Modulation Of Receptor Function Research Articles

Insights into differential modulation of receptor function by hinge region using novel agonistic lutropin receptor and inverse agonistic thyrotropin receptor antibodies

We report two antibodies, scFv 13B1 and MAb PD1.37, against the hinge regions of LHR and TSHR, respectively, which have similar epitopes but different effects on receptor function. While neither of them affected hormone binding, with marginal effects on hormone response, scFv 13B1 stimulated LHR in a dose-dependent manner, whereas MAb PD1.37 acted as an inverse agonist of TSHR. Moreover, PD1.37 could decrease the basal activity of hinge region CAMs, but had varied effects on those present in ECLs, whereas 13B1 was refractory to any CAMs in LHR. Using truncation mutants and peptide phage display, we compared the differential roles of the hinge region cysteine box-2/3 as well as the exoloops in the activation of these two homologus receptors.

NCS-1 associates with adenosine A2A receptors and modulates receptor function

Modulation of G protein-coupled receptor (GPCR) signaling by local changes in intracellular calcium concentration is an established function of Calmodulin (CaM) which is known to interact with many GPCRs. Less is known about the functional role of the closely related neuronal EF-hand Ca2+-sensor proteins that frequently associate with CaM targets with different functional outcome. In the present study we aimed to investigate if a target of CaM—the A2A adenosine receptor is able to associate with two other neuronal calcium binding proteins (nCaBPs), namely NCS-1 and caldendrin. Using bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments we show the existence of A2A—NCS-1 complexes in living cells whereas caldendrin did not associate with A2A receptors under the conditions tested. Interestingly, NCS-1 binding modulated downstream A2A receptor intracellular signaling in a Ca2+-dependent manner. Taken together this study provides further evidence that neuronal Ca2+-sensor proteins play an important role in modulation of GPCR signaling.

Roles of phosphoinositide-dependent kinase-1 in α1B-adrenoceptor phosphorylation and desensitization

The role of phosphoinositide-dependent protein kinase-1 (PDK-1) activity on α1B-adrenoceptor phosphorylation and function was explored using pharmacological inhibitors and expression of a dominant-negative mutant of this enzyme. Noradrenaline-, phorbol myristate acetate-, lysophosphatidic acid- and epidermal growth factor-mediated α1B-adrenoceptor phosphorylation were markedly reduced by the two inhibitors used: UCN-01 [(7-hydroxystaurosporine; (3R*,8S*, 9R*, 10R*,12R*)-2,3,9,10,11,12-hexahydro-3-hydroxy-9-methoxy-8-methyl-10-(methylamino)-8,12-epoxy-1H, 8H-2,7b,12a-triazadibenzo[a,g]-cyclonona[cde]triden-1-one)] and OSU-03012 [(2-amino-N-[4-[5-(2-phenanthrenyl)-3-trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide)]. A similar effect was observed in cells expressing a PDK-1 dominant-negative mutant. Phosphorylated PDK-1 (S241) and protein kinase C α (T497) were associated with cell membranes in the basal state which increased in response to the hormonal stimuli mentioned previously. UCN-01 essentially abolished phospho-PDK-1 membrane-association and markedly attenuated that of protein kinase C α. Consistent with the findings, UCN-01 reduced lysophosphatidic acid- and epidermal growth factor-induced α1B-adrenoceptor desensitization. Our data suggest that PDK-1 plays a permissive role in α1B-adrenoceptor desensitization and phosphorylation and participates in the formation of signaling complexes, which delicately modulate receptor function and regulation.

