Human Receptor Activity-modifying Protein Research Articles

Development of a Novel Assay for Direct Assessment of Selective Amylin Receptor Activation Reveals Novel Differences in Behavior of Selective and Nonselective Peptide Agonists.

Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs, indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rational drug development. To date, such understanding has been hampered by lack of direct, subtype-selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus-tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R), or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin, or DACRA peptides that was distinct at each receptor subtype. Of particular note, although both of the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviors at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalization of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases. SIGNIFICANCE STATEMENT: Amylin receptors (AMYRs) are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases.

Screening of potential GCMS derived antimigraine compound from the leaves of Abrus precatorius Linn to target “calcitonin gene related peptide” receptor using in silico analysis

Calcitonin receptor-like receptor (CRLR) is a human protein, that produces a calcitonin gene-related peptide receptor (CGRP) when associates with human receptor activity-modifying protein-1 (HRAMP1). CGRP is believed to be involved in triggering of migraine. Many strategies were employed to design antimigraine drug using various CGRP antagonist/ligands but most of them have failed due to their inability to reach the target “CGRP receptor” as they get metabolized before conferring their pharmacological action and exhibit toxic effect to the liver. The present study, evaluated the binding of 13 phyto-chemical compounds of “Abrus precatorius” identified through GCMS analysis against the protein CGRP using “Discovery Studio software”. The molecular docking study and ADME/T properties prediction were performed with the compounds using C-DOCKER module. The results showed that five lead compounds of A. precatorius may act as good inhibitors for migraine headache and the compounds can be re-designed and synthesized for better antimigraine activity.

Increased receptor activity-modifying protein 1 in the nervous system is sufficient to protect against autonomic dysregulation and hypertension

Calcitonin gene-related peptide (CGRP) can cause migraines, yet it is also a potent vasodilator that protects against hypertension. Given the emerging role of CGRP-targeted antibodies for migraine prevention, an important question is whether the protective actions of CGRP are mediated by vascular or neural CGRP receptors. To address this, we have characterized the cardiovascular phenotype of transgenic nestin/hRAMP1 mice that have selective elevation of a CGRP receptor subunit in the nervous system, human receptor activity-modifying protein 1 (hRAMP1). Nestin/hRAMP1 mice had relatively little hRAMP1 RNA in blood vessels and intravenous injection of CGRP caused a similar blood pressure decrease in transgenic and control mice. At baseline, nestin/hRAMP1 mice exhibited similar mean arterial pressure, heart rate, baroreflex sensitivity, and sympathetic vasomotor tone as control mice. We previously reported that expression of hRAMP1 in all tissues favorably improved autonomic regulation and attenuated hypertension induced by angiotensin II (Ang II). Similarly, in nestin/hRAMP1 mice, hypertension caused by Ang II or phenylephrine was greatly attenuated, and associated autonomic dysregulation and increased sympathetic vasomotor tone were diminished or abolished. We conclude that increased expression of neuronal CGRP receptors is sufficient to induce a protective change in cardiovascular autonomic regulation with implications for migraine therapy.

GW28-e0821 Transplantation of mesenchymal stem cells carrying the human receptor activity-modifying protein 1 gene improves cardiac function and inhibits neointimal proliferation in the carotid angioplasty and myocardial infarction rabbit model

we investigated the effects of MSCs overexpressing the human receptor activity-modifying protein 1 (hRAMP1) on heart function and artery repair in rabbit models of myocardial infarction (MI) reperfusion and carotid artery injury. MSCs transfected with a recombinant adenovirus containing the hRAMP1

CGRP receptor activity in mice with global expression of human receptor activity modifying protein 1.

