T-cell proliferation and cytokine production. TCR activation in response to host cells, rather than pathogens, results in autoimmune disorders such as rheumatoid arthritis and type 1 diabetes. TCR is thus a promising target for therapeutic inhibition.
      TCR is a complex of six different proteins (TCR α and β,and CD3 δ, ϵ, γ, and ζ), each of which possesses a single TMD. The TCR α- and β-subunits contain extracellular ligand recognition sites that bind to the MHC complex (
      • Cantrell D.
      T cell antigen receptor signal transduction pathways.
      ). Complex assembly is mediated by the TMDs (
      • Manolios N.
      • Bonifacino J.S.
      • Klausner R.D.
      Transmembrane helical interactions and the assembly of the T-cell receptor complex.
      ). First, heterodimers of TCRαβ, CD3δϵ, and CD3γϵ form (
      • Call M.E.
      • Pyrdol J.
      • Wiedmann M.
      • Wucherpfennig K.W.
      The organizing principle in the formation of the T cell receptor–CD3 complex.
      ). TCRα then binds to the CD3δϵ heterodimer via a critical lysine residue, and likewise TCRβ binds to the CD3γϵ heterodimer. The two CD3ζ subunits join the complex last and are phosphorylated to initiate a signaling cascade.
      Manolios et al. (
      • Manolios N.
      • Collier S.
      • Taylor J.
      • Pollard J.
      • Harrison L.C.
      • Bender V.
      T-cell antigen receptor transmembrane peptides modulate T-cell function and T cell-mediated disease.
      ) hypothesized that they could inhibit TCR signaling by blocking the formation of the full TCR complex. At the time, they knew that to interact with CD3δϵ TCRα required a stretch of eight amino acids containing a critical arginine and lysine (
      • Manolios N.
      • Kemp O.
      • Li Z.G.
      The T-cell antigen receptor-α and β-chains interact via distinct regions with Cd3 chains.
      ). Based on the central role of TCRα in complex assembly, the authors designed several short peptides based on the TCRα TMD (
      • Manolios N.
      • Collier S.
      • Taylor J.
      • Pollard J.
      • Harrison L.C.
      • Bender V.
      T-cell antigen receptor transmembrane peptides modulate T-cell function and T cell-mediated disease.
      ). The most active TCR inhibitor was derived from the central portion of the TCRα TMD and thus named “core peptide” (CP) (Table S1). CP treatment results in inhibition of CD3ζ phosphorylation and the downstream production of interleukin 2 (
      • Wang X.M.
      • Djordjevic J.T.
      • Kurosaka N.
      • Schibeci S.
      • Lee L.
      • Williamson P.
      • Manolios N.
      T-cell antigen receptor peptides inhibit signal transduction within the membrane bilayer.
      ), and it inhibits TCR in human cells in vitro (
      • Huynh N.T.
      • Ffrench R.A.
      • Boadle R.A.
      • Manolios N.
      Transmembrane T-cell receptor peptides inhibit B- and natural killer-cell function.
      ) and in mice (
      • Ali M.
      • De Planque M.R.
      • Huynh N.T.
      • Manolios N.
      • Separovic F.
      Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor.
      ,
      • Mahnke K.
      • Qian Y.
      • Knop J.
      • Enk A.H.
      Dendritic cells, engineered to secrete a T-cell receptor mimic peptide, induce antigen-specific immunosuppression in vivo.
      ). CP can even be applied topically to inhibit allergic response in mice and in humans, and it improved symptoms of a small number of psoriasis patients (
      • Göllner G.P.
      • Müller G.
      • Alt R.
      • Knop J.
      • Enk A.H.
      Therapeutic application of T cell receptor mimic peptides or cDNA in the treatment of T cell-mediated skin diseases.
      ).
      CP functions by competitively inhibiting formation of the TCR complex. This is evidenced by the fact that a cross-linked TCR complex is unaffected by CP (
      • Wang X.M.
      • Djordjevic J.T.
      • Kurosaka N.
      • Schibeci S.
      • Lee L.
      • Williamson P.
      • Manolios N.
      T-cell antigen receptor peptides inhibit signal transduction within the membrane bilayer.
      ). For CP to function, it must contain two hydrophobic faces interrupted by two opposing positively charged residues (
      • Ali M.