Regulation of human EGF receptor by lipids

The human epidermal growth factor receptor (EGFR) is a key representative of tyrosine kinase receptors, ubiquitous actors in cell signaling, proliferation, differentiation, and migration. Although the receptor is well-studied, a central issue remains: How does the compositional diversity and functional diversity of the surrounding membrane modulate receptor function? Reconstituting human EGFR into proteoliposomes of well-defined and controlled lipid compositions represents a minimal synthetic approach to systematically address this question. We show that lipid composition has little effect on ligand-binding properties of the EGFR but rather exerts a profound regulatory effect on kinase domain activation. Here, the ganglioside GM3 but not other related lipids strongly inhibited the autophosphorylation of the EGFR kinase domain. This inhibitory action of GM3 was only seen in liposomes compositionally poised to phase separate into coexisting liquid domains. The inhibition by GM3 was released by either removing the neuraminic acid of the GM3 headgroup or by mutating a membrane proximal lysine of EGFR (K642G). Our results demonstrate that GM3 exhibits the potential to regulate the allosteric structural transition from inactive to a signaling EGFR dimer, by preventing the autophosphorylation of the intracellular kinase domain in response to ligand binding.

Pharmacological Characterization of LY593093, an M1 Muscarinic Acetylcholine Receptor-Selective Partial Orthosteric Agonist

Alzheimer's disease and schizophrenia are characterized by expression of psychotic, affective, and cognitive symptoms. Currently, there is a lack of adequate treatment for the cognitive symptoms associated with these diseases. Cholinergic signaling and, in particular, M1 muscarinic acetylcholine receptor (m1AChR) signaling have been implicated in the regulation of multiple cognitive domains. Thus, the M1AChR has been identified as a therapeutic drug target for diseases, such as schizophrenia and Alzheimer's disease, that exhibit marked cognitive dysfunction as part of their clinical manifestation. Unfortunately, the development of selective M1 agonist medications has not been successful, mostly because of the highly conserved orthosteric acetylcholine binding site among the five muscarinic receptor subtypes. More recent efforts have focused on the development of allosteric M1AChR modulators that target regions of the receptor distinct from the orthosteric site that are less conserved between family members. However, orthosteric and allosteric ligands may differentially modulate receptor function and ultimately downstream signaling pathways. Thus, the need for highly selective M1AChR orthosteric agonists still exists, not only as a potential therapeutic but also as a pharmacological tool to better understand the physiologic consequences of M1AChR orthosteric activation. Here, we describe the novel, potent and selective M1AChR orthosteric partial agonist LY593093 [N-[(1R,2R)-6-({(1E)-1-[(4-fluorobenzyl)(methyl)amino]ethylidene})amino)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]biphenyl-4-carboxamide]. This compound demonstrates modest to no activity at the other muscarinic receptor subtypes, stimulates Gα(q)-coupled signaling events as well as β-arrestin recruitment, and displays significant efficacy in in vivo models of cognition.

Helix 12 Dynamics and Thyroid Hormone Receptor Activity: Experimental and Molecular Dynamics Studies of Ile280 Mutants

Nuclear hormone receptors (NRs) form a family of transcription factors that mediate cellular responses initiated by hormone binding. It is generally recognized that the structure and dynamics of the C-terminal helix 12 (H12) of NRs' ligand binding domain (LBD) are fundamental to the recognition of coactivators and corepressors that modulate receptor function. Here we study the role of three mutations in the I280 residue of H12 of thyroid hormone receptors using site-directed mutagenesis, functional assays, and molecular dynamics simulations. Although residues at position 280 do not interact with coactivators or with the ligand, we show that its mutations can selectively block coactivator and corepressor binding, and affect hormone binding affinity differently. Molecular dynamics simulations suggest that ligand affinity is reduced by indirectly displacing the ligand in the binding pocket, facilitating water penetration and ligand destabilization. Mutations I280R and I280K link H12 to the LBD by forming salt bridges with E457 in H12, stabilizing H12 in a conformation that blocks both corepressor and coactivator recruitment. The I280M mutation, in turn, blocks corepressor binding, but appears to enhance coactivator affinity, suggesting stabilization of H12 in agonist conformation.