CGRP is a potent vasodilator and nociceptive neuropeptide linked to migraine. CGRP receptors are heterodimers of receptor activity modifying protein 1 (RAMP1) and either calcitonin receptor-like receptor (CLR; forms canonical CGRP receptor) or calcitonin receptor (CT receptor; forms AMY1 receptor). The goal of this study was to test whether transgenic mice globally expressing human RAMP1 have increased CGRP receptor activity and whether the receptors are sensitive to human selective antagonist telcagepant. cAMP production was measured in primary cultures of aortic smooth muscle and trigeminal ganglia neurons from global hRAMP1 mice and non-transgenic littermates. Functional activity and inhibition were compared with clonal cell lines expressing combinations of CLR or CT receptors with RAMP1. Cultured smooth muscle from global hRAMP1 mice had a 10-fold greater CGRP-induced cAMP maximal response (Rmax) than non-transgenic littermates, with similar EC50 s. In contrast, cultured trigeminal ganglia from global hRAMP1 mice had a 40-fold leftward shift of the EC50 , with similar Rmax values as littermates. In both hRAMP1 cultures, telcagepant blocked CGRP-induced cAMP production, but was not effective in non-transgenic cultures. IC50 values were closer to those observed for CT receptor/hRAMP1 than CLR/hRAMP1 in clonal cell lines. Overexpression of hRAMP1 increases CGRP signalling by changing the maximal response or ligand sensitivity, depending on tissue type. Furthermore, telcagepant inhibited transgenic hRAMP1 CGRP receptors, but the degree of inhibition suggests that the transgenic mice are only partially humanized or both canonical CGRP and AMY1 receptors are functional in trigeminal ganglia neurons and vascular smooth muscle.

Featured Article: Transplantation of mesenchymal stem cells carrying the human receptor activity-modifying protein 1 gene improves cardiac function and inhibits neointimal proliferation in the carotid angioplasty and myocardial infarction rabbit model

Although transplanting mesenchymal stem cells (MSCs) can improve cardiac function and contribute to endothelial recovery in a damaged artery, natural MSCs may induce neointimal hyperplasia by directly or indirectly acting on vascular smooth muscle cells (VSMCs). Receptor activity-modifying protein 1 (RAMP1) is the component and the determinant of ligand specificity of calcitonin gene-related peptide (CGRP). It is recently reported that CGRP and its receptor involve the proliferation and the apoptosis in vivo and in vitro, and the exogenous RAMP1 enhances the antiproliferation effect of CGRP in VSMCs. Here, we investigated the effects of MSCs overexpressing the human receptor activity-modifying protein 1 (hRAMP1) on heart function and artery repair in rabbit models of myocardial infarction (MI) reperfusion and carotid artery injury. MSCs transfected with a recombinant adenovirus containing the hRAMP1 gene (EGFP-hRAMP1-MSCs) were injected into the rabbit models via the ear vein at 24 h after carotid artery injury and MI 7 days post-EGFP-hRAMP1-MSC transplantation. The cells that expressed both enhance green fluorescent protein (EGFP) and CD31 were detected in the neointima of the damaged artery via immunofluorescence. EGFP-hRAMP1 expression was observed in the injured artery and infarcted myocardium by western blot analysis, confirming that the engineered MSCs targeted the injured artery and infarcted myocardium and expressed hRAMP1 protein. Compared with the EGFP-MSCs group, the EGFP-hRAMP1-MSCs group had a significantly smaller infarcted area and improved cardiac function by 28 days after cell transplantation, as detected by triphenyltetrazolium chloride staining and echocardiography. Additionally, arterial hematoxylin-eosin staining revealed that the area of the neointima and the area ratio of intima/media were significantly decreased in the EGFP-hRAMP1-MSCs group. An immunohistological study showed that the expression of α-smooth muscle antigen and proliferating cell nuclear antigen in the neointima cells of the carotid artery of the EGFP-hRAMP1-MSCs group was approximately 50% lower than that of the EGFP-MSCs group, suggesting that hRAMP1 expression may inhibit VSMCs proliferation within the neointima. Therefore, compared with natural MSCs, EGFP-hRAMP1-engineered MSCs improved infarcted heart function and endothelial recovery from artery injury more efficiently, which will provide valuable information for the development of MSC-based therapy.

