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The role of receptor activity‐modifying proteins (RAMPs) in modulating the pharmacological effects of an amylin receptor selective agonist (NN1213) or the dual amylin–calcitonin receptor agonist (DACRA), salmon calcitonin (sCT), was tested in three RAMP KO mouse models, RAMP1, RAMP3 and RAMP1/3 KO. Male wild‐type (WT) and knockout (KO) littermate mice were fed a 45% high‐fat diet for 20 weeks prior to the 3‐week treatment period. A decrease in body weight after NN1213 was observed in all WT mice, whereas sCT had no effect. The absence of RAMP1 had no significant effect on NN1213 efficacy, and sCT was still inactive. However, the absence of RAMP3 impeded NN1213 efficacy but improved sCT efficacy. Similar results were observed in RAMP1/3 KO suggesting that the amylin receptor 3 (AMY3 = CTR + RAMP3) is necessary for NN1213's maximal action on body weight and food intake and that the lack of AMY3 allowed sCT to be active. These results suggest that the chronic use of DACRA such as sCT can have unfavourable effect on body weight loss in mice (which differs from the situation in rats), whereas the use of the amylin receptor selective agonist does not. AMY3 seems to play a crucial role in modulating the action of these two compounds, but in opposite directions. The assessment of a long‐term effect of amylin and DACRA in different rodent models is necessary to understand potential physiological beneficial and unfavourable effects on weight loss before its transition to clinical trials., AMY3 mediates the effect of the dual amylin receptor agonist NN1213.Subchronic sCT does not reduce body weight in WT mice.Absence of AMY3 allows sCT to be active.

Detection and transduction of environmental signals, constitute a prerequisite for successful parasite invasion; i.e., Leishmania transmission, survival, pathogenesis and disease manifestation and dissemination, with diverse molecules functioning as inter-cellular signaling ligands. Receptors [i.e., G protein-coupled receptors (GPCRs)] and their associated transduction mechanisms, well conserved through evolution, specialize in this function. However, canonical GPCR-related signal transduction systems have not been described in Leishmania, although orthologs, with reduced domains and function, have been identified in Trypanosomatidae. These inter-cellular communication means seem to be essential for multicellular and unicellular organism’s survival. GPCRs are flexible in their molecular architecture and may interact with the so-called receptor activity-modifying proteins (RAMPs), which modulate their function, changing GPCRs pharmacology, acting as chaperones and regulating signaling and/or trafficking in a receptor-dependent manner. In the skin, vasoactive- and neuro- peptides released in response to the noxious stimuli represented by the insect bite may trigger parasite physiological responses, for example, chemotaxis. For instance, in Leishmania (V.) braziliensis, sensory [Substance P, SP, chemoattractant] and autonomic [Vasoactive Intestinal Peptide, VIP, and Neuropeptide Y, NPY, chemorepellent] neuropeptides at physiological levels stimulate in vitro effects on parasite taxis. VIP and NPY chemotactic effects are impaired by their corresponding receptor antagonists, suggesting that the stimulated responses might be mediated by putative GPCRs (with essential conserved receptor domains); the effect of SP is blocked by [(D-Pro 2, D-Trp7,9]-Substance P (10-6 M)] suggesting that it might be mediated by neurokinin-1 transmembrane receptors. Additionally, vasoactive molecules like Calcitonin Gene-Related Peptide [CGRP] and Adrenomedullin [AM], exert a chemorepellent effect and increase the expression of a 24 kDa band recognized in western blot analysis by (human-)-RAMP-2 antibodies. In-silico search oriented towards GPCRs-like receptors and signaling cascades detected a RAMP-2-aligned sequence corresponding to Leishmania folylpolyglutamate synthase and a RAMP-3 aligned protein, a hypothetical Leishmania protein with yet unknown function, suggesting that in Leishmania, CGRP and AM activities may be modulated by RAMP- (-2) and (-3) homologs. The possible presence of proteins and molecules potentially involved in GPCRs cascades, i.e., RAMPs, signpost conservation of ancient signaling systems associated with responses, fundamental for cell survival, (i.e., taxis and migration) and may constitute an open field for description of pharmacophores against Leishmania parasites.

