Expression of the calcitonin receptor, calcitonin receptor-like receptor, and receptor activity modifying proteins during osteoclast differentiation

The expressions of the calcitonin receptor (CTR), the calcitonin receptor-like receptor (CLR), the receptor activity-modifying proteins (RAMP) 1-3, and of the receptor component protein (RCP) have been studied in mouse bone marrow macrophages (BMM) during osteoclast differentiation, induced by treatment with M-CSF and RANKL. Analyses of mRNA showed that CLR and RAMP1-3, but not CTR, were expressed in M-CSF stimulated BMM. RANKL gradually increased CTR mRNA, transiently enhanced CLR and transiently decreased RAMP1 mRNA, but did not affect RAMP2, RAMP3, or RCP mRNA. However, RANKL did not affect protein levels of CLR or RAMP1-3 as assessed by Western blots or FACS analyses, whereas immunocytochemistry showed enhanced CTR protein. Analyses of cAMP production showed that BMM cells expressed functional receptors for calcitonin gene-related peptide (CGRP), amylin, adrenomedullin, and intermedin, but not for calcitonin and calcitonin receptor stimulating peptide (CRSP), but that RANKL induced the expression of receptors for calcitonin and CRSP as well. Calcitonin, CGRP, amylin, adrenomedullin, intermedin, and CRSP all down regulated the CTR mRNA, but none of the peptides caused any effects on the expression of CLR or any of the RAMPs. Our data show that BMM cells express receptors for CGRP, amylin, adrenomedullin, and intermedin and that RANKL induces the formation of receptors for calcitonin and CRSP in these cells. We also show, for the first time, that the CTR is not only down regulated by signaling through the CTR but also by the peptides signaling through CLR/RAMPs.     

Adrenomedullin and related peptides: receptors and accessory proteins.

Adrenomedullin (AM), alpha- and beta-calcitonin gene-related peptide (CGRP), amylin and calcitonin (CT) are structurally and functionally related peptides. The structure of a receptor for CT (CTR) was elucidated in 1991 through molecular cloning, but the structures of the receptors for the other three peptides had yet to be elucidated. The discovery of receptor-activity-modifying proteins (RAMP) 1 and -2 and their co-expression with an orphan receptor, calcitonin receptor-like receptor (CRLR) has led to the elucidation of functional CGRP and AM receptors, respectively. RAMP1 and -3 which are co-expressed with CTR revealed two amylin receptor isotypes. Molecular interactions between CRLR and RAMPs are involved in their transport to the cell surface. Heterodimeric complexes between CRLR or CTR and RAMPs are required for ligand recognition.     

降钙素基因相关肽家族受体及其受体活性修饰蛋白

降钙素基因相关肽家族是一类多功能的激素家族 ,参与人体的多种生物学功能 ,与多种疾病有关。降钙素基因相关肽受体包括降钙素受体 (CTR)和降钙素受体样受体 (CRLR) ,CTR可以独自与降钙素结合 ,而CRLR必须与一组称作受体活性修饰蛋白 (RAMPs)的蛋白质共同作用才能发挥生物学功能。综述CTR的研究概况及CRLR与RAMPs相互作用的机制和表达调控 ,以期为人们设计新型药物提供参考。     

Molecular and functional characterization of adrenomedullin receptors in pufferfish

The receptors for the calcitonin gene-related peptide (CGRP)/adrenomedullin (AM) family peptides were characterized in the mefugu Takifugu obscurus, a euryhaline fugu species very close to Takifugu rubripes, which has as many as five adrenomedullin genes (AM1-5). CGRP and AM share a G protein-coupled core receptor called calcitonin receptor-like receptor (CLR), and the specificity of the CLR is determined by the interaction with receptor activity-modifying proteins (RAMPs). Through database mining, three CLRs (CLR1-3) and five RAMPs (RAMP1-5) were identified, and all of them were cloned by RT-PCR and characterized by functional expression in COS7 cells in every possible combination of CLR-RAMP. The following combinations generated cAMP in response to physiological concentrations of CGRP, AM1 (an ortholog of mammalian AM), AM2, and AM5: CLR1-RAMP1/4 (CGRP), CLR1-RAMP2/3/5 (AM1), CLR2-RAMP2 (AM1), CLR1-RAMP3 (AM2), and CLR1-RAMP3 (AM5). Their expressions were found by Northern blot analysis to be tissue specific and salinity dependent. For example, CLR1-RAMP5 and CLR1-RAMP2 are expressed specifically in the gill and kidney, respectively, suggesting their involvement in osmoregulation. Furthermore, relatively high levels of CLRs and RAMPs were found in the spleen and ovary, suggesting roles in the immune and female reproductive systems. Immunohistochemistry revealed that AM receptors of the following types are expressed in the locations, indicated in brackets, of the mefugu gill and kidney: CLR1-RAMP5 (interlamellar vessels), CLR2-RAMP2 (pillar cells), and CLR1-RAMP2 (apical side of renal proximal tubule cells).     