RE: “The influence of RAMP1 overexpression on CGRP-induced osteogenic differentiation in MG-63 cells in vitro: An experimental study”

The aim of this study was to elucidate the influence of receptor activity modifying protein 1 (RAMP1) overexpression on the expression and distribution of calcitonin receptor-like receptor (CRLR) in MG-63 cells. Our research also focused on whether RAMP1 overexpression enhanced the promoting effect of exogenous CGRP on osteogenic differentiation in MG-63 cells. We first constructed a eukaryotic expression vector containing human RAMP1 and stably transfected it into MG-63 cells. Real-time PCR and Western blotting were used to determine the expression levels of RAMP1 and CRLR mRNA and protein, respectively. Immunofluorescence analysis was employed to compare the distribution of CRLR in transfected cells. After treatment with CGRP, the extent of osteogenic differentiation was evaluated by simultaneous monitoring of alkaline phosphatase activity, the expression patterns of osteoblastic markers and mineralisation staining. We found that RAMP1 was more highly expressed in the transfected group compared with the control groups (P < 0.01). The CRLR expression was significantly higher than that in the control groups (P < 0.05). In addition, after 7 days of CGRP treatment to induce osteogenic differentiation, the expression of collagen I mRNA was markedly increased in the transfected group (P < 0.05). The transfected group exhibited more granular precipitation in the cytoplasm with alkaline phosphatase staining after 7 and 14 days of differentiation. When stained with Alizarin Red, cells overexpressing RAMP1 were darker and formed many mineralised nodules with clear boundaries and calcium deposition typical of mineralised bone matrix structures at 28 days post-induction of differentiation. The CGRP-induced ALP activity in the RAMP1 overexpression group was significantly higher 3, 6 and 9 days after induction than that in the two control groups (P < 0.05). RAMP1 overexpression promotes CRLR expression, localisation on the cell membrane and enhanced CGRP-mediated differentiation of MG-63 cells. This study contributes to a better understanding of the molecular mechanisms governing CGRP-induced MG-63 differentiation.

High‐yield membrane protein expression from E. coli using an engineered outer membrane protein F fusion

Obtaining high yields of membrane proteins necessary to perform detailed structural study is difficult due to poor solubility and variability in yields from heterologous expression systems. To address this issue, an Escherichia coli-based membrane protein overexpression system utilizing an engineered bacterial outer membrane protein F (pOmpF) fusion has been developed. Full-length human receptor activity-modifying protein 1 (RAMP1) was expressed using pOmpF, solubilized in FC15 and purified to homogeneity. Using circular dichroism and fluorescence spectroscopy, purified full-length RAMP1 is composed of approximately 90% α-helix, and retains its solubility and structure in FC15 over a wide range of temperatures (20-60°C). Thus, our approach provides a useful, complementary approach to achieve high-yield, full-length membrane protein overexpression for biophysical studies.

The influence of RAMP1 overexpression on CGRP‐induced osteogenic differentiation in MG‐63 cells in vitro: An experimental study

