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Intermedin (IMD) is a recently discovered peptide closely related to adrenomedullin. Its principal physiological activity is its role in the regulation of the cardiovascular system, where it exerts a potent hypotensive effect. In addition, data were recently provided showing that this peptide is able to exert a clearcut pro-angiogenic effect both in vitro and in vivo. IMD acts through the non-selective interaction with receptor complexes formed by the dimerization of calcitonin-like receptor (CLR) with the receptor activity-modifying proteins RAMP1, 2 or 3. Thus, in the present study, the role of CLR/RAMP complexes in mediating the pro-angiogenic effect induced by IMD on human umbilical vein endothelial cells (HUVECs) cultured on Matrigel was examined. Real-time PCR demonstrated the expression of IMD, CLR/RAMP1 and CLR/RAMP2 (but not CLR/RAMP3) mRNA in HUVECs. IMD exerted a significant in vitro angiogenic action, specifically triggered by the binding of the peptide to CLR/RAMP complexes. Both CLR/RAMP1 and CLR/RAMP2 appeared to mediate the pro-angiogenic effect, which was associated with a significant increase of vascular endothelial growth factor (VEGF) mRNA expression 18 h following IMD administration, indicating that the observed pro-angiogenic effects are related, at least in part, to an increased synthesis of this growth factor promoted by the peptide. Western blot analysis, however, showed a significant increase of VEGF receptor-2 phosphorylation as early as 5 min following IMD administration, indicating that IMD induces a pro-angiogenic response in human vascular endothelial cells not only via CLR/RAMP-induced release of VEGF, but also during signal initiation and propagation by transactivating the VEGF receptor-2 machinery.

Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [(125)I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser(449) to Ser(467) were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.

The role of receptor activity modifying protein 1 (RAMP1) in forming receptors with the calcitonin receptor-like receptor (CLR) and the calcitonin receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1., (Copyright 2009 Elsevier Inc. All rights reserved.)

The three receptor activity-modifying proteins (RAMPs1, -2, and -3) associate with a wide variety of G protein-coupled receptors (GPCRs), including calcitonin receptor-like receptor (CRLR). In this study, we used flow cytometry to measure RAMP translocation to the cell surface as a marker of RAMP-receptor interaction. Because VPAC2 does not interact with RAMPs, although, like CRLR, it is a Family B peptide hormone receptor, we constructed a set of chimeric CRLR/VPAC2 receptors to evaluate the trafficking interactions between CRLR domains and each RAMP. We found that CRLR regions extending from transmembrane domain 1 (TM1) through TM5 are necessary and sufficient for the transport of RAMPs to the plasma membrane. In addition, the extracellular N-terminal domain of CRLR, its 3rd intracellular loop and/or TM6 were also important for the cell-surface translocation of RAMP2, but not RAMP1 or RAMP3. Other regions within CRLR were not involved in trafficking interactions with RAMPs. These findings provide new insight into the trafficking interactions between accessory proteins such as RAMPs and their receptor partners.

Intermedin (IMD), also called adrenomedullin 2 (ADM2), is a 47-amino acid peptide belonging to the calcitonin/calcitonin gene-related peptide (CGRP) family. IMD has similar or more potent vasodilatory and hypotensive actions compared with adrenomedullin (ADM) and CGRP. This study was designed to explore the role of IMD and its receptor in the pathogenesis of spontaneous hypertension and cardiac hypertrophy. Radioimmunoassay was employed to determine plasma immunoreactive IMD concentration and tissue immunoreactive IMD levels in the myocardium and aorta as well as cAMP concentration in the cardiovascular tissues in 13-week-old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). The mRNA expression of IMD, its receptor, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP)) were determined by semi-quantitative RT-PCR. Protein levels of CRLR and RAMPs were assayed by Western blotting. Our results showed that immunoreactive IMD concentration was enhanced in the SHR myocardium, aortas and plasma. Both the mRNA and protein levels of IMD, as well as those of CRLR and RAMP 1-3 were upregulated in SHRs. IMD affected cAMP generation in the myocardium and aorta, which were not attenuated by prior addition of either CGRP(8-37) or ADM(22-52) alone. These results indicate that the elevation of IMD and its receptor in the cardiovascular tissue may play an important role in the pathogenesis of spontaneous hypertension.

Receptor activity modifying protein 1 (RAMP1) is an integral component of several receptors including the calcitonin gene-related peptide (CGRP) receptor. It forms a complex with the calcitonin receptor-like receptor (CLR) and is required for receptor trafficking and ligand binding. The N-terminus of RAMP1 comprises three helices. The current study investigated regions of RAMP1 important for CGRP or CLR interactions by alanine mutagenesis. Modeling suggested the second and third helices were important in protein-protein interactions. Most of the conserved residues in the N-terminus (M48, W56, Y66, P85, N66, H97, F101, D113, P114, P115), together with a further 13 residues spread throughout three helices of RAMP1, were mutated to alanine and coexpressed with CLR in Cos 7 cells. None of the mutations significantly reduced RAMP expression. Of the nine mutants from helix 1, only M48A had any effect, producing a modest reduction in trafficking of CLR to the cell surface. In helix 2 Y66A almost completely abolished CLR trafficking; L69A and T73A reduced the potency of CGRP to produce cAMP. In helix 3, H97A abolished CLR trafficking; P85A, N86A, and F101A had caused modest reductions in CLR trafficking and also reduced the potency of CGRP on cAMP production. F93A caused a modest reduction in CLR trafficking alone and L94A increased cAMP production. The data are consistent with a CLR recognition site particularly involving Y66 and H97, with lesser roles for adjacent residues in helix 3. L69 and T73 may contribute to a CGRP recognition site in helix 2 also involving nearby residues.

Receptor activity-modifying protein (RAMP)-2 and -3 chaperone calcitonin receptor-like receptor (CRLR) to the plasma membrane, where together they form heterodimeric adrenomedullin (AM) receptors. We investigated the contributions made by His residues situated in the RAMP extracellular domain to AM receptor trafficking and receptor signaling by co-expressing hCRLR and V5-tagged-hRAMP2 or -3 mutants in which a His residue was substituted with Ala in HEK-293 cells. Flow cytometric analysis revealed that hRAMP2-H71A mediated normal hCRLR surface delivery, but the resultant heterodimers showed significantly diminished [(125)I]AM binding and AM-evoked cAMP production. Expression of hRAMP2-H124A and -H127A impaired surface delivery of hCRLR, which impaired or abolishing AM binding and receptor signaling. Although hRAMP3-H97A mediated full surface delivery of hCRLR, the resultant heterodimers showed impaired AM binding and signaling. Other His residues appeared uninvolved in hCRLR-related functions. Thus, the His residues of hRAMP2 and -3 differentially govern AM receptor function.