      • Salam N.K.
      • Amon M.
      • Bender V.
      • Hibbs D.E.
      • Manolios N.
      T-cell antigen receptor-α chain transmembrane peptides: correlation between structure and function.
      ), and the same sequence constraints apply even if d-amino acids are used (Table S1) (
      • Gerber D.
      • Quintana F.J.
      • Bloch I.
      • Cohen I.R.
      • Shai Y.
      d-Enantiomer peptide of the TCRα transmembrane domain inhibits T-cell activation in vitro in vivo.
      ). These two residues (i.e. lysine and arginine) are thought to interact with negatively-charged amino acids in the CD3δϵ and ζζ dimers (
      • Call M.E.
      • Pyrdol J.
      • Wiedmann M.
      • Wucherpfennig K.W.
      The organizing principle in the formation of the T cell receptor–CD3 complex.
      ), whereas the hydrophobic faces are thought to interact with lipids. This suggests stringent structural requirements for signaling activation.
      An often-overlooked practical aspect of TMD peptide development is membrane partitioning. Obviously, for CP to interact with the TCR TMD, it must adopt a transmembrane state. This requires delivery from aqueous solution into the cell membrane. The high hydrophobicity of most membrane peptides makes this a nontrivial step. Indeed, proper delivery is one of the determining factors for the ability of TMD peptides to act on their receptors (
      • Raguine L.
      • Ali M.
      • Bender V.
      • Diefenbach E.
      • Doddareddy M.R.
      • Hibbs D.
      • Manolios N.
      Alanine scan of an immunosuppressive peptide (CP): analysis of structure–function relationships.
      ). Delivery may be enhanced by post-translational modifications such as palmitoylation (
      • Manolios N.
      • Collier S.
      • Taylor J.
      • Pollard J.
      • Harrison L.C.
      • Bender V.
      T-cell antigen receptor transmembrane peptides modulate T-cell function and T cell-mediated disease.
      ,
      • Ali M.
      • Amon M.
      • Bender V.
      • Manolios N.
      Hydrophobic transmembrane–peptide lipid conjugations enhance membrane binding and functional activity in T-cells.
      ,
      • Amon M.A.
      • Ali M.
      • Bender V.
      • Chan Y.N.
      • Toth I.
      • Manolios N.
      Lipidation and glycosylation of a T cell antigen receptor (TCR) transmembrane hydrophobic peptide dramatically enhances in vitro in vivo function.
      ,
      • Ali M.
      • Manolios N.
      Peptide delivery systems.
      ). However, delivery systems may affect the ability of peptides to work properly. For example, CP is typically delivered to cells in DMSO, whereas in vivo CP is delivered in squamous oil, a less toxic hydrophobic medium. When comparing how strongly peptides inhibit TCR in different model systems, Collier et al. (
      • Collier S.
      • Bolte A.
      • Manolios N.
      Discrepancy in CD3-transmembrane peptide activity between in vitro in vivo T-cell inhibition.
      ) found that peptides dissolved in oil before being administered to mice inhibited TCR much more effectively than peptides dissolved in DMSO before being administered to cells. Collier et al. (
      • Collier S.
      • Bolte A.
      • Manolios N.
      Discrepancy in CD3-transmembrane peptide activity between in vitro in vivo T-cell inhibition.
      ) attribute this different response to the difference in solvent. Thus, delivery may dramatically affect the ability of TMD peptides to interact with their receptors (see under “FGFR3”).

      Neuropilin-1

      Semaphorins are a diverse class of small extracellular proteins that regulate cell morphology by promoting or inhibiting axon guidance and motility (
      • Alto L.T.
      • Terman J.R.
      Semaphorins and their signaling mechanisms.
      ). Of their many members, Sema3A in particular has been singled out as a key inhibitor of axon growth through its interactions with the single-pass membrane receptor, neuropilin-1 (NRP1) (
      • He Z.
      • Tessier-Lavigne M.
      Neuropilin is a receptor for the axonal chemorepellent semaphorin III.
      ,
      • Kolodkin A.L.
      • Matthes D.J.
      • Goodman C.S.
      The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules.
      ). Although initially studied for its role in semaphorin signaling in the nervous system, misregulation of NRP1 has also been implicated in cancer via its role as a co-receptor for vascular endothelial growth factor (
      • Soker S.