The aim of this study was to elucidate the influence of receptor activity modifying protein 1 (RAMP1) overexpression on the expression and distribution of calcitonin receptor-like receptor (CRLR) in MG-63 cells. Our research also focused on whether RAMP1 overexpression enhanced the promoting effect of exogenous CGRP on osteogenic differentiation in MG-63 cells. We first constructed a eukaryotic expression vector containing human RAMP1 and stably transfected it into MG-63 cells. Real-time PCR and Western blotting were used to determine the expression levels of RAMP1 and CRLR mRNA and protein, respectively. Immunofluorescence analysis was employed to compare the distribution of CRLR in transfected cells. After treatment with CGRP, the extent of osteogenic differentiation was evaluated by simultaneous monitoring of alkaline phosphatase activity, the expression patterns of osteoblastic markers and mineralisation staining. We found that RAMP1 was more highly expressed in the transfected group compared with the control groups (P < 0.01). The CRLR expression was significantly higher than that in the control groups (P < 0.05). In addition, after 7 days of CGRP treatment to induce osteogenic differentiation, the expression of collagen I mRNA was markedly increased in the transfected group (P < 0.05). The transfected group exhibited more granular precipitation in the cytoplasm with alkaline phosphatase staining after 7 and 14 days of differentiation. When stained with Alizarin Red, cells overexpressing RAMP1 were darker and formed many mineralised nodules with clear boundaries and calcium deposition typical of mineralised bone matrix structures at 28 days post-induction of differentiation. The CGRP-induced ALP activity in the RAMP1 overexpression group was significantly higher 3, 6 and 9 days after induction than that in the two control groups (P < 0.05). RAMP1 overexpression promotes CRLR expression, localisation on the cell membrane and enhanced CGRP-mediated differentiation of MG-63 cells. This study contributes to a better understanding of the molecular mechanisms governing CGRP-induced MG-63 differentiation.

HRAMP1 MODIFIED MSCS IMPROVE CARDIAC FUNCTION AND INHIBIT NEOINTIMAL PROLIFERATION IN THE CAROTID ANGIOPLASTY AND MYOCARDIAL INFARCTION RABBIT MODEL

ObjectivesAlthough transplanting mesenchymal stem cells (MSCs) can improve cardiac function and contribute to endothelial recovery in a damaged artery, natural MSCs may induce neointimal hyperplasia by directly or indirectly acting...

Effects of hRAMP1 modified mesenchymal stem cells on restenosis and heart function in rabbit model of carotid angioplasty and myocardial infarction

To observe the effects of mesenchymal stem cells (MSC) modified by human receptor activity-modifying protein 1 (hRAMP1) on restenosis and cardiac function post-myocardial infarction (MI) and explore the therapeutic safety for gene modification of MSC. The double-injury rabbit model with MI reperfusion and sacculus damaged atherosclerotic carotid were established according to the previous study. MSC were transfected with adenovirus vector with enhanced green fluorescence protein (EGFP) and then introduced into rabbit model. The animals were randomly divided into Ad-EGFP-hRAMP1-MSC transfection group (hRAMP1-MSC group, n = 24) and Ad-EGFP-MSC transfection group (MSC group, n = 24), and PBS transfection group (C group, n = 24). At Day 28 post-transplantation, the injured carotid arteries and infarction myocardium were harvested to detect the expression of EGFP-positive MSC and assess organization morphology by hematoxylin and eosin, triphenyltetrazolium chloride or immunohistochemical stains and heart function by echocardiography. On flow cytometry, most cells expressed CD29 and CD90 while few ones expressed CD45. MSC with EGFP and a continuous expression of CD31 were found in intima of damaged carotid of both hRAMP1-MSC and MSC, but the expression of EGFP was not found in the control group. At Day 28 post-transplantation, the improvement of heart function and the decrease of infarct size were found in the hRAMP1-MSC and MSC groups compared with that in the control group(EF: 60.6% ± 1.5%,50.8% ± 3.2% vs 38.2% ± 2.0%, infarct size: 20.7% ± 1.4%, 33.2% ± 3.7% vs 35.6% ± 2.7%, all P < 0.05), especially much higher in hRAMP1-MSC group. At Day 28 post-transplantation, the area of intima hyperplasia and the rate of neointima and media in the hRAMP1-MSC group were lower than those in the MSC and C groups (0.15 ± 0.05 and 0.33 ± 0.08 vs 0.77 ± 0.11, 0.24 ± 0.07 and 0.51 ± 0.11 vs 1.09 ± 0.23, all P < 0.05). Also the expression of α-SMA was found in the hyperplasia intima. The expression of proliferating cell nuclear antigen significantly decreased in the hRAMP1-MSC group than those of the MSC and control groups (0.120 ± 0.028 vs 0.366 ± 0.013 and 0.627 ± 0.049, both P < 0.05). And it was much lower in the MSC group than that in the control group. Compared with MSC alone, hRAMP1-modified MSC have the potency not only of more improvement on cardiac function and the recovery of damaged endothelium, but also of significant inhibition of the proliferation of vascular smooth muscle cells. The recombinant hRAMP1 adenovirus vectors do not affect the differentiation potential MSC into endothelial cells.