Receptor activity-modifying proteins (RAMPs) interact with G-protein-coupled receptors (GPCRs) to modify their functions, imparting significant implications upon their physiological and therapeutic potentials. A resurging interest in identifying RAMP-GPCR interactions has recently been fueled by coevolution studies and orthogonal technological screening platforms. These new studies reveal previously unrecognized RAMP-interacting GPCRs, many of which expand beyond Class B GPCRs. The consequences of these interactions on GPCR function and physiology lays the foundation for new molecular therapeutic targets, as evidenced by the recent success of Erenumab. Here, we highlight recent papers that uncovered novel RAMP-GPCR interactions, human RAMP-GPCR disease-causing mutations, and RAMP-related human pathologies, paving the way for a new era of RAMP-targeted drug development.

Knockout technologies provide insights into physiological roles of genes. Studies initiated into endocrinology of heteromeric G protein-coupled receptors included deletion of receptor activity modifying protein-3, an accessory protein that alters ligand selectivity of calcitonin and calcitonin-like receptors. Initially, deletion of Ramp3-/- appeared phenotypically silent, but it has emerged that mice have a high bone mass phenotype, and more subtle alterations to angiogenesis, amylin homeostasis, and a small proportion of the effects of adrenomedullin on cardiovascular and lymphatic systems. Here we explore in detail, effects of Ramp3-/- deletion on skeletal growth/development, bone mass and response of bone to mechanical loading mimicking exercise. Mouse pups lacking RAMP3 are healthy and viable, having accelerated development of the skeleton as assessed by degree of mineralisation of specific bones, and by microCT measurements. Specifically, we observed that neonates and young mice have increased bone volume and mineralisation in hindlimbs and vertebrae and increased thickness of bone trabeculae. These changes are associated with increased osteoblast numbers and bone apposition rate in Ramp3-/- mice, and increased cell proliferation in epiphyseal growth plates. Effects persist for some weeks after birth, but differences in gross bone mass between RAMP3 and WT mice lose significance in older animals although architectural differences persist. Responses of bones of 17-week old mice to mechanical loading that mimics effects of vigorous exercise is increased significantly in Ramp3-/- mice by 30% compared with WT control mice. Studies on cultured osteoblasts from Ramp3-/- mice indicate interactions between mRNA expression of RAMPs1 and 3, but not RAMP2 and 3. Our preliminary data shows that Ramp3-/- osteoblasts had increased expression β-catenin, a component of the canonical Wnt signalling pathway known to regulate skeletal homeostasis and mechanosensitivity. Given interactions of RAMPs with both calcitonin and calcitonin-like receptors to alter ligand selectivity, and with other GPCRs to change trafficking or ligand bias, it is not clear whether the bone phenotype of Ramp3-/- mice is due to alterations in signalling mediated by one or more GPCRS. However, as antagonists of RAMP-interacting receptors are growing in availability, there appears the likelihood that manipulation of the RAMP3 signalling system could provide anabolic effects therapeutically.

OBJECTIVE: To study the role of adrenomedullin (ADM) in hyperoxia-induced lung injury by examining the effect of ADM on the expression of calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 2 (RAMP2), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB) in human pulmonary microvascular endothelial cells (HPMECs) under different experimental conditions. METHODS: HPMECs were randomly divided into an air group and a hyperoxia group (n=3 each).The HPMECs in the hyperoxia group were cultured in an atmosphere of 92% O(2 )(3 L/minute) +5% CO(2). RT-qPCR and Western blot were used to measure the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB. Other HPMECs were divided into a non-interference group and an interference group (n=3 each), and the mRNA and protein expression levels of ADM, ERK1/2, and PKB were measured after the HPMECs in the interference group were transfected with ADM siRNA. RESULTS: Compared with the air group, the hyperoxia group had significant increases in the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB (P