“Molecular Switches” on mGluR Allosteric Ligands That Modulate Modes of Pharmacology

G-protein-coupled receptors (GPCRs) represent the largest class of drug targets, accounting for more than 40% of marketed drugs; however, discovery efforts for many GPCRs have failed to provide viable drug candidates. Historically, drug discovery efforts have focused on developing ligands that act at the orthosteric site of the endogenous agonist. Recently, efforts have focused on functional assay paradigms and the discovery of ligands that act at allosteric sites to modulate receptor function in either a positive, negative, or neutral manner. Allosteric modulators have numerous advantages over orthosteric ligands, including high subtype selectivity; the ability to mimic physiological conditions; the lack of densensitization, downregulation, and internalization; and reduced side effects. Despite these virtues, challenging issues have now arisen for allosteric modulators of metabotropic glutamate receptors (mGluRs): shallow SAR, ligand-directed trafficking, and the identification of subtle "molecular switches" that modulate the modes of pharmacology. Here, we will discuss the impact of modest structural changes to multiple mGluR allosteric ligands scaffolds that unexpectedly modulate pharmacology and raise concerns over metabolism and the pharmacology of metabolites.

Allosteric Modulation of Family C G-Protein-Coupled Receptors: from Molecular Insights to Therapeutic Perspectives

Allosteric receptor modulation is an attractive concept in drug targeting because it offers important potential advantages over conventional orthosteric agonism or antagonism. Allosteric ligands modulate receptor function by binding to a site distinct from the recognition site for the endogenous agonist. They often have no effect on their own and therefore act only in conjunction with physiological receptor activation. This article reviews the current status of allosteric modulation at family C G-protein coupled receptors in the light of their specific structural features on the one hand and current concepts in receptor theory on the other hand. Family C G-protein-coupled receptors are characterized by a large extracellular domain containing the orthosteric agonist binding site known as the "venus flytrap module" because of its bilobal structure and the dynamics of its activation mechanism. Mutational analysis and chimeric constructs have revealed that allosteric modulators of the calcium-sensing, metabotropic glutamate and GABA(B) receptors bind to the seven transmembrane domain, through which they modify signal transduction after receptor activation. This is in contrast to taste-enhancing molecules, which bind to different parts of sweet and umami receptors. The complexity of interactions between orthosteric and allosteric ligands is revealed by a number of adequate biochemical and electrophysiological assay systems. Many allosteric family C GPCR modulators show in vivo efficacy in behavioral models for a variety of clinical indications. The positive allosteric calcium sensing receptor modulator cinacalcet is the first drug of this type to enter the market and therefore provides proof of principle in humans.

Sialyl Lewis X modification of the epidermal growth factor receptor regulates receptor function during airway epithelial wound repair

Epidermal growth factor receptor (EGFR) is a major regulator of airway epithelial cell (AEC) functions such as migration, proliferation and differentiation, which play an essential role in epithelial repair. EGFR is a glycoprotein with 12 potential N-glycosylation sites in its extracellular domain. Glycosylation of EGFR has been shown to modulate its function. Previously, our laboratory demonstrated an important role of the carbohydrate structure sialyl Lewis x (sLe(x)) in airway epithelial repair. To examine whether an sLe(x) decoration of EGFR can modulate receptor function during AEC repair. Primary normal human bronchial epithelial (NHBE) cells were cultured in vitro. Co-localization of sLe(x) and EGFR was examined using confocal microscopy. Expressions of RNA and protein were analysed using RT-PCR and Western blotting. The final step in the synthesis of sLe(x) was catalysed by a specific alpha-1,3-fucosyltransferase (FucT-IV). To evaluate the role of sLe(x) in EGFR activation, a knockdown of the FucT-IV gene with small interfering RNA (siRNA) and an inhibitory anti-sLe(x) antibody (KM-93) was used. We demonstrated a co-localization of sLe(x) with EGFR on NHBE cells using confocal microscopy. Using a blocking antibody for sLe(x) after a mechanical injury, we observed a reduction in EGFR phosphorylation and epithelial repair following injury. FucT-IV demonstrates a temporal expression coordinate with epithelial repair. Down-regulation of FucT-IV expression in NHBE by specific siRNA suppressed sLe(x) expression. The use of FucT-IV siRNA significantly reduced phosphorylation of EGFR and prevented epithelial repair. An immunohistochemical analysis of human normal and asthmatic airways showed a significant reduction in sLe(x) and tyrosine-phosphorylated EGFR (pY(845)-EGFR) in the epithelium of asthmatic subjects compared with that of normal subjects. The present data demonstrate that sLe(x), in association with EGFR, in NHBE is coordinate with repair. This glycosylation is important in modulating EGFR activity to affect the repair of normal primary AEC.