Characterization of the single transmembrane domain of human receptor activity-modifying protein 3 in adrenomedullin receptor internalization

Two receptor activity-modifying proteins (RAMP2 and RAMP3) enable calcitonin receptor-like receptor (CLR) to function as two heterodimeric receptors (CLR/RAMP2 and CLR/RAMP3) for adrenomedullin (AM), a potent cardiovascular protective peptide. Following AM stimulation, both receptors undergo rapid internalization through a clathrin-dependent pathway, after which CLR/RAMP3, but not CLR/RAMP2, can be recycled to the cell surface for resensitization. However, human (h)RAMP3 mediates CLR internalization much less efficiently than does hRAMP2. Therefore, the molecular basis of the single transmembrane domain (TMD) and the intracellular domain of hRAMP3 during AM receptor internalization was investigated by transiently transfecting various RAMP chimeras and mutants into HEK-293 cells stably expressing hCLR. Flow cytometric analysis revealed that substituting the RAMP3 TMD with that of RAMP2 markedly enhanced AM-induced internalization of CLR. However, this replacement did not enhance the cell surface expression of CLR, [125I]AM binding affinity or AM-induced cAMP response. More detailed analyses showed that substituting the Thr130–Val131 sequence in the RAMP3 TMD with the corresponding sequence (Ile157–Pro158) from RAMP2 significantly enhanced AM-mediated CLR internalization. In contrast, substituting the RAMP3 target sequence with Ala130–Ala131 did not significantly affect CLR internalization. Thus, the RAMP3 TMD participates in the negative regulation of CLR/RAMP3 internalization, and the aforementioned introduction of the Ile–Pro sequence into the RAMP3 TMD may be a strategy for promoting receptor internalization/resensitization.

Effect of adenovirus mediated human RAMP1 gene on proliferation and apoptosis of VSMCs

ObjectiveTo investigate the effect of adenovirus mediated human receptor activity modifying protein-1 (RAMP1) gene on Proliferation and Apoptosis of Vascular Smooth Muscle Cells (VSMCs) derived from thoracic aortic of rabbits.MethodsVSMCs,...

Regarding Article “Receptor Activity-Modifying Protein-1 Augments Cerebrovascular Responses to Calcitonin Gene-Related Peptide and Inhibits Angiotensin II-Induced Vascular Dysfunction”