Functional relevance of G-protein-coupled-receptor-associated proteins,exemplified by receptor-activity-modifying proteins (RAMPs)

The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.     

RAMPs and G protein coupled receptors

RAMPs (receptor activity-modifying proteins) were discovered in 1998 as accessory proteins needed to the functionnal activity of CGRP (calcitonin gene-related peptide) receptors. Three RAMPs generated by three different genes are known in human, rat and mice. The coding sequences of such genes are described, but as yet, regulation sequences are unknown. RAMPs interact with GPCR (G protein-coupled receptors) of class II. In the case of the calcitonin/CGRP peptide family, RAMPs determine the functionnal specificity of the receptor, glycosylate and translocate the receptor to the cell surface. CGRP receptors are observed in presence of the RAMP1/calcitonin receptor-like receptor (CRLR), but the association of RAMP2 or RAMP3 with CRLR generates an adrenomedullin receptor. The calcitonin receptor (CTR) is translocated alone to the cell surface, but interactions of RAMPs with CTR forms amylin receptors. If RAMPs can interact with glucagon, parathyroid hormone and VIP/PACAP (vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide (VPACR1)) receptors, the functionnal specificity of these receptors remains unaltered. However, the complex VPACR1/RAMP2 enhances specifically the phosphoinoside signaling pathway.     

Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y(1), Y(2), Y(4), Y(5) and y(6). All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.     

Novel calcitonin-(8-32)-sensitive adrenomedullin receptors derived from co-expression of calcitonin receptor with receptor activity-modifying proteins

We tested whether heterodimers comprised of calcitonin (CT) receptor lacking the 16-amino acid insert in intracellular domain 1 (CTR(I1-)) and receptor activity-modifying protein (RAMP) can function not only as calcitonin gene-related peptide (CGRP) receptors but also as adrenomedullin (AM) receptors. Whether transfected alone or together with RAMP, human (h)CTR(I1-) appeared mainly at the surface of HEK-293 cells. Expression of CTR(I1-) alone led to significant increases in cAMP in response to hCGRP or hAM, though both peptides remained about 100-fold less potent than hCT. However, the apparent potency of AM, like that of CGRP, approached that of CT when CTR(I1-) was co-expressed with RAMP. CGRP- or AM-evoked cAMP production was strongly inhibited by salmon CT-(8-32), a selective amylin receptor antagonist, but not by hCGRP-(8-37) or hAM-(22-52), antagonists of CGRP and AM receptors, respectively. Moreover, the inhibitory effects of CT-(8-32) were much stronger in cells co-expressing CTR(I1-) and RAMP than in cells expressing CTR(I1-) alone. Co-expression of CTR(I1-) with RAMP thus appears to produce functional CT-(8-32)-sensitive AM receptors.     

受体活性修饰蛋白研究进展

受体活性修饰蛋白(receptor activity-modifying proteins,RAMPs)属于单跨膜蛋白家族,分三个结构域,RAMP的N端和跨膜区决定本身的功能和受体表型,胞内C端对于配体的信号传导和受体循环有重要作用。目前发现有三个成员:RAMP1、RAMP2和RAMP3。RAMPs通过改变G蛋白偶联受体的糖基化,作用于配体结合区域来调节受体表型。RAMP1与降钙素受体样受体(calcitonin receptor like receptor,CRLR)结合表现出降钙素基因相关肽(calcitonin gene-related peptide,CGRP)受体表型:RAMP2和RAMP3与CRLR结合则对肾上腺髓质素(adrenomedullin,AM)表现高亲和力,与降钙素受体(calcitonin receptor,CTR)结合则作为胰淀粉样酶(amylin,AMY)受体。由此可见,RAMPs不仅调节受体与配体结合,还影响细胞内的蛋白相互作用调节细胞内信号传导来影响细胞的增殖、迁移、分化等生物学特性。RAMPs还对心血管系统的病理生理有重要调节作用。     