      • Takashima S.
      • Miao H.Q.
      • Neufeld G.
      • Klagsbrun M.
      Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.
      ). How NRP1 signals is not fully understood, as it does not possess an intracellular kinase domain or obligate partner. It is thus thought to form dynamic complexes with other co-receptors such as plexins (
      • Sawma P.
      • Roth L.
      • Blanchard C.
      • Bagnard D.
      • Crémel G.
      • Bouveret E.
      • Duneau J.-P.
      • Sturgis J.N.
      • Hubert P.
      Evidence for new homotypic and heterotypic interactions between transmembrane helices of proteins involved in receptor tyrosine kinase and neuropilin signaling.
      ,
      • Sharma A.
      • Verhaagen J.
      • Harvey A.R.
      Receptor complexes for each of the Class 3 semaphorins.
      ). The determinants for complex formation, however, remain elusive.
      Roth et al. (
      • Roth L.
      • Nasarre C.
      • Dirrig-Grosch S.
      • Aunis D.
      • Crémel G.
      • Hubert P.
      • Bagnard D.
      Transmembrane domain interactions control biological functions of neuropilin-1.
      ) set out to test whether a TMD peptide could inhibit NRP1 signaling and provide insights into Sema3A-induced signaling. Using a modified TOXCAT assay (
      • Russ W.P.
      • Engelman D.M.
      TOXCAT: a measure of transmembrane helix association in a biological membrane.
      ), they found that the TMD of NRP1 strongly dimerizes—a finding that agrees with simulations (
      • Aci-Sèche S.
      • Sawma P.
      • Hubert P.
      • Sturgis J.N.
      • Bagnard D.
      • Jacob L.
      • Genest M.
      • Garnier N.
      Transmembrane recognition of the semaphorin co-receptors neuropilin 1 and plexin A1: coarse-grained simulations.
      ). They then synthesized a peptide composed of the NRP1 TMD called “membrane-targeting peptide,” MTP-NRP1 (Table S1), to interfere with the interaction. MTP-NRP1 inhibits NRP1 by competitively inhibiting its GXXXG-mediated dimerization (
      • Roth L.
      • Nasarre C.
      • Dirrig-Grosch S.
      • Aunis D.
      • Crémel G.
      • Hubert P.
      • Bagnard D.
      Transmembrane domain interactions control biological functions of neuropilin-1.
      ).
      Blocking NRP1 dimerization has in vitro and in vivo effects. Sema3A induces growth cone collapse in COS cells, a fibroblast-like simian cell line, expressing NRP1. Pre-treatment with MTP-NRP1 blocks this effect, presumably because it prevents the receptor from forming dimers (
      • Roth L.
      • Nasarre C.
      • Dirrig-Grosch S.
      • Aunis D.
      • Crémel G.
      • Hubert P.
      • Bagnard D.
      Transmembrane domain interactions control biological functions of neuropilin-1.
      ). This allows axons to grow even in the presence of the inhibitory Sema3A. Thus, treatment with MTP-NRP1 can facilitate innervation of bioprosthetics (
      • Kuchler-Bopp S.
      • Bagnard D.
      • Van-Der-Heyden M.
      • Idoux-Gillet Y.
      • Strub M.
      • Gegout H.
      • Lesot H.
      • Benkirane-Jessel N.
      • Keller L.
      Semaphorin 3A receptor inhibitor as a novel therapeutic to promote innervation of bioengineered teeth.
      ). Promisingly, MTP-NRP1 also inhibits the growth of tumors in mice by blocking VEGFR signaling (
      • Nasarre C.
      • Roth M.
      • Jacob L.
      • Roth L.
      • Koncina E.
      • Thien A.
      • Labourdette G.
      • Poulet P.
      • Hubert P.
      • Crémel G.
      • Roussel G.
      • Aunis D.
      • Bagnard D.
      Peptide-based interference of the transmembrane domain of neuropilin-1 inhibits glioma growth in vivo.
      ,
      • Arpel A.
      • Gamper C.
      • Spenlé C.
      • Fernandez A.
      • Jacob L.
      • Baumlin N.
      • Laquerriere P.
      • Orend G.
      • Crémel G.
      • Bagnard D. <