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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.
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 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 (
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) (
). 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.
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 (
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 (
). 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 (
). 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 (
). 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 (
). 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 (
) 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 (
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 (
). 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 (
). 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 (
). 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.
). 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 (
). 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.
). 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 (
). 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 (
). 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 (
), 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 (
). 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 (
) 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 (
). 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 (
). 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 (
). 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 (
) 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 (
). 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 (
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 (
). 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 (
). 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.
. 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 (
). 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 (
) 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 (
) 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 (
). 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 (
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.
“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” (
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).
Many receptors cluster upon ligand stimulation. Examples include the following: immune receptors such as the B-cell (
). 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 (
). 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.
), 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 (
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 (
) 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
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.
). 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 (
). 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 (
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 (
) 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 (
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 (
) 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 (
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.
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 (
), 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. (
). 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 (
). 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. (
) 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 (
). 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 (
). 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 (
), 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 (
). 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 (
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 (
). 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 (
). 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.
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 (
). 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 (
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.
). 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 (
). 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 (
). 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 (
). 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 (
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.
). 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 (
) 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) (
), 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. (
) 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.
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 (
). 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.
The T-cell receptor (TCR) is found on T lymphocytes and is required for cell-mediated adaptive immune response (
). 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 (
). 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.
) 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 (
). 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 (
), 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 (
). 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. (
). 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 (
). 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 (
). 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 (
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 (