Deletions of genes encoding calcitonin/alpha-CGRP, amylin and calcitonin receptor have given new and unexpected insights into the function of calcitonin receptors and calcitonin receptor-like receptors in bone

It has been suggested that skeletal nerves fibers may play important roles in neuro-osteogenic interactions. This view is partly based upon information obtained from immunohistochemical studies, chemical and surgical denervation experiments and clinical observations in patients with stroke and spinal cord injury, indicating the presence of a network of nerve fibers in the skeleton and that defective signalling in skeletal nerve fibers affects remodelling of bone. This view is also supported by data showing that functional receptors for signalling molecules in skeletal nerve fibers are expressed in bone cells and that activation of these receptors leads to profound effects on bone forming osteoblasts and bone resorbing osteoclasts. Convincing evidence for a role of neuronal signalling in bone metabolism has been provided by gene deletion approaches in which it has been shown that leptin-sensitive and neuropeptide Y-sensitive receptors in hypothalamus are important for bone remodelling in mice. Recently, gene deletion experiments have shown that calcitonin gene-related peptide (CGRP), one of the neuropeptides present in skeletal nerve fibers, is an important physiological regulator of bone formation at the level of osteoblast activity. CGRP belongs to the calcitonin (CT) family of peptides also including CT, amylin and adrenomedullin, as well as the recently described intermedin and calcitonin receptor-stimulating peptide. These peptides utilize two seven transmembrane G protein-coupled receptors - the calcitonin receptor (CTR) and the calcitonin receptor- like receptor (CRLR) - which can dimerize with three different single transmembrane proteins, making up the RAMP family. Associations between RAMPs and either CTR or CRLR give rise to seven distinct, molecularly characterized, receptors for CT, CGRP, amylin and adrenomedullin. Deletions of the genes for ligands in the CT family of peptides and for one of the receptors have revealed unexpected findings that have changed our view on the role of these peptides in bone remodelling. It was anticipated that deletions of the CT/alpha-CGRP and CTR genes would lead to bone loss, since CT has been shown to inhibit bone resorption in vitro and in vivo and has been used to treat patients with excessive bone resorption. Surprisingly, it was found that CT/alpha-CGRP-/- and CTR+/- mice have increased bone mass due to increased bone formation. Mice with deletion of the amylin gene, however, exhibited bone loss due to enhanced bone resorption. Selective deletion of the alpha-CGRP gene also leads to bone loss, but due to decreased bone formation. Thus, our understanding of the role of the CT family of peptides has been changed dramatically and much more data have to be gained before we fully understand the roles these peptides have in bone biology.     

Selective CGRP and adrenomedullin peptide binding by tethered RAMP‐calcitonin receptor‐like receptor extracellular domain fusion proteins

Calcitonin gene‐related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor‐like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP‐CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1‐CLR ECD fusion purified as a monomer, whereas the RAMP2‐CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27‐37) and AM(37‐52) fragments were identified as the minimal ECD complex binding regions. The CGRP C‐terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C‐terminal amide group was essential for ECD binding. Alanine‐scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries.     

Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM₁ and AM₂, respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM₂/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM₂ potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM₂ and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM₂ and CGRP receptors.     

Identification and pharmacological characterization of domains involved in binding of CGRP receptor antagonists to the calcitonin-like receptor

The calcitonin-like receptor (CLR) and the calcitonin receptor (CTR) interact with receptor activity-modifying protein 1 (RAMP1) at the cell surface to form heterodimeric receptor complexes. CLR and CTR are members of the class II (family B) G-protein-coupled receptors (GPCR) and bind calcitonin gene-related peptide (CGRP) with similar affinities when coexpressed with RAMP1. The observation that various nonpeptide CGRP receptor antagonists display a higher affinity for the CLR/RAMP1 complex than for CTR/RAMP1 provided an opportunity to investigate the molecular determinants of the differential receptor affinities of these antagonists. A chimeric receptor approach was utilized to identify key domains within CLR responsible for conferring high-affinity antagonist binding. Initial chimera experiments implicated distinct regions within CLR as responsible for the affinities of structurally diverse CGRP receptor antagonists. Dissection of these key regions implicated amino acids 37-63 located in the amino terminus of CLR as responsible for the high-affinity interaction of one structural class, while transmembrane domain (TM) 7 was responsible for the interaction of a second class of antagonist. A unique binding interaction in the amino terminus of CLR is consistent with the observation that these compounds also interact with the extracellular region of RAMP1 and could suggest the formation of a binding pocket between the two proteins. Conversely, a compound which interacted with TM7 did not display a similar RAMP1 dependence, suggesting an allosteric mechanism of antagonism. Collectively, these data provide insight into two alternative mechanisms of antagonism for this unique heterodimeric receptor complex.     