Objective: Rheumatoid arthritis (RA) is characterized by hyperplasia of fibro-blast-like synoviocytes (FLSs), in part due to apoptosis resistance. Adrenomedullin, an anti-apoptotic peptide, is secreted more by RA than osteoarthritis FLSs. Adrenomedullin binds to a heterodimeric functional receptor, of calcitonin receptor-like receptor (CRLR) coupled with a receptor activity-modifying protein-2 (RAMP-2), which is also overexpressed by rheumatoid synoviocytes. Since adrenomedullin decreases RA FLS apoptosis, possibly contributing to the development of pannus, study of adrenomedullin and its receptor genes might reveal a linkage and association in French Caucasian RA trio families., Methods: Within each of 100 families, one RA-affected patient and both parents underwent genotyping for polymorphisms of adrenomedullin, CRLR and RAMP-2, by PCR-restricted fragment-length polymorphism (RFLP) or Taqman 5' allelic discrimination assay. Statistical analysis relied on the transmission disequilibrium test, the affected family-based controls and the genotype relative risk. Haplotypes of CRLR were inferred, and linkage and association studies were performed., Results: No significant transmission disequilibrium or association between the three genes and RA was observed. CRLR haplotypes revealed two major haplotypes, but no significant linkage with RA., Conclusion: Our findings provided no significant linkage or association of adrenomedullin and CRLR-RAMP-2 genes with RA in the studied trio families. The two CRLR polymorphisms rs3771076 and rs3771084 should be investigated in larger samples.

Adrenomedullin (AM) is a peptide involved both in the pathogenesis of cardiovascular diseases and in circulatory homeostasis. The high-affinity AM receptor is composed of receptor activity-modifying protein 2 or 3 (RAMP2 or -3) and the GPCR calcitonin receptor-like receptor. Testing our hypothesis that RAMP2 is a key determinant of the effects of AM on the vasculature, we generated and analyzed mice lacking RAMP2. Similar to AM-/- embryos, RAMP2-/- embryos died in utero at midgestation due to vascular fragility that led to severe edema and hemorrhage. Vascular ECs in RAMP2-/- embryos were severely deformed and detached from the basement membrane. In addition, the abnormally thin arterial walls of these mice had a severe disruption of their typically multilayer structure. Expression of tight junction, adherence junction, and basement membrane molecules by ECs was diminished in RAMP2-/- embryos, leading to paracellular leakage and likely contributing to the severe edema observed. In adult RAMP2+/- mice, reduced RAMP2 expression led to vascular hyperpermeability and impaired neovascularization. Conversely, ECs overexpressing RAMP2 had enhanced capillary formation, firmer tight junctions, and reduced vascular permeability. Our findings in human cells and in mice demonstrate that RAMP2 is a key determinant of the effects of AM on the vasculature and is essential for angiogenesis and vascular integrity.

Growth of blood and lymphatic vessels is essential in the developing embryo and in the pathogenesis of human diseases such as cancer, but the signaling pathways that regulate vessel growth and function are not yet well characterized. In this issue of the JCI, studies by Fritz-Six et al. and Ichikawa-Shindo et al. demonstrate that loss of signaling by the adrenomedullin peptide results in embryonic edema and death (see the related articles beginning on pages 40 and 29, respectively). Remarkably, this phenotype is attributed to defects in lymphatic vessels by one group and to defects in blood vessels by the other. In addition to defining what I believe to be a novel angiogenic signaling pathway, these studies demonstrate the intricate molecular, genetic, and physiologic relationships between blood and lymphatic vessels that must be considered by investigators of vascular biology.

This study investigated the levels of adrenomedullin (AM) and the gene expression of AM, calcitonin receptor-like receptor (CRLR), receptor activity-modifying proteins (RAMPs), and receptor-coupling protein (RCP) in the testis, ventral prostate, seminal vesicle, and epididymis in rats aged 3, 12, and 20 months by radioimmunoassay and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The results indicate an age-related increase in AM and its messenger RNA (mRNA) levels in the testis and decrease in the sex accessory glands. The gene expression of CRLR and RCP decreased only in the sex accessory glands. The changes in the gene expression of RAMPs suggest that the major increase was in CGRP receptors in the testis, whereas the major decreases in the ventral prostate and the seminal vesicles might be CGRP and AM receptors, respectively. The decreases in these receptors were similar in the epididymis. The results suggest possible roles of AM in the male reproductive system during aging.

Calcitonin-like receptor (CL-R) is a functional CGRP1-receptor when complexed with RAMP1 or an adrenomedullin-receptor or when complexed with RAMP2 or RAMP3. This study was carried out 1. to set up a method to examine the relative quantity of mRNA of CL-R, RAMP1, RAMP2 and RAMP3 in human coronary (CA), pulmonary (PA) and middle cerebral arteries (MCA), and 2. to examine the level of mRNA expression in cerebra- and cardiovascular diseases. The method was validated with respect to the use of postmortem tissue and we compared beta-actin and GAPDH as housekeeping genes. There was no time-dependent change in total RNA and level of mRNA for p-actin or GAPDH could be detected in vessels removed from 1 and 5 days post mortem. The expression of beta-actin appears lower in coronary artery than in pulmonary artery and middle cerebral artery with no significant difference for GAPDH; both worked well. There were some differences in mRNA expression for CL-R (higher) and RAMP3 (lower) in middle cerebral artery compared to coronary artery and pulmonary artery. There was no significant difference in mRNA for RAMP1 and RAMP2 in the three types of arteries. We did not observe any difference in mRNA for CL-R and RAMPs in arteries from patients with hemorrhagic stroke, arteriosclerosis and acute myocardial infarction when compared to patients without these diagnoses. Thus the mRNA expression seems to be unaltered in these disorders.

Objective: To investigate the changes of pulmonary IMD and its receptor system-calcitonin receptor-like receptor (CL) and receptor activity modifying proteins (RAMPs) mRNA in acute lung injury(ALI) induced by oleic acid of rats., Methods: Contents of IMD in plasma and lung homogenates were measured by radioimmunoassay(RIA). The lung mRNA of IMD, CL and RAMPs was determined by semi-quantitative RT-PCR., Results: Compared with control group, in ALI group, the contents of IMD1-53 in plasma and lung homogenates were decreased by 20.8% and 74.5% (all P < 0.05) , respectively. Furthermore , it was found that the levels of IMD, CL, RAMP1 and RAMP2 mRNA in lung were decreased by 30%, 38%, 26% and 37.9% (all P < 0. 05) , respectively. The levels of CL , RAMP1 or RAMP2 mRNA were positively correlated with down-regulations of IMD mRNA in ALI. However, compared with control group, the maximum binding capacity of IMD1-53 to plasma membranes was significantly increased in ALI group, and the affinity of IMD1-53 for its receptor almost had no change., Conclusion: The amount of IMD 1-53 is down-regulated and IMD receptor system also down-regulated in Oliec acid induced ALI of rats. These changes suggest that IMD and its receptor system probably are involved in the development of ALI.