Adrenomedullin (AM), a member of the calcitonin peptide superfamily, is a peptide involved in both the pathogenesis of cardiovascular diseases and circulatory homeostasis. Its receptor, calcitonin receptor-like receptor (CLR), associates with an accessory protein, receptor activity-modifying protein (RAMP). Depending upon which the three RAMP isoforms (RAMP1-3) it interacts with, CLR functions as a receptor for AM or other calcitonin family peptides. AM knockout mice (-/-) died mid-gestation due to abnormalities in vascular development. We found that phenotypes similar to AM-/- were reproduced only in RAMP2-/- mice. We generated endothelial cell-specific RAMP2 knockout mice (E-RAMP2-/-) and found most E-RAMP2-/- mice died perinatally. In surviving adults, vasculitis and organ fibrosis occurred spontaneously. We next generated drug-inducible cardiac myocyte-specific RAMP2-/- (DI-C-RAMP2-/-) mice, which exhibited dilated cardiomyopathy-like heart failure with cardiac dilatation and myofibril disruption. DI-C-RAMP2-/- hearts also showed changes in mitochondrial structure and downregulation of mitochondria-related genes involved in oxidative phosphorylation and β-oxidation. In contrast to RAMP2-/- mice, RAMP3-/- mice were born with no major abnormalities. In adult RAMP3-/- mice, postnatal angiogenesis was normal, but drainage of subcutaneous lymphatic vessels was delayed. RAMP3-/- mice also showed more severe interstitial edema than in wild-type mice in a tail lymphedema model. These findings show that the AM-RAMP system is a key determinant of cardiovascular integrity and homeostasis from prenatal stages through adulthood. The AM-RAMP2 system mainly regulates vascular development and homeostasis, while the AM-RAMP3 system mainly regulates lymphatic function in adults. The AM-RAMP system may thus have therapeutic potential for the treatment of cardiovascular diseases.

Objective Melanocortin 2 receptor accessory protein 2 (MRAP2) has a critical role in energy homeostasis. Although MRAP2 has been shown to regulates a number of GPCRs involved in metabolism, the key neurons responsible for the phenotype of gross obesity in MRAP2 deficient animals are unclear. Furthermore, to date, all the murine MRAP2 models involve the prenatal deletion of MRAP2. Methods To target Melanocortin 4 receptor (MC4R)-expressing neurons in the hypothalamic paraventricular nucleus (PVN), we performed stereotaxic surgery using AAV to selectively overexpress MRAP2 postnatally in adult Mc4r-cre mice. We assessed energy homeostasis, glucose metabolism, core body temperature, and response to MC3R/MC4R agonist MTII. Results Mc4r-crePVN-MRAP2 female mice on a standard chow diet had less age-related weight gain and improved glucose/insulin profile compared to control Mc4r-crePVN-GFP mice. These changes were associated with a reduction in food intake and increased energy expenditure. In contrast, Mc4r-crePVN-MRAP2 male mice showed no improvement on a chow diet, but improvement of energy and glucose metabolism was observed following high fat diet (HFD) feeding. In addition, an increase in core body temperature was found in both females fed on standard chow diet and males fed on HFD. Mc4r-crePVN-MRAP2 female and male mice showed increased neuronal activation in the PVN compared to controls, with further increase in neuronal activation post MTII treatment in females. Conclusions Our data indicate a site-specific role for MRAP2 in PVN MC4R-expressing neurons in potentiating MC4R neuronal activation at baseline conditions in the regulation of food intake and energy expenditure., Highlights • Postnatal overexpression of MRAP2 regulates energy balance, thermogenesis and glucose metabolism. • Overexpression of MRAP2 in MC4R expressing neurons increases PVN neuronal activation. • There is a sex difference in extent of metabolic protection, with a more marked lean phenotype in females.