HomeStrokeVol. 42, No. 2Regarding Article “Receptor Activity-Modifying Protein-1 Augments Cerebrovascular Responses to Calcitonin Gene-Related Peptide and Inhibits Angiotensin II-Induced Vascular Dysfunction” Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBRegarding Article “Receptor Activity-Modifying Protein-1 Augments Cerebrovascular Responses to Calcitonin Gene-Related Peptide and Inhibits Angiotensin II-Induced Vascular Dysfunction” Kazushi Tsuda, MD, FAHA Kazushi TsudaKazushi Tsuda Cardiovascular and Metabolic Research Center Kansai University of Health Sciences Osaka, Japan (Tsuda) Search for more papers by this author Originally published30 Dec 2010https://doi.org/10.1161/STROKEAHA.110.604272Stroke. 2011;42:e24Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2010: Previous Version 1 To the Editor:We read with great interest the recent article by Dr Chrissobolis and colleagues1 dealing with the cerebrovascular responses in the transgenic mice with overexpression of the receptor activity-modifying protein-1 (RAMP1) for calcitonin gene-related peptide (CGRP) receptors. The results of their study demonstrated that the responses to CGRP in carotid and basilar arteries in vitro as well as cerebral arterioles in vivo were selectively enhanced in human RAMP1 transgenic mice compared with controls. In addition, the authors presented that angiotensin II-induced oxidative stress and endothelial dysfunction was prevented after expression of RAMP1. The authors propose the hypothesis that RAMP1 may be an important mediator of vascular protection and a new therapeutic target in vascular disease.Several studies have reported that enhanced activity of the sympathetic nervous system might actively participate in the pathogenesis of vascular dysfunction. In a study we presented previously, the changes in norepinephrine release induced by CGRP was investigated in the rat central nervous system.2 In an in vitro study, we showed that CGRP inhibited the stimulation-evoked norepinephrine release in a dose-dependent manner. It was also demonstrated that a dihydropyridine-sensitive calcium channel agonist, Bay K 8644, significantly reversed the inhibitory effect of CGRP on norepinephrine release, indicating that CGRP might partially interact with dihydropyridine-sensitive calcium channels and modulate intracellular calcium mobilization. Furthermore, we showed that the inhibitory action on CGRP on norepinephrine release was significantly attenuated in spontaneously hypertensive rats compared with normotensive rats.3 In the peripheral tissues, Ohhashi and Jacobowitz4 observed that CGRP might have reduced the electric stimulation-induced contraction of rat vas deferens, suggesting that CGRP might inhibit norepinephrine release during adrenergic nerve stimulation. It can be speculated that the sympatholytic action of CGRP might be a defense against vascular dysfunction. Because angiotensin II may stimulate the sympathetic neurotransmission in the central nervous system,5 we would like to know whether changes in sympathetic nervous activity might be associated with the magnitude of the RAMP1 overexpression or whether CGRP might suppress the angiotensin II-induced sympathetic hyperactivity in the study of Dr Chrissobolis and colleagues. Further studies should be performed to assess more thoroughly the interactions between CGRP and the sympathetic nervous system and their role in the protective effect against the angiotensin II-induced vascular dysfunction in the RAMP1 transgenic mice.Kazushi Tsuda, MD, FAHA Cardiovascular and Metabolic Research Center Kansai University of Health Sciences Osaka, JapanDisclosuresNone.FootnotesStroke welcomes Letters to the Editor and will publish them, if suitable, as space permits. Letters must reference a Stroke published-ahead-of-print article or an article printed within the past 3 weeks. The maximum length is 750 words including no more than 5 references and 3 authors. Please submit letters typed double-spaced. Letters may be shortened or edited. Include a completed copyright transfer agreement form (available online at http://stroke.ahajournals.org and http://submit-stroke.ahajournals.org).

Receptor Activity-Modifying Protein-1 Augments Cerebrovascular Responses to Calcitonin Gene-Related Peptide and Inhibits Angiotensin II-Induced Vascular Dysfunction

Receptors for calcitonin gene-related peptide (CGRP) are composed of the calcitonin-like receptor in association with receptor activity-modifying protein-1 (RAMP1). CGRP is an extremely potent vasodilator and may protect against vascular disease through other mechanisms. We tested the hypothesis that overexpression of RAMP1 enhances vascular effects of CGRP using transgenic mice with ubiquitous expression of human RAMP1. Because angiotensin II (Ang II) is a key mediator of vascular disease, we also tested the hypothesis that RAMP1 protects against Ang II-induced vascular dysfunction. Responses to CGRP in carotid and basilar arteries in vitro as well as cerebral arterioles in vivo were selectively enhanced in human RAMP1 transgenic mice compared to littermate controls (P<0.05), and this effect was prevented by a CGRP receptor antagonist (P<0.05). Thus, vascular responses to CGRP are normally RAMP1-limited. Responses of carotid arteries were examined in vitro after overnight incubation with vehicle or Ang II. In arteries from control mice, Ang II selectively impaired responses to the endothelium-dependent agonist acetylcholine by ≈50% (P<0.05) via a superoxide-mediated mechanism. In contrast, Ang II did not impair responses to acetylcholine in human RAMP1 transgenic mice. RAMP1 overexpression increases CGRP-induced vasodilation and protects against Ang II-induced endothelial dysfunction. These findings suggest that RAMP1 may be a new therapeutic target to regulate CGRP-mediated effects during disease including pathophysiological states in which Ang II plays a major role.