Adrenomedullin (AM) is a multifunctional peptide hormone, which plays a significant role in vasodilation and angiogenesis, implicating it in hypertension as well as in carcinogenesis. AM exerts its effects via the calcitonin receptor-like receptor (CRLR, now known as CL) complexed with either receptor activity modifying protein (RAMP) 2 or 3. We have investigated the effect of AM on immortalized human microvascular endothelial cells 1, since endothelial cells are a major source as well as a target of AM actions in vivo. Cells treated with AM showed elevated cAMP in a time (5-45 min)-dependent and dose (10(-6)-10(-14) M)-dependent manner. Pre-treatment with the AM receptor antagonist AM(22-52) partially suppressed the AM-induced increase in cAMP levels. An increase in extracellular signal-regulated kinase 1/2 phosphorylation was observed after 5 min of treatment with 10(-8) M AM. This phosphorylation was specific, since we were able to block the AM-induced effect with 1 microM U0126, a specific mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor. Using real-time PCR, we were able to show for the first time that AM upregulates peptide and mRNA expression of vascular endothelial growth factor (VEGF). However, AM treatment of cells did not result in increased cell proliferation. Instead, we observed that AM and VEGF induced cell migration, which could be inhibited by the AM(22-52) and anti-VEGF antibody respectively. AM also significantly elevated mRNA levels of CL (after 2 and 24 h treatment) and RAMP2 (after 1 and 24 h treatment). The upregulation of the AM receptor at two time points reflects possibly different cellular responses to short- and long-term exposure to AM.

Adrenomedullin (ADM) has been shown to be present in the human and rat male reproductive systems. This study demonstrates the expression of ADM in the rat testis and its effect on the secretion of testosterone. Whole testicular extracts had 5.43 +/- 0.42 fmol of immunoreactive ADM per milligram of protein and 84 +/- 8 fg of ADM mRNA per picogram of Actb (beta-actin) mRNA. Immunocytochemical studies showed positive ADM immunostaining in the Leydig cells and in the Sertoli cells. Gel filtration chromatography of testicular extracts showed two peaks, with the predominant one eluting at the position of the ADM precursor. Furthermore, the testis was shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity modifying protein 1 (Ramp1), Ramp2, and Ramp3. These account for the specific binding of ADM to the testis, which was partially inhibited by human ADM (22-52) and by human calcitonin gene-related peptide (8-37), the ADM and calcitonin gene-related peptide receptor antagonists, respectively. Administration of ADM to testicular blocks in vitro resulted in a dose-dependent inhibition of hCG-stimulated release of testosterone, which was abolished by the administration of ADM (22-52). Our results suggest a paracrine effect of ADM on testicular steroidogenesis.

This study was undertaken to determine AM expression in carbon tetrachloride (CCl4)-induced liver cirrhosis developed with peritoneal ascites. Sprague-Dawley rats received subcutaneous injections of CCl4 twice weekly in olive oil (1:1, 0.3 ml per kg body weight) for 6 or 12 weeks until ascites developed, or saline in olive oil as control. At 6 weeks, fibrosis developed and at 12 weeks cirrhosis developed with ascites formation. At both 6 and 12 weeks, increases in plasma renin and AM were evident, as was the gene expression of AM. At 12 weeks after CCl4 injection, the gene expression of calcitonin-like-receptor (CRLR) and receptor activity modifying proteins (RAMP1, RAMP2 and RAMP3) were all elevated when compared to the control. The results suggest that liver cirrhosis increases mRNA expressions of AM, CRLR and RAMP1, RAMP2 and RAMP3 and that the increase in AM gene expression precedes the development of cirrhosis. The increase in AM synthesis as reflected by an increase in AM gene expression, together with a lack of increase in AM peptide at both 6 and 12 weeks may suggest an elevation of AM release. Given the potent vasodilatory action of AM, the increase in the synthesis and release of AM in the cirrhotic liver may also contribute to peripheral vasodilatation in liver cirrhosis.

Calcitonin receptors (CTRs) dimerize with receptor activity-modifying proteins (RAMPs) to generate high-affinity amylin (AMY) receptors; however, the relative contribution of individual RAMP domains to the formation of AMY receptors is poorly understood. We have used chimeras between RAMP1 and RAMP2 that specifically exchanged the N-terminal, transmembrane, or C-terminal domain and examined these in assays of [(125)I]amylin binding or peptide-induced cAMP signaling in COS-7 cells transiently transfected with wild-type or chimeric RAMPs and human CTRa. The specificity of peptides in competition for [(125)I]amylin binding was principally dictated by the N-terminal domain present in the chimeras; however, the maximal level of binding induced was dictated by the transmembrane domain present. This extended previous data (Zumpe et al., 2000) to provide a distinction between the transmembrane domain and the C terminus in this function. In contrast to the effects on binding, each of the RAMP domains played a role in the signaling phenotype of the receptors. In particular, the potency of calcitonin gene-related peptide (CGRP) was most influenced by the C-terminal domain present, in which the presence of the RAMP1 C-terminal domain led to increased potency over CTRa alone, whereas chimeras with the RAMP2 C-terminal domain did not induce increased CGRP potency. The data provide additional support for the importance of the N terminus in determining binding affinity but reveal a prominent role of the transmembrane domain in the strength of amylin binding and a unique role for the C terminus in signaling by peptides to stimulate cAMP production.

Calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene related peptide (CGRP) and intermedin. Although CGRP is widely expressed in the nervous system, less is known about the localization of CLR and RAMP1. To localize these proteins, we raised antibodies to CLR and RAMP1. Antibodies specifically interacted with CLR and RAMP1 in HEK cells coexpressing rat CLR and RAMP1, determined by Western blotting and immunofluorescence. Fluorescent CGRP specifically bound to the surface of these cells and CGRP, CLR, and RAMP1 internalized into the same endosomes. CLR was prominently localized in nerve fibers of the myenteric and submucosal plexuses, muscularis externa and lamina propria of the gastrointestinal tract, and in the dorsal horn of the spinal cord of rats. CLR was detected at low levels in the soma of enteric, dorsal root ganglia (DRG), and spinal neurons. RAMP1 was also localized to enteric and DRG neurons and the dorsal horn. CLR and RAMP1 were detected in perivascular nerves and arterial smooth muscle. Nerve fibers containing CGRP and intermedin were closely associated with CLR fibers in the gastrointestinal tract and dorsal horn, and CGRP and CLR colocalized in DRG neurons. Thus, CLR and RAMP1 may mediate the effects of CGRP and intermedin in the nervous system. However, mRNA encoding RAMP2 and RAMP3 was also detected in the gastrointestinal tract, DRG, and dorsal horn, suggesting that CLR may associate with other RAMPs in these tissues to form a receptor for additional peptides such as adrenomedullin., ((c) 2005 Wiley-Liss, Inc.)