Antagonists of the P2X7 receptor (P2X7R) have the potential to treat diseases where neuroinflammation is present such as depression, chronic pain and Alzheimer's disease. We recently developed a structural hybrid (C1; 1-((adamantan-1-yl)methyl)-2-cyano-3-(quinolin-5-yl)guanidine) of a purported competitive P2X7R antagonist (C2; 2-cyano-1-((1S)-1-phenylethyl)-3-(quinolin-5-yl)guanidine) and a likely negative allosteric modulator (NAM) of the P2X7R (C3; N-((adamantan-1-yl)methyl)-2-chloro-5-methoxybenzamide). Here we aimed to pharmacologically characterize C1, to gain insights into how select structural components impact antagonist interaction with the P2X7R. A second aim was to examine the role of the peptide LL-37, an apparent activator of the P2X7R, and compare the ability of multiple P2X7R antagonists to block its effects. Compounds 1, 2 and 3 were characterised using washout, Schild and receptor protection studies, all using dye uptake assays in HEK293 cells expressing the P2X7R. LL-37 was examined in the same HEK293 cells and THP-1 monocytes. Compounds 2 and 3 acted as a BzATP-competitive antagonist and NAM of the P2X7R respectively. Compound 1 was a slowly reversible NAM of the P2X7R suggesting the incorporation of an appropriately positioned adamantane promotes binding to the allosteric site of the P2X7R. LL-37 was shown to potentiate the ability of ATP to induce dye uptake at low concentrations (1–3 μg mL-1) or induce dye uptake alone at higher concentrations (10–20 μg mL-1). None of the P2X7R antagonists studied were able to block LL-37-induced dye uptake bringing in to question the ability of current P2X7R antagonists to inhibit the inflammatory action of LL-37 in vivo.

Adrenomedullin (AM) is a peptide hormone with multiple physiological functions, which are regulated by its receptor activity–modifying proteins, RAMP2 and RAMP3. We previously reported that AM or RAMP2 knockout (KO) (AM–/–, RAMP2–/–) is embryonically lethal in mice, whereas RAMP3–/– mice are apparently normal. AM, RAMP2, and RAMP3 are all highly expressed in the heart; however, their functions there are not fully understood. Here, we analyzed the pathophysiological functions of the AM-RAMP2 and AM-RAMP3 systems in hearts subjected to cardiovascular stress. Cardiomyocyte-specific RAMP2–/– (C-RAMP2–/–) and RAMP3–/– showed no apparent heart failure at base line. After 1 week of transverse aortic constriction (TAC), however, C-RAMP2–/– exhibited significant cardiac hypertrophy, decreased ejection fraction, and increased fibrosis compared with wild-type mice. Both dP/dtmax and dP/dtmin were significantly reduced in C-RAMP2–/–, indicating reduced ventricular contractility and relaxation. Exposing C-RAMP2–/– cardiomyocytes to isoproterenol enhanced their hypertrophy and oxidative stress compared with wild-type cells. C-RAMP2–/– cardiomyocytes also contained fewer viable mitochondria and showed reduced mitochondrial membrane potential and respiratory capacity. RAMP3–/– also showed reduced systolic function and enhanced fibrosis after TAC, but those only became apparent after 4 weeks. A reduction in cardiac lymphatic vessels was the characteristic feature in RAMP3–/–. These observations indicate the AM-RAMP2 system is necessary for early adaptation to cardiovascular stress through regulation of cardiac mitochondria. AM-RAMP3 is necessary for later adaptation through regulation of lymphatic vessels. The AM-RAMP2 and AM-RAMP3 systems thus play separate critical roles in the maintenance of cardiovascular homeostasis against cardiovascular stress.

Receptor activity-modifying proteins (RAMPs) are a family of three single span transmembrane proteins in humans that interact with many GPCRs and can modulate their function. RAMPs were discovered as key components of the calcitonin gene-related peptide and adrenomedullin receptors. They are required for transport of this class B GPCR, calcitonin receptor-like receptor (CLR), to the cell surface and determine its peptide ligand binding preferences. Soon thereafter RAMPs were shown to modulate the binding of calcitonin and amylin peptides to the related calcitonin receptor (CTR) and in the years since an ever-growing number of RAMP-interacting receptors have been identified including most if not all of the fifteen class B GPCRs and several GPCRs from other families. Studies of CLR, CTR, and a handful of other GPCRs revealed that RAMPs are able to modulate various aspects of receptor function including trafficking, ligand binding, and signaling. Here, we review RAMP interactions and functions with an emphasis on class B receptors for which our understanding is most advanced. A key focus is to discuss recent evidence that RAMPs serve as endogenous allosteric modulators of CLR and CTR. We discuss structural studies of RAMP-CLR complexes and CTR and biochemical and pharmacological studies that collectively have significantly expanded our understanding of the mechanistic basis for RAMP modulation of these class B GPCRs. Last, we consider the implications of these findings for drug development targeting RAMP-CLR/CTR complexes.