Galectins: regulators of acute and chronic inflammation

Galectins, beta-galactoside-binding animal lectins, are differentially expressed by various immune cells as well as a wide range of other cell types. Extracellularly, galectins are able to exhibit bivalent or multivalent interactions with cell-surface glycans on various immune cells and exert various effects. These include cytokine and mediator production, cell adhesion, apoptosis, and chemoattraction. In addition, they can form lattices with cell-surface glycoprotein receptors, resulting in modulation of receptor functions, including clustering and endocytosis. Intracellularly, galectins can participate in signaling pathways and modulate biologic responses. These include apoptosis, cell differentiation, and cell migration. Thus, a large body of literature indicates that galectins play important roles in the immune and inflammatory responses through regulating the homeostasis and functions of immune cells. The use of mice deficient in individual galectins has provided additional evidence for the contributions of these proteins to these responses. Current research indicates that galectins play important roles in the development of acute inflammation as well as chronic inflammation associated with allergies, autoimmune diseases, atherosclerosis, infectious processes, and cancer. Thus, recombinant proteins or specific galectin inhibitors may be used as therapeutic agents for inflammatory diseases.

The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+‐binding protein 2 modulates receptor function

Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

Special issue on neuron–glia interactions

Special issue on neuron–glia interactions

Features of Central Neurotransmission in Animals in Conditions of Dietary Magnesium Deficiency and After Its Correction

Magnesium is important in the regulation of neurotransmitter metabolism and the modulation of receptor function in the CNS, including neurotransmitters and receptors involved in the pathogenesis of many mental disorders. The aim of the present work was to perform a pharmacological evaluation of the central mechanisms of action of magnesium salts in the clofelin, phenamine, arecoline, nicotine, apomorphine, and 5-hydroxytryptophan tests in conditions of dietary magnesium deficiency. After reaching the magnesium deficiency state, animals were given oral (via tube) magnesium L-asparaginate and magnesium chloride lone and in combination with vitamin B(6), as well as the reference agent Magne B6. Our assessments of phenamine stereotypy in magnesium-deficient animals showed reductions in the latent period by an average of 14.89% and a significant increase in the duration of phenamine stereotypy by an average of 19.44% (from 268.23 +/- 8.17 to 320.36 +/- 19.90 min) as compared with intact rats. Studies of hyperkinesia induced by 5-hydroxytryptophan showed a two-fold reduction in its extent in the magnesium-deficient group (p </= 0.05). Administration of arecoline to magnesium-deficient animals resulted in a statistically significant increase in the latent period from a mean of 92.75 +/- 19.35 to 245.17 +/- 121.86 sec, with a reduction in the duration of tremor from an average of 1175.58 +/- 127.87 to 703.83 +/- 89.33 sec (p </= 0.05) as compared with intact rats. In terms of its influence on the hypothermic effects of clofelin and apomorphine and the convulsive effect of nicotine, there were no significant differences between the intact group and the magnesium-deficiency animals. Administration of magnesium salts compensated for the magnesium deficiency in plasma and erythrocytes, which was accompanied by recovery of measures in the phenamine, arecoline, and 5-HT tests to levels typical of intact controls. There was a tendency for magnesium L-asparaginate and magnesium chloride combined with pyridoxine to have greater activity, and the efficacies of these treatments was no less than that of reference agent Magne B6. Thus, dietary magnesium deficiency led to impairment of neurotransmission in central serotoninergic, M-cholinergic, and noradrenergic structures and administration of magnesium salts reversed these changes.

Crystal structure and association behaviour of the GluR2 amino-terminal domain

Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1- and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanisms by which the ATD guides subfamily-specific receptor assembly.