Calcitonin (CT) receptors dimerize with receptor activity-modifying proteins (RAMPs) to create high-affinity amylin (AMY) receptors, but there is no reliable means of pharmacologically distinguishing these receptors. We used agonists and antagonists to define their pharmacology, expressing the CT(a) receptor alone or with RAMPs in COS-7 cells and measuring cAMP accumulation. Intermedin short, otherwise known as adrenomedullin 2, mirrored the action of alpha CGRP, being a weak agonist at CT(a), AMY(2a), and AMY(3a) receptors but considerably more potent at AMY(1a) receptors. Likewise, the linear calcitonin gene-related peptide (CGRP) analogs (Cys(ACM)(2,7))h alpha CGRP and (Cys(Et)(2,7))h alpha CGRP were only effective at AMY(1a) receptors, but they were partial agonists. As previously observed in COS-7 cells, there was little induction of the AMY(2a) receptor phenotype; thus, AMY(2a) was not examined further in this study. The antagonist peptide salmon calcitonin(8-32) (sCT(8-32)) did not discriminate strongly between CT and AMY receptors; however, AC187 was a more effective antagonist of AMY responses at AMY receptors, and AC413 additionally showed modest selectivity for AMY(1a) over AMY(3a) receptors. CGRP(8-37) also demonstrated receptor-dependent effects. CGRP(8-37) more effectively antagonized AMY at AMY(1a) than AMY(3a) receptors, although it was only a weak antagonist of both, but it did not inhibit responses at the CT(a) receptor. Low CGRP(8-37) affinity and agonism by linear CGRP analogs at AMY(1a) are the classic signature of a CGRP2 receptor. Our data indicate that careful use of combinations of agonists and antagonists may allow pharmacological discrimination of CT(a), AMY(1a), and AMY(3a) receptors, providing a means to delineate the physiological significance of these receptors.

The calcitonin receptor-like receptor (CLR) requires the associated receptor activity-modifying protein (RAMP)1 to reveal a calcitonin gene-related peptide (CGRP) receptor. Here, the subdomain of the CLR that associates with RAMP1 has been identified in chimeras between the CLR and the parathyroid hormone (PTH) receptor 1 (PTHR). The PTHR alone does not interact with RAMP1. RAMP1 requires the CLR for its transport to the cell surface. Thus, receptor-dependent RAMP1 delivery to the plasma membrane and coimmunoprecipitation from the cell surface were used as measures for receptor/RAMP1 interaction. Several chimeric CLR-PTHR included the N-terminal amino acids 23-60 of the CLR transported RAMP1 to the surface of COS-7 cells much like the intact CLR. Moreover, RAMP1 coimmunoprecipitated with these receptors from the cell surface. A CLR deletion mutant, consisting of the N-terminal extracellular domain, the first transmembrane domain, and the C-terminal intracellular region, revealed the same results. Cyclic AMP was stimulated by CGRP in CLR/RAMP1 expressing cells (58 +/- 19-fold, EC(50) = 0.12 +/- 0.03 nM) and by PTH-related protein in cells expressing the PTHR (50 +/- 10-fold, EC(50) = 0.25 +/- 0.03 nM) or a PTHR with the N-terminal amino acids 23-60 of the CLR (23 +/- 5-fold, EC(50) > 1000 nM). Other chimeric CLR-PTHR were inactive. In conclusion, structural elements in the extreme N-terminus of the CLR between amino acids 23-60 are required and sufficient for CLR/RAMP1 cotransport to the plasma membrane and heterodimerization.

Specific adrenomedullin receptors have been identified as calcitonin receptor-like receptor (CRLR)/receptor activity-modifying proteins (RAMP2 and RAMP3) complexes. Although we have demonstrated that adrenomedullin is increased in volume overload-induced cardiac hypertrophy, it remains unknown whether the adrenomedullin receptor is altered or not. This study sought to investigate the significance of intracardiac adrenomedullin and its receptor system in volume overload-induced cardiac hypertrophy. Left ventricular adrenomedullin levels were higher in aortocaval shunt (ACS) rats than in controls (+58%). The left ventricular gene expressions of adrenomedullin, CRLR, RAMP2 and RAMP3 were increased (+27%, +76%, +108% and +131%, respectively) and the left ventricular collagen gene expressions were also increased (type I: +138%, type III: +87%). The left ventricular adrenomedullin level correlated with the gene expression of type III collagen (R=0.42). These results suggest that intracardiac adrenomedullin and its receptor system are upregulated and may participate in the regulation of cardiac remodeling in volume overload-induced cardiac hypertrophy.

Adrenomedullin (AM) expressed by and secreted from a variety of cells plays pluripotent roles in an autocrine/paracrine fashion. The present study was undertaken to explore the expression of AM and its receptor genes in adipose tissues, their changes during the development of obesity, and the process of preadipocyte differentiation. Both mature adipocytes and stromal vascular cells constituting adipose tissue expressed AM transcript. AM and its receptor component [calcitonin receptor-like receptor and receptor activity-modifying protein-2 (CRLR/RAMP2)] mRNAs were expressed in a variety of rat adipose tissues, including epididymal, mesenteric, retroperitoneal, and subcutaneous adipose tissue. AM mRNA levels in rat and human epididymal adipose tissue were about one-tenth of those in the kidney. Steady-state mRNA levels of AM and CRLR/RAMP2 in epididymal, mesenteric, and retroperitoneal adipose tissues in rats fed a high-fat diet for 4 wk were far greater than those in rats with normal diet accompanied by increased plasma AM levels, whereas steady-state AM mRNA levels conversely decreased in other organs, such as kidney and liver. AM mRNA expressed in a mouse preadipocyte cell line (3T3-L1) transiently decreased by day 3, returned to basal level by day 6, and then increased by day 9 during preadipocyte differentiation, which paralleled AM secretion from the cells. However, the addition of either exogenous AM or AM receptor antagonist calcitonin gene-related peptide-(8-37), to block endogenous AM did not affect lipid droplet accumulation during preadipocyte differentiation. The present study demonstrates for the first time that AM and its receptor component (CRLR/RAMP2) mRNAs were concomitantly expressed in various adipose tissues, whose tissue-specific upregulation was induced during the development of obesity. These data suggest that AM may act as a new member of adipokines, although its functional role, as well as its pathophysiological significance in obesity, remains to be determined.

Adrenomedullin (ADM) is a potent stimulator of osteoblastic activity and promotes bone growth in vivo. ADM receptors are formed by heterodimerization of the CRLR and a RAMP2 or RAMP3 molecule. Since glucocorticoid responsive elements were recently identified in the human CRLR promoter and that glucocorticoids exert a major action in bones, we investigated the acute effect of dexamethasone (Dex) treatment on ADM receptor components in osteoblastic cell types: the MC3T3-E1 cells and calvaria-derived osteoblastic cells. Changes in expression of CRLR and RAMPs molecules were evaluated at mRNA levels using RT-PCR and at protein levels by Western blot analysis. We found that Dex increased expression of RAMP1 and RAMP2 mRNA in a time-dependent but dose-independent manner, while RAMP3 was unchanged. In contrast, Dex decreased the CRLR mRNA expression and these changes were reflected at protein levels. We suggest that Dex, in osteoblastic cells, altered ADM receptor by inhibition of CRLR expression and consequently could impair the ADM anabolic effect on bone. Our findings could explain in part, the detrimental side effects observed at bone level during glucocorticoid therapy.