The melanocortin-4 receptor (MC4R) is a class A G protein-coupled receptor (GPCR), essential for regulation of appetite and metabolism. Pathogenic inactivating MC4R mutations are the most frequent cause of monogenic obesity, a growing medical and socioeconomic problem worldwide. The MC4R mediates either ligand-independent or ligand-dependent signaling. Agonists such as α-melanocyte-stimulating hormone (α-MSH) induce anorexigenic effects, in contrast to the endogenous inverse agonist agouti-related peptide (AgRP), which causes orexigenic effects by suppressing high basal signaling activity. Agonist action triggers the binding of different subtypes of G proteins and arrestins, leading to concomitant induction of diverse intracellular signaling cascades. An increasing number of experimental studies have unraveled molecular properties and mechanisms of MC4R signal transduction related to physiological and pathophysiological aspects. In addition, the MC4R crystal structure was recently determined at 2.75 Å resolution in an inactive state bound with a peptide antagonist. Underpinned by structural homology models of MC4R complexes simulating a presumably active-state conformation compared to the structure of the inactive state, we here briefly summarize the current understanding and key players involved in the MC4R switching process between different activity states. Finally, these perspectives highlight the complexity and plasticity in MC4R signaling regulation and identify gaps in our current knowledge.

The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor–like class B G protein–coupled receptor (CLR) and a variable receptor activity–modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM(1) (RAMP2:CLR) and AM(2) (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP–CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM(2) receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment–bound RAMP1–CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple β-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM(1) and AM(2) receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

The calcitonin receptor-like receptor (CLR) is a class B G protein-coupled receptor (GPCR) that forms the basis of three pharmacologically distinct receptors, the calcitonin gene-related peptide (CGRP) receptor, and two adrenomedullin (AM) receptors. These three receptors are created by CLR interacting with three receptor activity-modifying proteins (RAMPs). Class B GPCRs have an N-terminal extracellular domain (ECD) and transmembrane bundle that are both important for binding endogenous ligands. These two domains are joined together by a stretch of amino acids that is referred to as the "stalk". Studies of other class B GPCRs suggest that the stalk may act as hinge, allowing the ECD to adopt multiple conformations. It is unclear what the role of the stalk is within CLR and whether RAMPs can influence its function. Therefore, this study investigated the role of this region using an alanine scan. Effects of mutations were measured with all three RAMPs through cell surface expression, cAMP production and, in select cases, radioligand binding and total cell expression assays. Most mutants did not affect expression or cAMP signaling. CLR C127A, N140A, F142A, and L144A impaired cell surface expression with all three RAMPs. T125A decreased the potency of all peptides at all receptors. N128A, V135A, and L139A showed ligand-dependent effects. While the stalk appears to play a role in CLR function, the effect of RAMPs on this region seems limited, in contrast to their effects on the structure of CLR in other receptor regions.

Background The pancreatic- and brain-derived hormone amylin promotes negative energy balance and is receiving increasing attention as a promising obesity therapeutic. However, the neurobiological substrates mediating amylin's effects are not fully characterized. We postulated that amylin acts in the lateral dorsal tegmental nucleus (LDTg), an understudied neural processing hub for reward and homeostatic feeding signals. Methods We used immunohistochemical and quantitative polymerase chain reaction analyses to examine expression of the amylin receptor complex in rat LDTg tissue. Behavioral experiments were performed to examine the mechanisms underlying the hypophagic effects of amylin receptor activation in the LDTg. Results Immunohistochemical and quantitative polymerase chain reaction analyses show expression of the amylin receptor complex in the LDTg. Activation of LDTg amylin receptors by the agonist salmon calcitonin dose-dependently reduces body weight, food intake, and motivated feeding behaviors. Acute pharmacological studies and longer-term adeno-associated viral knockdown experiments indicate that LDTg amylin receptor signaling is physiologically and potentially preclinically relevant for energy balance control. Finally, immunohistochemical data indicate that LDTg amylin receptors are expressed on gamma-aminobutyric acidergic neurons, and behavioral results suggest that local gamma-aminobutyric acid receptor signaling mediates the hypophagia after LDTg amylin receptor activation. Conclusions These findings identify the LDTg as a novel nucleus with therapeutic potential in mediating amylin's effects on energy balance through gamma-aminobutyric acid receptor signaling.