Investigation of human follicle stimulating hormone residency in membrane microdomains

The human follicle stimulating hormone receptor (hFSHR) is a G protein‐coupled receptor involved in reproduction in both males and females. hFSHR is involved in complex networks of signaling molecules beyond the G proteins and understanding the ability of the receptor to participate in many signaling pathways is of significant interest for those looking to modulate receptor function. One possibility is that microdomains in the plasma membrane, such as lipid rafts, play an important role in signal transduction. The aim of this study was to determine the role of plasma membrane microdomains in hFSHR function. To analyze this, lipid rafts were disrupted using the cholesterol withdrawing agent methyl‐β ‐cylcodextrin (MβCD) and hFSH activation of MAP kinase pathways was investigated. Removal of membrane cholesterol caused the loss of hFSH induced p38‐MAP kinase activation but did not alter p42/44 MAP kinase activation. To investigate further the residency of hFSHR in lipid rafts, co‐immunoprecipitation experiments were performed to determine if caveolin‐1 interacted with hFSHR. Caveolin‐1 was overexpressed in cells stably transfected with hFSHR. A monoclonal antibody against hFSHR was used to immunoprecipitate the receptor and caveolin‐1 was shown to co‐immunoprecipitate. Although the physiological role of this interaction has not been determined, it presents a new opportunity to study hFSHR function.

Modulation of receptor sensitivity: possible therapeutic target?

Ischaemic preconditioning and post-conditioning are cardioprotective interventions that salvage ischaemic myocardium and reduce infarct size. Yet this cardioprotective effect is not the sole response of the heart to ischaemic preconditioning and post-conditioning. It was known that protein kinase C activation in the signalling cascade of ischaemic preconditioning increased the affinity of the adenosine A(2b) receptor so that much lower concentrations of adenosine caused A(2b) receptor-dependent signalling. In this issue of the British Journal of Pharmacology, these cardioprotective interventions are shown to block desensitization of surface receptors on the sarcolemma of the cardiomyocyte and this receptor effect is divorced from any cardioprotection. Modulating receptor function through signalling pathways is a novel idea but, currently, whether these observations have any clinical relevance is not known. Additional investigations are warranted to determine whether this effect on receptors can be generalized to other surface receptors, and whether the effect can be harnessed to improve treatment of the patient with acute myocardial infarction.

Colchicine: A Novel Positive Allosteric Modulator of the Human 5-Hydroxytryptamine3AReceptor

The actions of colchicine were examined with the two-electrode voltage-clamp technique and radioligand binding assays in mouse and human 5-hydroxytryptamine(3A) receptors (5-HT(3A)Rs) expressed in Xenopus laevis oocytes. Colchicine inhibited 5-hydroxytryptamine (5-HT)-evoked currents in oocytes expressing mouse 5-HT(3A)Rs, with an IC(50) of 59.5 +/- 3 microM. In contrast to the mouse receptor, coapplication of colchicine with 5-HT (<1 microM) strongly enhanced 5-HT-evoked currents in oocytes expressing human 5-HT(3A)Rs. Colchicine applied alone did not induce a detectable current. In the presence of 0.5 microM 5-HT, the potentiation was concentration-dependent and reached the maximum (approximately 100%) when 750 microM colchicine was applied. However, colchicine-dependent inhibition can be observed at 5-HT concentrations > 1 microM. In oocyte membranes expressing mouse or human receptors, binding studies with colchicine (25 nM-1 mM) revealed no displacement of 1-methyl-N-((1R,3r,5S)-9-methyl-9 azabicyclo [3.3.1]nonan-3yl)-1H-indazole-3 carboxamide ([(3)H]BRL-43694), suggesting that actions of colchicine do not occur at the ligand binding domain. Functional effects of colchicine on both receptors occurred in the absence of preincubation and after cold temperature incubation, suggesting that the microtubule-depolymerizing effects of colchicine play no role in modulation of receptor function. Studies with interspecies chimeric receptors demonstrated that the distal one third of the N terminus is responsible for the bidirectional modulation by colchicine. Collectively, these results suggest that colchicine modulates receptor function through loops C and/or F through a gating mechanism.