Vascular calcification is a common finding in many cardiovascular diseases. Paracrine/autocrine changes in calcified vessels, and the secreted factors participate in and play an important role in the progress of calcification. Adrenomedullin (ADM) is a potent vasodilator peptide secreted by vascular smooth muscle cells (VSMCs) and vascular endothelial cells. Recently, receptor activity-modifying proteins (RAMPs) have been shown to transport calcitonin receptor-like receptor (CRLR) to the cell surface to present either as CGRP receptor or ADM receptor. In this work, we explored the production of ADM, alterations and significance of ADM mRNA and its receptor system components--CRLR and RAMPs mRNA in calcified VSMCs. Our results showed that calcium content, 45Ca2+ uptake and alkaline phosphatases (ALPs) activity in calcified VSMCs were increased, respectively, compared with control VSMCs. Content of ADM in medium was increased by 99% (p < 0.01). Furthermore, it was found that the levels of ADM, CRLR, RAMP2 and RAMP3 mRNA in calcified cells were elevated, respectively, compared with that of control. The elevated levels of CRLR, RAMP2 and RAMP3 mRNA were significant correlation with ADM mRNA (r = 0.83, 0.92 and 0.93, respectively, all p's < 0.01) in calcified VSMCs. The results show that calcified VSMCs generate an increased amount of ADM, up-regulate gene expressions of ADM and its receptor system components--CRLR, RAMP2 and RAMP3, suggesting an important role of ADM and its receptor system in the regulation of vascular calcification.

Objective: Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a calcitonin gene related peptide (CGRP) receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an adrenomedullin(ADM) receptor. As CGRP and ADM may play a beneficial role in heart failure, this study aimed at the question whether RAMPs mRNAs are changed in heart failure., Methods: Semi-quantitative reverse transcription-PCR (RT-PCR) was used to detect and quantify the mRNAs of RAMP1 and RAMP3 in the atria of heart failing patients., Results: It was found that the expressions of RAMP1, RAMP2 and RAMP3 mRNAs increased with the worsening of heart function, but the expressions of RAMP1 and RAMP2 mRNA decreased at level IV of heart failure., Conclusion: The above results demonstrated in the atria of heart failure patients an up-regulation of CGRP receptor by an increase of RAMP1 in association with CRLR and an up-regulation of ADM receptor by an increase of RAMP2 expression in association with CRLR, thus suggesting that CGRP and ADM receptors be playing a functional role in compensating the chronic heart failure in human.

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.

Objective: To explore the production of ADM, changes and significance of adrenomedullin (ADM) mRNA and ADM receptor system-calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) mRNA in calcified myocardium and aorta of rats., Methods: Contents of ADM in plasma, myocardium and vessel were measured by radioimmunoassay (RIA). The amount of ADM, CRLR and RAMPs mRNA was determined by semi-quantitative RT-PCR., Results: The contents of calcium in calcified myocardium and aorta were increased by 342% and 606% (all P<0.01), respectively, and alkaline phosphatase activities in calcified myocardium and vessel were increased by 66.5% and 82.7% (all P<0.01), respectively, compared with the control. Contents of ADM in plasma, myocardium and aorta were increased by 58 % (P<0.01), 14.3%(P<0.01) and 27.8%(P<0.05). Furthermore, it was found that the levels of ADM, CRLR and RAMP2 mRNA in calcified myocardium were elevated by 90.6% 157.5% and 119.6%(all P<0.01), respectively, but RAMP3 mRNA was decreased by 14.1 %(P<0.01). The levels of ADM, CRLR, RAMP2 and RAMP3 mRNA in calcified aorta were elevated by 37.7%(P<0.01), 41.4%(P<0.01),60.1%(P<0.05) and 13%(P<0.01) respectively, compared with the control. The elevated levels of CRLR, RAMP2 or RAMP3 mRNA were positively correlated with up regulations of ADM mRNA in calcified myocardium and aorta., Conclusion: Calcified myocardium and aorta may generate an increased amount of ADM, different alterations of gene expressions of ADM and its receptor system. These RESULTS suggest that ADM and its receptor system might participate in the regulation of calcification in heart and vessel.

Adrenomedullin (ADM) is a potent vasodilatory peptide which regulates blood pressure, cell growth and bone formation. Our work was aimed to explore the production of ADM, changes and pathophysiological significance of ADM mRNA and ADM receptor components--calcitonin receptor like receptor (CRLR) and receptor activity modifying proteins (RAMPs) mRNA in calcified myocardium and aorta of rats induced by Vitamin D3 plus nicotine. Contents of ADM in plasma, myocardium and aorta were measured by radioimmunoassay (RIA). The amount of ADM, CRLR and RAMPs mRNA was determined by semi-quantitative RT-PCR. The calcium content and alkaline phosphatase activity in myocardium and aorta of rats were measured. The results showed that the contents of calcium in calcified myocardium and aorta were increased by 3.5- and 6-fold (all P < 0.01), respectively, and alkaline phosphatases activity in calcified myocardium and aorta were increased by 66.5 and 82.7% (all P < 0.01 ), respectively, compared with control. Contents of ADM in plasma, myocardium and aorta were increased by 58% (P < 0.01), 14.3% (P < 0.01) and 27.8% P < 0.05). Furthermore, it was found that the amount of ADM, CRLR and RAMP2 mRNA in calcified myocardium was elevated by 90.6, 157.5 and 119.6% (all P < 0.01), RAMP3 mRNA was decreased by 14.1% (P < 0.01), respectively, compared with control. The amount of ADM, CRLR, RAMP2 and RAMP3 mRNA in calcified aorta was elevated by 37.7% (P < 0.01), 41.4% (P < 0.01), 60.1% (P < 0.05) and 13% P < 0.01), respectively, compared with control. The elevated level of CRLR and RAMP2 mRNA were in positive correlation with that of ADM mRNA (r = 0.992 and 0.882, respectively, P < 0.01) in calcified myocardium. The elevated level of CRLR and RAMP3 mRNA were also in positive correlation with that of ADM mRNA (r = 0.727, P < 0.05 and 0.816, P < 0.01, respectively) in calcified aorta. These results demonstrated that calcified myocardium and aorta generated an increased amount of ADM, up-regulated gene expressions of ADM, CRLR and RAMP2 mRNA. While the alteration of RAMP3 mRNA in calcified myocardium and aorta was different. These suggested that ADM and its receptor system might involve in the regulation of calcification in heart and aorta., (Copyright 2004 Elsevier Inc.)

The pancreatic hormone amylin (AMY) and the AMY-receptor-agonist salmon-calcitonin (sCT) reduce short-term food-intake after binding to the area postrema (AP), a circumventricular organ (CVO) lacking blood-brain-barrier characteristics. AMY has also been proposed to induce drinking via another CVO, the subfornical organ (SFO). In cellular systems, AMY-binding is generated by interaction of calcitonin-receptor a/b (CT((a))/CT((b))) with receptor-activity modifying proteins (RAMPs). By using in situ hybridization, the codistribution of CT((a))/CT((b)) with RAMP1-3 and c-fos was mapped in CVOs of rats. AMY and sCT induced c-fos within the SFO which contained CT((a)) and/or CT((b)) and RAMP1/2 mRNA. AMY and sCT also activated AP neurons, which express the CT((a)), but not the CT((b)), receptor and RAMP2/3 mRNA. These data emphasize the important role of these structures as primary targets for circulating AMY.