Ghrelin is the only known circulating orexigenic hormone. It is primarily secreted by the stomach and acts at its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), in the hypothalamus to signal hunger and promote food intake. The melanocortin receptor accessory protein 2 (MRAP2) was previously shown to regulate energy homeostasis through the modulation of the activity of the melanocortin-4 receptor and prokineticin receptors. In this study we identify MRAP2 as a partner of ghrelin-GHSR1a signaling. We show that MRAP2 interacts with GHSR1a and potentiates ghrelin-stimulated signaling both in vitro and in vivo. We demonstrate that in the absence of MRAP2, fasting fails to activate agouti-related protein neurons. In addition, we show that the orexigenic effect of ghrelin is lost in mice lacking MRAP2. Our results suggest that MRAP2 is an important modulator of the energy homeostasis machinery that operates through the regulation of multiple GPCRs throughout the hypothalamus., Melanocortin receptor accessory protein 2 (MRAP2) is an adaptor protein that contributes to melanocortin-4 receptor and prokineticin receptor 1 signalling. Here the authors show that MRAP2 also regulates ghrelin receptor signalling in the hypothalamus and starvation sensing in mice.

Unidirectional flow of oviductal fluid from the ovarian to uterine side of the ampulla plays a significant role in successful pregnancy, and is produced by ciliary beating. Various systems regulate ciliary beating, such as paracrine, autocrine, and endocrine. We hypothesized that Adrenomedullin (ADM)-a peptide hormone that acts via its receptors, which are complexes of Calcitonin receptor-like receptor (CRLR) and Receptor activity-modifying protein (RAMP) 2 or 3 - promotes oviductal fluid flow in the ampulla of bovine oviducts. First, we examined the expression of ADM, CRLR, RAMP2, and RAMP3 mRNAs in isolated epithelial cells throughout the estrous cycle, and the localization of ADM receptor protein constituents in the ampulla. RAMP2 expression was significantly higher in the follicular phase. Furthermore, RAMP2 protein was detected only in ciliated cells, whereas CRLR and RAMP3 were detected in all epithelial cells. The effects of ADM and an ADM antagonist on fluid-flow speed were examined using microbeads in ampullary tissue. ADM antagonist decreased bead transport speed, and this decrease was reversed by ADM. In addition, ADM recovered the bead transport speed that decreased in the absence of calcium. Overall, our results suggest that ADM contributes to the regulation of oviductal fluid flow in ampulla.

We used a multiplexed suspension bead array immunoassay to map GPCR-receptor activity–modifying protein (RAMP) complexes., Receptor activity–modifying proteins (RAMPs) have been shown to modulate the functions of several G protein–coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

Autocommentary to: Cottrell GS, Soubrane CH, Hounshell JA, Lin H, Owenson V, Rigby M, Cox PJ, Barker BS, Ottolini M, Ince S, Bauer, CC, Perez-Reyes E, Patel MK, Stevens EB, Stephens GJ (2018) CACHD1 is an 2-like protein that modulates CaV3 voltage-gated calcium channel activity J Neurosci 38:9186-9201.