Calcitonin receptor-like receptor (CLR) influences posttranslational events of receptor activity-modifying proteins (RAMPs)

Adrenomedullins (AM) form a multifunctional subfamily of the calcitonin gene-related peptide (CGRP) superfamily, the members of which exert their physiological roles through a 1:1 combination of calcitonin receptor-like receptors (CLRs) and receptor activity-modifying proteins (RAMPs). It has been shown that RAMPs can modify the biochemical properties of CLRs; for example, RAMP escorts CLR to the plasma membrane, affects glycosylation state of CLR, and transforms the ligand selectivity of CLR, but on the other hand the effects of CLRs on the biochemical and functional properties of the partner RAMPs are not well established. In this study, using pufferfish (mefugu, mf) homolog, we revealed that mfCLR1 could affect the post-translational modification and trafficking pathway of mfRAMP1. In addition, mfCLRs boosted mfRAMP1, mfRAMP2b, and mfRAMP3 translocation to cell surface. We further revealed that mfRAMPs, except mfRAMP1 and mfRAMP3, could be expressed as multimers on the plasma membrane. However, only monomeric form of mfRAMP2a, mfRAMP4, and mfRAMP5 could heteromerize with mfCLR1 but not with mfCLR2 or mfCLR3, which was consistent with their abilities to induce cAMP response. Collectively our results indicate that the glycosylation, subcellular trafficking, and pharmacological properties of the components of RAMP-CLR receptor complexes are regulated in an interdependent manner.     

Multiple ramp domains are required for generation of amylin receptor phenotype from the calcitonin receptor gene product

Calcitonin (CT), calcitonin gene-related peptide (CGRP), amylin, and adrenomedullin constitute a family of structurally related peptides that signal via either the calcitonin receptor-like receptor or the CT receptor, with receptor phenotype determined by coexpression of one of the three receptor activity-modifying proteins (RAMPs). The nature of the interaction between the receptor and RAMP was investigated using chimeras between RAMP1 and RAMP2 where the amino-terminal domain of RAMP1 was attached to the transmembrane domain and carboxy terminus of RAMP2 and called RAMP1/2, and vice versa for RAMP2/1. Cotransfection of wild-type or chimeric RAMPs with the insert-negative isoform of the human CT receptor (hCTR(I1-)) into COS-7 cells resulted in the expression of (125)I-rat amylin binding sites. Highest specific binding was observed when either RAMP1 or RAMP2/1 were cotransfected, indicating the importance of the RAMP transmembrane domain and/or carboxy terminus for the degree to which amylin receptors are expressed. In contrast, the phenotype generated was primarily determined by the amino terminus, with similar RAMP1- and RAMP1/2-induced receptor phenotypes that had higher affinity for human CGRPalpha and lower affinity for human calcitonin than the RAMP2- and RAMP2/1-induced receptors.     

The role of the extracellular loops of the CGRP receptor, a family B GPCR

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.     

Aspartate(69) of the calcitonin-like receptor is required for its functional expression together with receptor-activity-modifying proteins 1 and -2