Crystal Structure of the Ectodomain Complex of the CGRP Receptor, a Class-B GPCR, Reveals the Site of Drug Antagonism

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

Receptor Activity-Modifying Protein 1 Increases Baroreflex Sensitivity and Attenuates Angiotensin-Induced Hypertension

Calcitonin gene-related peptide (CGRP) is a powerful vasodilator that interacts with the autonomic nervous system. A subunit of the CGRP receptor complex, receptor activity-modifying protein 1 (RAMP1), is required for trafficking of the receptor to the cell surface and high-affinity binding to CGRP. We hypothesized that upregulation of RAMP1 would favorably enhance autonomic regulation and attenuate hypertension. Blood pressure, heart rate, and locomotor activity were measured by radiotelemetry in transgenic mice with ubiquitous expression of human RAMP1 (hRAMP1) and littermate controls. Compared with control mice, hRAMP1 mice exhibited similar mean arterial pressure, a lower mean heart rate, increased heart rate variability, reduced blood pressure variability, and increased baroreflex sensitivity (2.83+/-0.20 versus 1.49+/-0.10 ms/mm Hg in controls; P<0.05). In control mice, infusion of angiotensin II (Ang-II) increased mean arterial pressure from 118+/-2 mm Hg to 153+/-4 and 174+/-6 mm Hg after 7 and 14 days of infusion, respectively (P<0.05). In contrast, Ang-II hypertension was markedly attenuated in hRAMP1 mice with corresponding values of mean arterial pressure of 111+/-2, 119+/-2, and 132+/-3 mm Hg. Ang-II induced decreases in baroreflex sensitivity and heart rate variability, and increases in blood pressure variability observed in control mice were also abrogated or reversed in hRAMP1 mice (P<0.05). Moreover, during the Ang-II infusion, the pressor response to the CGRP receptor antagonist CGRP(8-37) was significantly greater (P<0.05) in hRAMP1 mice (+30+/-2 mm Hg) than in control mice (+19+/-2 mm Hg), confirming a significantly greater antihypertensive action of endogenous CGRP in hRAMP1 mice. We conclude that RAMP1 overexpression attenuates Ang-II-induced hypertension and induces a protective change in cardiovascular autonomic regulation.

Functional characterization of two human receptor activity-modifying protein 3 variants