Coexpression of receptor activity-modifying proteins (RAMPs) with calcitonin receptor 2 (CTR2) or calcitonin receptor-like receptor (CRLR) leads to the formation of four functional heterodimeric receptors for human calcitonin gene-related peptide (hCGRP). In this study, we transfected hCGRP receptors into human embryonic kidney 293 cells and examined their pharmacological profiles using three dominant-negative (DN) RAMP mutants and various hCGRPalpha analogs. Fluorescence-activated cell-sorting analysis revealed that their cotransfection with CTR2 induced cell surface expression of all three RAMPs, and the three CTR2/RAMP heterodimers mediated equivalent levels of cAMP production in response to hCGRPalpha that were approximately 50-fold greater than were seen with CTR2 alone. By contrast, [Tyr0]hCGRPalpha binding and signaling were markedly weaker with CTR2/RAMP2 or -3 than with CTR2/RAMP1 or CRLR/RAMP1; likewise, 125I-[His10]hCGRPalpha bound most potently to CTR2/RAMP1. When CTR2 was coexpressed with DN RAMP1 or -2, hCGRPalpha-evoked responses were similar to those seen with CTR2 alone, despite the expression of both CTR2 and DN RAMP at the cell surface. But coexpression of DN RAMP3 with CTR2 significantly diminished hCGRPalpha signaling compared with that seen with CTR2 alone, indicating that DN RAMP3 is able to function as a negative regulator of CTR2 function. Competition experiments showed the relative agonist sensitivity of the four receptors to be hCGRPalpha > [Tyr0]hCGRPalpha > [Cys(Et)2,7]hCGRPalpha > [Cys(ACM)2,7]hCGRPalpha. Of the linear analogs, [Cys(ACM)2,7]hCGRPalpha (ACM, acetylmethoxy) enhanced cAMP formation only via CTR2/RAMP1, whereas [Cys(Et2,7)]hCGRPalpha acted via CRLR/RAMP1 and somewhat less potently via CTR2/RAMP1. Thus, among the three CGRP8-37-insensitive receptors, CTR2/RAMP1 is most sensitive to the two linear analogs, suggesting that it could be classified as a CGRP2 receptor. Moreover, the combined use of iodinated CGRPalpha analogs may be useful for defining the CGRP1 receptor.

The receptor-activity-modifying protein (RAMP) 1 is a single-transmembrane-domain protein associated with the calcitonin-like receptor (CLR) to reveal a calcitonin gene-related peptide (CGRP) receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys(27) in myc-tagged human (h) RAMP1 was deleted (hRAMP1Delta1), and Cys(40), Cys(57), Cys(72), Cys(82) and Cys(104) were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Delta1 or -C82A, cell surface expression, [(125)I]halphaCGRP binding and cAMP formation in response to halphaCGRP were similar to those of hCLR/myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24+/-7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [(125)I]halphaCGRP binding of hCLR/myc-hRAMP1-C72A was 13+/-3% of hCLR/myc-hRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A- and -C104A-expressing cells. Maximal cAMP stimulation by halphaCGRP in hCLR/myc-hRAMP1-C40A- and -C72A-expressing cells was 14+/-1% and 33+/-2% of that of the hCLR/myc-hRAMP1 with comparable EC(50). But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP receptor function was not affected by the deletion of Cys(27) or the substitution of Cys(82) by Ala in hRAMP1, but it was impaired by the substitution of Cys(40), Cys(57), Cys(72) and Cys(104) by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.

Calcitonin gene-related peptide (CGRP) and its related peptide, adrenomedullin (AM), are potent smooth muscle relaxants in a variety of tissues. The CGRP has been reported to play an important role in maintaining uterine relaxation during pregnancy. We have previously reported that CGRP-induced uterine relaxation was gestationally regulated. Calcitonin receptor-like receptor (CRLR), a seven-domain transmembrane protein functions as CGRP-A receptor, in association with receptor activity-modifying protein (RAMP) 1, a single-domain transmembrane protein, whereas CRLR and RAMP2 or RAMP3 constitute a receptor for AM. In the present investigation, we examined the mRNA expression of CRLR, RAMP1, RAMP2, and RAMP3 in rat uterus (n = 8) by reverse transcriptional analysis and polymerase chain reaction to assess the changes in the expression of CGRP-A- and AM-receptor components during pregnancy and labor and by steroid hormone treatments in adult ovariectomized rats. The changes in mRNA are expressed relative to the 18S mRNA in the uterus of rats at various stages: nonpregnant, pregnant on Day 18, spontaneous labor at term, Day 2 postpartum, and in pregnant rats on treatment with RU486. Ovariectomized rats treated for 3 days twice daily s.c. with estradiol-17beta (2.5 microg/injection), progesterone (2 mg/injection), and the combination of estradiol-17beta and progesterone (same doses as above) were also examined for the expression of various receptor components. Results showed that mRNA expression of the receptor components was significantly higher (P < 0.001 for CRLR, P < 0.01 for RAMP1, P < 0.05 for RAMP2, and P < 0.01 for RAMP3) in pregnant compared to nonpregnant rats. Except for RAMP3, expression of all the other three genes decreased significantly (P < 0.05) during labor. A progesterone antagonist, RU486 significantly decreased (P < 0.01 for CRLR, P < 0.05 for RAMP1, RAMP2, and RAMP3) all the receptor components during pregnancy. In adult ovariectomized rats, progesterone caused significant increases in CRLR (P < 0.001), RAMP1 (P < 0.05), and RAMP2 (P < 0.01). Levels of RAMP3 were unaffected by the progesterone treatment. Estradiol-17beta treatment decreased all of the four receptor components significantly (P < 0.01 for CRLR, P < 0.05 for RAMP1, RAMP2, and RAMP3). Our results demonstrate that both CGRP and AM may play a role in uterine quiescence during pregnancy and that their receptor components are regulated by the steroid hormones.

The neuropeptide, adrenomedullin, acts in the central nervous system (CNS) to regulate blood pressure, at least partly through an adrenomedullin receptor which is composed of receptor activity modifying protein 2 (RAMP-2) and calcitonin receptor-like receptor (CRLR). We used in situ hybridization to localize RAMP-2 mRNA throughout the brain, and we performed reverse transcription-polymerase chain reaction to detect CRLR mRNA in the brain. We found that RAMP-2 mRNA is expressed in numerous areas, including autonomic nuclei such as the paraventricular, supraoptic, arcuate and ventromedial nuclei, as well as the nucleus of the solitary tract (NTS), area postrema and dorsal motor nucleus of the vagus. Many regions expressing RAMP-2 mRNA also express low levels of CRLR mRNA. We examined changes in the mRNA expression of RAMP-2 and preproadrenomedullin in the brain in response to blood pressure manipulations. Rats received intravenous infusions of nitroprusside or phenylephrine to decrease or increase blood pressure, respectively. Decreased blood pressure elicited an increase in RAMP-2 mRNA expression in the NTS and a decrease in preproadrenomedullin mRNA expression in the paraventricular nucleus (PVN). Increased blood pressure elicited a decrease in RAMP-2 mRNA expression in the PVN and NTS. The CNS distribution and modulation of adrenomedullin signalling components by changes in blood pressure provide anatomical and physiological evidence for a homeostatic role for adrenomedullin in the brain.