Investigations of Ligand-gated Ion Channels Using Caged Neurotransmitters Reveal Mechanism-based Approaches to Modulating Receptor Function

Recognizing the problem of rapidly equilibrating membrane-bound proteins on cell surfaces with activating ligands due to the water layer that tightly adheres to the surface of spherical objects (1), we began to develop caged (2) neurotransmitters (3,4). A caged neurotransmitter can be equilibrated with receptors on a cell surface before photolysis releases the neurotransmitter in the microsecond time region (3,4). Using caged neurotransmitters in whole-cell current recordings enables the rate constants for the opening (kop) and closing (kcl) of the receptor channel, and thus the channel-opening equilibrium constant (Phe−1), to be measured without interference from diffusional delays or receptor desensitization.The rate constants and the channel-opening equilibrium constant of the excitatory nicotinic acetylcholine receptor (5) are altered in the presence of noncompetitive inhibitors, such as cocaine, as are those of a mutated inhibitory gamma-aminobutyric acid (GABA) receptor (6) linked to epilepsy (7). Based on the mechanisms of the two receptors, we developed compounds that bind with higher affinity to the open-channel than to the closed-channel conformation of each receptor, alleviating (8,9) the inhibition of the acetylcholine receptor and potentiating (10) the function of a mutated GABAA receptor linked to epilepsy.1. Landau & Lifshitz (1959) Fluid Mechanics (Pergamon, Oxford)2. Kaplan et al. (1978) Biochemistry 17, 19293. Milburn et al. (1989) Biochemistry 28, 514. Shembekar et al. (2007) Biochemistry 46, 54795. Hess et al. (2000) Proc. Natl. Acad. Sci. USA 97, 138956. Ramakrishnan. & Hess (2004) Biochemistry 43, 75347. Baulac et al. (2001) Nature Genetics 28, 468. Hess et al. (2003) Biochemistry 42, 61069. Chen et al. (2004) Biochemistry 43, 1014910. Krivoshein & Hess (2006) Biochemistry 45, 11632

Modulation of the Dimer Interface at Ionotropic Glutamate-Like Receptor δ2 by d-Serine and Extracellular Calcium

GluRdelta2 is a member of the iGluR family, but despite a prominent role in cerebellar synaptic plasticity, this receptor does not appear to function as an ion channel. Endogenous ligands that modulate the activity of native GluRdelta2 in the cerebellum have not been identified, but two candidate modulators are d-serine and extracellular calcium. Taking advantage of known crystal structures and spontaneously active GluRdelta2 receptors containing the lurcher mutation (GluRdelta2(Lc)), we investigated the mechanism by which calcium and d-serine regulate the activity of GluRdelta2(Lc). Our data suggest that calcium binding stabilizes the dimer interface formed between two agonist-binding domains and increases GluRdelta2(Lc) currents. The data further suggest that d-serine binding induces rearrangements at the dimer interface to diminish GluRdelta2(Lc) currents by a mechanism that resembles desensitization at AMPA and kainate receptors. Thus, we propose that calcium and d-serine binding have opposing effects on the stability of the dimer interface. Furthermore, the effects of calcium are observed at concentrations that are within the physiological range, suggesting that the ability of native GluRdelta2 to respond to ligand binding may be modulated by extracellular calcium. These findings place GluRdelta2 among AMPA and kainate receptors, where the dimer interface is not only a biologically important site for functional regulation, but also an important target for exogenous and endogenous ligands that modulate receptor function.

Modulating receptor function through RAMPs: can they represent drug targets in themselves?

G protein-coupled receptors (GPCRs) are successfully exploited as drug targets. As our understanding of how distinct GPCR subtypes can be generated expands, so do possibilities for therapeutic intervention via these receptors. Receptor activity-modifying proteins (RAMPs) are excellent examples of proteins that enhance diversity in GPCR function. They facilitate the creation of binding pockets, controlling the pharmacology of some GPCRs. Moreover, they have the ability to regulate cell-surface trafficking, internalisation and signalling of GPCRs, creating novel opportunities for drug discovery. RAMPs could be directly targeted by drugs, or advantage could be taken of unique RAMP/GPCR interfaces for generating highly selective ligands.