Adrenomedullin (AM) and amylin are involved in angiogenesis/lymphangiogenesis and glucose homeostasis/food intake, respectively. They activate receptor activity-modifying protein (RAMP)/G protein-coupled receptor (GPCR) complexes. RAMP3 with the calcitonin receptor-like receptor (CLR) forms the AM 2 receptor, whereas when paired with the calcitonin receptor AMY 3 receptors are formed. RAMP3 interacts with other GPCRs although the consequences of these interactions are poorly understood. Therefore, variations in the RAMP3 sequence, such as single nucleotide polymorphisms or mutations could be relevant to human health. Variants of RAMP3 have been identified. In particular, analysis of AK222469 (Homo sapiens mRNA for receptor (calcitonin) activity-modifying protein 3 precursor variant) revealed several nucleotide differences, three of which encoded amino acid changes (Cys40Trp, Phe100Ser, Leu147Pro). Trp56Arg RAMP3 is a polymorphic variant of human RAMP3 at a conserved amino acid position. To determine their function we used wild-type (WT) human RAMP3 as a template for introducing amino acid mutations. Mutant or WT RAMP3 function was determined in Cos-7 cells with CLR or the calcitonin receptor (CT (a)). Cys40Trp/Phe100Ser/Leu147Pro RAMP3 was functionally compromised, with reduced AM and amylin potency at the respective AM 2 and AMY 3(a) receptor complexes. Cys40Trp and Phe100Ser mutations contributed to this phenotype, unlike Leu147Pro. Reduced cell-surface expression of mutant receptor complexes probably explains the functional data. In contrast, Trp56Arg RAMP3 was WT in phenotype. This study provides insight into the role of these residues in RAMP3. The existence of AK222469 in the human population has implications for the function of RAMP3/GPCR complexes, particularly AM and amylin receptors.

Haplotype-based case–control study of receptor (calcitonin) activity-modifying protein-1 gene in cerebral infarction

Calcitonin gene-related peptide (CGRP) receptor is a complex molecule that consists of calcitonin receptor-like receptor and receptor activity-modifying protein-1 (RAMP1). It was recently reported that RAMP1-deficient mice (RAMP1(-/-)) showed inflammatory responses with a transiently significant increase in serum CGRP levels and proinflammatory cytokines when compared with RAMP1(+/+) mice. The aim of this study was to investigate the relationship between the human RAMP1 gene and cerebral infarction (CI) using single-nucleotide polymorphisms (SNPs) in a Japanese population. We selected six SNPs in the human RAMP1 gene (rs3754701, rs3769048, rs7557078, rs1584243, rs10199956 and rs7590387) and performed a case-control study using each SNP and haplotype in 171 CI patients and 234 controls. There were no significant differences in overall distribution of genotype and allele frequencies of the SNPs between the CI and control groups. However, there was a significant difference in overall distribution between the CI and control groups (P<0.001) in the haplotype-based case-control study with the combinations of rs3754701-rs3769048-rs7590387. The T-A-C susceptibility haplotype for CI was significantly more frequent than in the control group (P=0.0024). The results suggest that the T-A-C haplotype is a genetic marker for CI, and that RAMP1 or neighbouring genes are associated with increased susceptibility to CI.

Evidence for a Protective Role for Receptor Activity Modifying Protein‐1 (RAMP1) in Angiotensin II‐Induced Endothelial Dysfunction

Angiotensin II (Ang II) plays a major role in hypertension and causes vascular oxidative stress and endothelial dysfunction. Calcitonin gene‐related peptide (CGRP), a potent vasodilator, reportedly has antioxidant properties in vascular cells in culture. CGRP receptors consist of a calcitonin‐like receptor and a receptor activity modifying protein‐1 (RAMP1). The aim of this study was to test if RAMP1 overexpression protects against Ang II‐induced endothelial dysfunction. Transgenic mice overexpressing human RAMP1 (hRAMP1) in most tissues (including blood vessels) were generated using a RAMP1 transgene under control of the CX1 promoter. Responses of carotid artery were examined in vitro following 22 hour incubation with vehicle or Ang II (10 nmol/L). Maximum relaxation to the endothelium‐dependent vasodilator acetylcholine (ACh) was impaired by ~40% in Ang II‐treated vs vehicle‐treated vessels from control mice (P&lt;0.05). In contrast, in hRAMP1 mice, maximum relaxation to ACh was similar in vehicle and Ang II‐treated vessels (P&gt;0.05), suggesting RAMP1 overexpression protects against Ang II‐induced endothelial dysfunction. Responses to nitroprusside were similar in both groups of mice and unaffected by Ang II treatment (P&gt;0.05). RAMP1 may represent a therapeutic target to protect endothelial function in conditions where Ang II plays a major role, including hypertension.