The calcitonin-like receptor (CLR) associated with receptor-activity-modifying proteins (RAMP) 1 or -2 recognizes calcitonin gene-related peptide (CGRP) and adrenomedullin (AM), respectively. The amino acid sequence CNRTWDGWLCW corresponding to residues 64-74 in the extracellular N-terminus of the CLR is conserved. The Asp(69) (D(69)) is present in all family B1 G-protein-coupled receptors. Here the D(69) of a V5-tagged mouse CLR has been mutated to Ala (A), Glu (E), and Asn (N). The function of the intact and the mutant CLR was investigated in COS-7 cells coexpressing myc-tagged mouse RAMP1 or -2. In CLR/RAMP1 and -2 expressing cells CGRP and AM stimulated cAMP formation with an EC(50) of 0.17 and 0.50 nM, respectively. The expression of the D69A, D69E, and D69N mutants at the cell surface was comparable to that of the intact CLR. cAMP stimulation by CGRP and AM was abolished in the D69A mutant. With the D69E mutant the EC(50) of CGRP and AM were 1000-fold higher than those with the intact CLR. With the D69N mutant the EC(50) of CGRP was 0.48 nM and that of AM 0.44 nM, but the maximal cAMP formation was reduced to 24% and to 12% of cells with the intact CLR. Co-immunoprecipitation of RAMP1 with the CLR, indicating complex formation, was reduced with the D69A, D69N, and D69E mutants. RAMP2 co-precipitated with the mutant receptors indistinguishable from the intact CLR. In conclusion, mutation of D69 to N, E or A in the CLR did not affect its expression at the cell surface, but impaired or abolished the CGRP and AM receptor function in the presence of RAMP1 and -2, respectively.     

Cardiovascular effects of exogenous adrenomedullin and CGRP in Ramp and Calcrl deficient mice

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are potent vasodilator peptides and serve as ligands for the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CLR/Calcrl). Three GPCR accessory proteins called receptor activity-modifying proteins (RAMPs) modify the ligand binding affinity of the receptor such that the CLR/RAMP1 heterodimer preferably binds CGRP, while CLR/RAMP2 and CLR/RAMP3 have a stronger affinity for AM. Here we determine the contribution of each of the three RAMPs to blood pressure control in response to exogenous AM and CGRP by measuring the blood pressure of mice with genetic reduction or deletion of the receptor components. Thus, the cardiovascular response of Ramp1^−/−, Ramp2^+/−, Ramp3^−/−, Ramp1^−/−/Ramp3^−/− double-knockout (dKO), and Calcrl^+/− mice to AM and CGRP were compared to wildtype mice. While under anesthesia, Ramp1^−/− male mice had significantly higher basal blood pressure than wildtype males; a difference which was not present in female mice. Additionally, anesthetized Ramp1^−/−, Ramp3^−/−, and Calcrl^+/− male mice exhibited significantly higher basal blood pressure than females of the same genotype. The hypotensive response to intravenously injected AM was greatly attenuated in Ramp1^−/− mice, and to a lesser extent in Ramp3^−/− and Calcrl^+/− mice. However, Ramp1^−/−/Ramp3^−/− dKO mice retained some hypotensive response to AM. These results suggest that the hypotensive effect of AM is primarily mediated through the CLR/RAMP1 heterodimer, but that AM signaling via CLR/RAMP2 and CLR/RAMP3 also contributes to some hypotensive action. On the other hand, CGRP’s hypotensive activity seems to be predominantly through the CLR/RAMP1 heterodimer. With this knowledge, therapeutic AM or CGRP peptides could be designed to cause less hypotension while maintaining canonical receptor-RAMP mediated signaling.     

Novel receptor partners and function of receptor activity-modifying proteins

The receptor activity-modifying proteins (RAMPs) comprise a family of three accessory proteins that heterodimerize with the calcitonin receptor-like receptor (CL receptor) or with the calcitonin receptor (CTR) to generate different receptor phenotypes. However, RAMPs are more widely distributed across cell and tissue types than the CTR and CL receptor, suggesting additional roles for RAMPs in cellular processes. We have investigated the potential for RAMP interaction with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor and the CTR. Using immunofluorescence confocal microscopy, we demonstrate, for the first time, that RAMPs interact with at least four additional receptors, the VPAC1 vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor with all three RAMPs; the glucagon and PTH1 parathyroid hormone receptors with RAMP2; and the PTH2 receptor with RAMP3. Unlike the interaction of RAMPs with the CL receptor or the CTR, VPAC1R-RAMP complexes do not show altered phenotypic behavior compared with the VPAC1R alone, as determined using radioligand binding in COS-7 cells. However, the VPAC1R-RAMP2 heterodimer displays a significant enhancement of agonist-mediated phosphoinositide hydrolysis with no change in cAMP stimulation compared with the VPAC1R alone. Our findings identify a new functional consequence of RAMP-receptor interaction, suggesting that RAMPs play a more general role in modulating cell signaling through other GPCRs than is currently appreciated.