The calcitonin gene-related peptide (CGRP) family of neuropeptides, consists of CGRP, adrenomedullin, amylin, and calcitonin. The receptors consist of either calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) which for function needs an accessory protein, receptor activity-modifying proteins (RAMPs). CGRP has a pivotal role in primary headaches but the role of the other members of the CGRP family of peptides in headaches is not known. Here, we describe the expression of these molecules in the trigeminal ganglion (TG) to understand more on their possible role(s). Single or double immunohistochemistry were applied on frozen sections of rat TG using primary antibodies against CGRP, procalcitonin, calcitonin, adrenomedullin, amylin, RAMP1/2/3, CLR, and CTR. In addition, mRNA expression was measured by quantitative qPCR on TGs. CGRP and calcitonin showed rich expression in the cytoplasm of small to medium-sized neurons, and co-localized sometimes. Procalcitonin was observed in the glial cells. Immunoreactive fibers storing both CGRP and calcitonin were also observed. Adrenomedullin immunoreactivity was found in the satellite glial cells and in fibers, probably the myelinating Schwann cells. Amylin was found in the cytoplasm in many TG neurons. Levels of mRNA expression for adrenomedullin, amylin, CLR, RAMP1, RAMP2, RAMP3, and CTR were measured using qPCR. The experiments verified the expression of mRNA in the TG with the exception of CTR, which was above the limit of detection indicating little or no mRNA expression. In addition to the well-known CGRP receptor (CLR/RAMP1) and the receptor for calcitonin—CTR, we propose that other receptors exist in the rat TG: adrenomedullin receptor AM2 (CLR/RAMP3) in mainly the satellite glial cells, amylin receptors AMY1 (CTR/RAMP1) in mainly neurons, and AMY3 (CTR/RAMP3) in the satellite glial cells. It is important to compare peptides and receptors side-by-side in studies to help address questions of actions resulting from cross-reactivity between receptors. Several of the diverse biological actions of the CGRP family of peptides are clinically relevant. Our findings demonstrate the specific ligand and receptor sites in the rat trigeminal ganglion, highlighting recognition mechanisms to facilitate drug development.

Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR–RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine.

Receptor activity modifying proteins (RAMPs) associate with G-protein-coupled receptors (GPCRs) at the plasma membrane and together bind a variety of peptide ligands, serving as a communication interface between the extracellular and intracellular environments. The collection of RAMP-interacting GPCRs continues to expand and now consists of GPCRs from families A, B, and C, suggesting that RAMP activity is extremely prevalent. RAMP association with GPCRs can regulate GPCR function by altering ligand binding, receptor trafficking and desensitization, and downstream signaling pathways. Here, we elaborate on these RAMP-dependent mechanisms of GPCR regulation, which provide opportunities for pharmacological intervention.

Adrenomedullin (AM) and their receptor components, calcitonin-receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP1, RMP2 and RAMP3) are widely expressed in the central nervous system, including cerebellum. We have shown that AM binding sites are altered in cerebellum during hypertension, suggesting a role for cerebellar adrenomedullinergic system in blood pressure regulation. To further evaluate the role of AM in cerebellum, we assessed the expression of AM, RAMP1, RAMP2, RAMP3 and CRLR in the cerebellar vermis of 8 and 16week old spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. In addition, the effect of microinjection of AM into rat cerebellar vermis on arterial blood pressure (BP) was determined. Animals were sacrificed by decapitation and cerebellar vermis was dissected for quantification of AM, CRLR, RAMP1, RAMP2 and RAMP3 expression using western blot analysis. Another group of male, 16week old SHR and WKY rats was anesthetized, and a cannula was implanted in the cerebellar vermis. Following recovery AM (0.02 to 200pmol/5μL) or vehicle was injected into cerebellar vermis. BP was determined, before and after treatments, by non-invasive plethysmography. In addition, to establish the receptor subtype involved in AM action in vivo, animals received microinjections of AM22-52 (200pmol/5μL), an AM1 receptor antagonist, or the CGRP1 receptor antagonist, CGRP8-37 (200pmol/5μL) into the cerebellar vermis, administered simultaneously with AM or vehicle microinjection. Cannulation was verified post mortem with the in situ injection of a dye solution. Our findings demonstrated that the expression of CRLR, RAMP1 and RAMP3 was higher in cerebellum of SHR rats, while AM and RAMP2 expression was lower than those of WKY rats, both in 8 and 16week old rats. In vivo microinjection of AM into the cerebellar vermis caused a profound, dose dependent, hypotensive effect in SHR but not in normotensive WKY rats. Coinjections of a putative AM receptor antagonist, AM22-52 abolished the decreases in mean arterial pressure (MAP) evoked by AM, showing that AM acts through its AM1 receptor in the vermis to reduce MAP. These findings demonstrate a dysregulation of cerebellar AM-system during hypertension, and suggest that cerebellar AM plays an important role in the regulation of BP. Likewise; they constitute a novel mechanism of BP control which has not been described so far.