Both adrenomedullin (AM) and angiotensin II (Ang II) are locally-acting hormones in the cardiac ventricles. Previously we reported that AM inhibits Ang II-induced hypertrophy of cultured rat neonatal cardiomyocytes. In this study, we examined whether Ang II affects the gene expression of the AM receptor components of calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein (RAMP) in rat cardiomyocytes. The mRNA levels of RAMP1 and RAMP3 were significantly elevated following 24-h treatment with Ang II without a change of those of RAMP2 and CRLR. AM increased the intracellular cAMP level and the cAMP accumulation by AM was significantly amplified by the 24-h preincubation with Ang II. The effects of Ang II on RAMP1 and RAMP3 expression were abolished by an Ang II type 1 (AT1) receptor antagonist, but not by an AT2 receptor antagonist. Thus, Ang II modulates gene expression of the AM receptor components via AT1 receptor, suggesting alteration of AM actions by Ang II in cultured rat cardiomyocytes.

Calcitonin gene-related peptide (CGRP), one of the most potent endogenous vasodilators known, has been implicated in vascular adaptations and placental function during pregnancy. The present study was aimed to investigate mRNA expression of CGRP-A receptor components, calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP(1)) in the rat placenta. Immunohistochemical staining of rat placentas obtained on day 18 of pregnancy using polyclonal anti-CRLR and RAMP(1) antibodies revealed that labelling was specifically concentrated in the cytotrophoblast and syncytium in labyrinth, trophoblastic giant cells and basophilic cells in trophospongial cell layer, and endothelium and smooth muscle cells in fetal vessels. The intensity of staining was reduced in all these cells except in the syncytium in placentas obtained during labour. RT-PCR analysis showed that mRNA expression of CRLR and RAMP(1) was significantly higher in the rat placenta from gestation day 17 to day 22, than during labour. During pregnancy, 17beta-estradiol inhibits, while progesterone stimulates, placental mRNA and proteins for CRLR and RAMP(1). Antiestrogen, ICI 182780, increased, whereas antiprogesterone, RU 486, inhibited the expression of both CRLR and RAMP(1). In summary, we demonstrate the presence and cellular localization of CRLR and RAMP(1) in the rat placenta. Elevated placental CRLR and RAMP(1) may be involved in CGRP-related increases in blood flow and therefore fetal growth and decreases at term labour may help minimize the blood loss.

Adrenomedullin is a hypotensive peptide secreted from various cells. Recently, we found that adrenomedullin, but not calcitonin gene-related peptide (CGRP), stimulates histamine release from rat peritoneal mast cells. In the present studies, we investigated the expression of mRNA for calcitonin-receptor-like receptor (CRLR) and receptor-activity modifying proteins (RAMPs), the components of proposed adrenomedullin receptors, in rat peritoneal mast cells by reverse transcription-polymerase chain reaction (RT-PCR). Results revealed that mRNA for CRLR, RAMP2 and RAMP3 was expressed in rat peritoneal mast cells, whereas mRNA for RAMP1 was not. These data suggest that adrenomedullin might stimulate histamine release via its proposed receptor (CRLR/RAMP2 or 3), rather than via the CGRP receptor (CRLR/RAMP1).

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.

When co-expressed with receptor activity-modifying protein (RAMP) 2, calcitonin receptor-like receptor (CRLR) functions as an adrenomedullin (AM) receptor (CRLR/RAMP2). In the present study, we examined the function of the cysteine (C) residues in the extracellular loops of human (h)CRLR (C212, C225 and C282) and in the extracellular domain of hRAMP2 (C68, C84, C99 and C131). Using site-directed mutagenesis, the cysteine residues were substituted, one at a time, with alanine (A). Co-expression in HEK293 cells of hRAMP2 with the hCRLR C212A or C282A mutant significantly reduced the 50% of effective concentration (EC50) for AM-evoked cyclic adenosine monophosphate (cAMP) production, despite full cell surface expression of the receptor heterodimer. Co-expression of the C225A mutant had no effect on [125I]AM binding or receptor signaling. These results suggest that the cysteine residues in the first (C212) and the second (C282) extracellular loops form a disulfide bond that is important for stabilizing the receptor in the correct conformation for ligand binding and activation. Cells expressing hCRLR with an hRAMP2 mutant (C68A, C84A, C99A or C131A) showed no specific AM binding or AM-stimulated cAMP accumulation. Though abundant in the intracellular compartment, these receptors were not detected at the cell surface, suggesting that all four cysteine residues are essential for efficient transport to the plasma membrane. Cysteine residues in the extracellular loops of hCRLR and in the extracellular domain of hRAMP2 thus appear to play distinct roles in the cell surface expression and function of the receptor heterodimer.

Adrenomedullin (AM), calcitonin gene-related peptide (CGRP), amylin (AMY) and calcitonin (CT) are members of the CGRP/CT superfamily of peptides. Among them, AM and CGRP are reported to share a core receptor, the calcitonin receptor-like receptor (CRLR), and the specificity of the CRLR is determined by the expression levels of receptor-activity-modifying proteins (RAMPs). In the case of AMY, co-expression of the calcitonin receptor (CTR) and RAMPs was recently reported to form its specific receptor. However, detailed analysis of the receptor specificity of the CRLR and CTR in the presence of RAMPs has so far been performed mainly in the human system. Thus, we cloned cDNAs encoding porcine CRLR, RAMP1, RAMP2 and RAMP3 precursors from a porcine lung and hypothalamus cDNA library, and determined their sequences. Then, porcine RAMPs, CRLR and CTR were expressed in COS-7 or porcine vascular smooth muscle cells, and the resulting receptor complexes were analyzed by the cyclic adenosine 3,5-monophosphate (cAMP) production assay. The specificity of CRLR was clearly determined by the expression of RAMPs; RAMP1 converted CRLR to CGRP receptor, while RAMP2 and RAMP3 converted it to AM receptor, but the affinity of CTR for AMY was not increased by the expression of any known RAMPs. In contrast to previous findings, porcine CTR and RAMP did not appear to form an AMY receptor having sufficient affinity and specificity for the physiological interaction.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are potent vasodilators in humans and improved myocardial ischemia is observed after CGRP administration. Receptors for CGRP and ADM were already identified in heart. Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a CGRP receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an ADM receptor. As CGRP and ADM may play a beneficial role in heart failure, we investigated whether the CGRP and ADM receptors are upregulated in chronic heart failure. We have used semi-quantitative RT-PCR and Western-blot analysis to detect and quantify the mRNA and the protein of RAMP1 and RAMP3 in both atria and ventricles of failing hearts 6 months after aortic banding in rats. Our results showed for the first time an up-regulation of RAMP1 and RAMP3 mRNAs and proteins in this model of cardiac failure. No change was observed in mRNAs coding for CRLR, RAMP2, RDC1 (canine orphan receptor), and ADM. The present results suggested after congestive heart failure in adult rats, an up-regulation of the CGRP receptor (by an increase in RAMP1 that is associated with CRLR) in atria and ventricles and of ADM receptor (by increased RAMP3 expression that is associated with CRLR) in atria. These findings support a functional role for CGRP and ADM receptors to compensate the chronic heart failure in rats., ((c) 2002 Elsevier Science (USA).)