Cadmium is an endocrine disruptor and inhibits corticosteroid production, but the mechanisms are far from clear. We tested the hypothesis that sublethal exposure to environmentally realistic levels of cadmium impairs cortisol production by disrupting the melanocortin 2 receptor (MC2R) signaling in rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of cadmium (0.75 or 2.0 μg/L) in a flow-through system for 7 d and subjected to an acute secondary stressor to evoke a cortisol response. Cadmium exposure for 7 d did not affect plasma cortisol concentrations, but head kidney mc2r mRNA levels were higher than in control fish. The cortisol stress performance to a secondary-stressor was attenuated in the cadmium groups, and this corresponded with transient reduction in transcript abundance of mc2r and the gene encoding its accessory protein MRAP1 but not MRAP2 in the head kidney. Furthermore, in vivo cadmium exposure attenuated the adrenocorticotropic hormone (ACTH)-, but not 8-br-cAMP-stimulated cortisol production in head kidney slices ex vivo. This corresponded with reduced transcript abundance of mc2r and mrap1, but not mrap2 in these tissue slices. Also, reporter assays with CHO cells transiently transfected with rainbow trout mc2r and zebrafish mrap1 revealed a dose-independent inhibition in ACTH-stimulated luciferase activity by cadmium. Collectively, waterborne exposure to environmentally realistic concentration of cadmium compromises the stressor-induced cortisol response, and a mode of action involves the disruption of MC2R signaling in rainbow trout.

Melanocortin 4 receptor (MC4R), which is a member of the G protein-coupled receptor (GPCR) family, mediates regulation of energy homeostasis upon the binding of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS). Melanocortin 2 receptor accessory protein 2 (MRAP2) modulates the function of MC4R. We performed cDNA cloning of cat MC4R and MRAP2 and characterized their amino acid sequences, mRNA expression patterns in cat tissues, protein–protein interactions, and functions. We found high sequence homology (>88%) with other mammalian MC4R and MRAP2 encoding 332 and 206 amino acid residues, respectively. Reverse transcription-polymerase chain reaction analysis revealed that cat MC4R and MRAP2 mRNA were expressed highly in the CNS. In CHO-K1 cells transfected with cat MC4R, stimulation with α-MSH increased intracellular cyclic adenosine monophosphate (cAMP) concentration in a dose-dependent manner. Furthermore, the presence of MRAP2 enhanced the cat MC4R-mediated cAMP production. These results suggested that cat MC4R acts as a neuronal mediator in the CNS and that its function is modulated by MRAP2. In addition, our NanoBiT study showed the dynamics of their interactions in living cells; stimulation with α-MSH slightly affected the interaction between MC4R and MRAP2, and did not affect MC4R homodimerization, suggesting that they interact in the basal state and that structural change of MC4R by activation may affect the interaction between MC4R and MRAP2.

Graphical abstract, Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs.

Angiotensin II (AngII)-activated epidermal growth factor receptor has been implicated in abdominal aortic aneurysm (AAA) development. In vascular smooth muscle cells (VSMCs), AngII activates epidermal growth factor receptor via a metalloproteinase, ADAM17 (a disintegrin and metalloproteinase domain 17). We hypothesized that AngII-dependent AAA development would be prevented in mice lacking ADAM17 in VSMCs. To test this concept, control and VSMC ADAM17-deficient mice were cotreated with AngII and a lysyl oxidase inhibitor, β-aminopropionitrile, to induce AAA. We found that 52.4% of control mice did not survive because of aortic rupture. All other surviving control mice developed AAA and demonstrated enhanced expression of ADAM17 in the AAA lesions. In contrast, all AngII and β-aminopropionitrile-treated VSMC ADAM17-deficient mice survived and showed reduction in external/internal diameters (51%/28%, respectively). VSMC ADAM17 deficiency was associated with lack of epidermal growth factor receptor activation, interleukin-6 induction, endoplasmic reticulum/oxidative stress, and matrix deposition in the abdominal aorta of treated mice. However, both VSMC ADAM17-deficient and control mice treated with AngII and β-aminopropionitrile developed comparable levels of hypertension. Treatment of C57Bl/6 mice with an ADAM17 inhibitory antibody but not with control IgG also prevented AAA development. In conclusion, VSMC ADAM17 silencing or systemic ADAM17 inhibition seems to protect mice from AAA formation. The mechanism seems to involve suppression of epidermal growth factor receptor activation.