Receptor activity modifying protein-3 (RAMP-3) has been shown to complex with the calcitonin receptor-like receptor, establishing a functional receptor for adrenomedullin (AM). AM exhibits potent antiproliferative and antimigratory effects on rat mesangial cells (RMCs). In this study we investigated the effect of platelet-derived growth factor (PDGF) on RAMP-3 expression in RMCs. We show here that PDGF-BB stimulates RAMP-3 mRNA expression in a concentration-dependent manner. Pretreatment with actinomycin-D and alpha-amanitin demonstrates that this effect is independent of new RNA synthesis. Furthermore, PDGF increased the half-life of RAMP-3 mRNA from 66.5 to 331.6 min. Using selective inhibitors, our results also indicate that the increase in RAMP-3 mRNA is mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK and p38 MAPK dependent. PDGF also caused a corresponding elevation in membrane-associated RAMP-3 protein. Associated with this increase, PDGF pretreatment led to a significantly higher AM-mediated adenylate cyclase activity, suggesting a functional consequence for the PDGF-induced increase in RAMP-3 expression. Taken together, these data identify PDGF-dependent regulation of RAMP-3 expression as a possible mechanism for modulating the responsiveness of the mesangial cell to AM.

When coexpressed with receptor activity-modifying protein (RAMP)2 or -3, calcitonin receptor-like receptor (CRLR) functions as an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Coexpression of rat (r)CRLR with rRAMP deletion mutants in HEK293T cells revealed that deletion of residues 93-99 from rRAMP2 or residues 58-64 from rRAMP3 significantly inhibits high-affinity [125I]AM binding and AM-evoked cAMP production, despite full cell surface expression of the receptor heterodimer. Apparently, these two seven-residue segments are key determinants of high-affinity agonist binding to rAM receptors and of receptor functionality. Consequently, their deletion yields peptides that are able to serve as negative regulators of AM receptor function.

Calcitonin gene-related peptide and adrenomedullin belong to a structurally related neuropeptide family and are potent vasodilators expressed in the trigeminovascular system. The molecular identity of receptors for these proteins has only recently been elucidated. Central to functional binding of these neuropeptides is the G-protein-coupled receptor, the calcitonin receptor-like receptor (CRLR), whose cell surface expression and pharmacology is determined by coexpression of a receptor activity-modifying protein (RAMP). CRLR combined with RAMP binds calcitonin gene-related peptide with high affinity, whereas CRLR coexpression with RAMP2 or -3 confers high-affinity binding of adrenomedullin. The authors investigated the expression of these receptor components in human cerebral vasculature to further characterize neuropeptide receptor content and the potential functions of these receptors. Localization has been carried out using specific antisera raised against immunogenic peptide sequences that were subsequently applied using modern immunohistochemical techniques and confocal microscopy. The results are the first to show the presence of these receptor component proteins in human middle meningeal, middle cerebral, pial, and superficial temporal vessels, and confirm that both calcitonin gene-related peptide and adrenomedullin receptors may arise from the coassembly of RAMPs with CRLR in these vessel types. These novel data advance the understanding of the molecular function of the trigeminovascular system, its potential role in vascular headache disorders such as migraine, and may lead to possible ways in which future synthetic ligands may be applied to manage these disorders.

The discovery of receptor-activity-modifying proteins (RAMP) revealed a new principle for the function of G protein-coupled receptors. The initially orphan calcitonin receptor-like receptor (CRLR) was identified as a CGRP receptor when coexpressed with RAMP1. The same receptor is specific for adrenomedullin (ADM) in the presence of RAMP2. Calcitonin receptors (CTR) with 60% homology to the CRLR predominantly recognize calcitonin in the absence of RAMP. An amylin/CGRP receptor was recognized when a calcitonin receptor (CTR) was coexpressed with RAMP1. In the presence of RAMP3, the CTR only interacts with amylin. Noncovalent association of the RAMP with the CRLR or the CTR reveals heterodimeric RAMP/receptor complexes at the cell surface. Thus, two Class II G protein-coupled receptors, the CRLR and CTR, associate with three RAMP to form high affinity receptors for CGRP, ADM, or amylin. Here, the molecular composition and the functional properties of these receptors is reviewed.

Adrenomedullin is known to inhibit cell proliferation in cultured rat vascular smooth muscle cells, through a cAMP-dependent process. The calcitonin receptor-like receptor could function as an adrenomedullin receptor when co-expressed with receptor activity-modifying protein 2. To determine whether vascular adrenomedullin receptor components, the calcitonin receptor-like receptor and the receptor activity-modifying protein 2, phenotypically change during in vitro culture conditions, we examined the expression of adrenomedullin receptor components, adrenomedullin-induced cAMP production, and the inhibition of cell proliferation in culture rat vascular smooth muscle cells during serial passages. The results demonstrated that the receptor activity-modifying protein 2 and calcitonin receptor-like receptor mRNAs increased in a passage-dependent manner in rat vascular smooth muscle cells. Furthermore, the responses of both the elevation of cAMP and the inhibition of cell proliferation became larger in vascular smooth muscle cells with an increasing number of passages. The results suggest that the increase in functional AM receptor during phenotypic change may in part contribute to the development of vascular lesions, such as in atherosclerosis., (Copyright 2001 Academic Press.)

Adrenomedullin (AM) has vasodilator and diuretic actions, similarly to natriuretic peptides. AM receptor complexes are composed of calcitonin receptor-like receptor (CRLR) and receptor-activity modifying protein-2 (RAMP2), or CRLR and RAMP3. We aimed to know whether gene expression of AM and AM receptor complexes are regulated in kidneys under pathophysiological conditions. Expression of AM, RAMP2, RAMP3 and CRLR mRNA was studied in the remnant kidney of rats with renal mass ablation using competitive quantitative RT-PCR techniques. Partial cloning was performed to determine the rat RAMP3 nucleotide sequence. In normal rat kidneys, expression levels of RAMP2, RAMP3, CRLR and AM mRNAs were 26.5 +/- 1.9 mmol/mole of GAPDH, 7.7 +/- 0.9 mmol/mole of GAPDH, 3.6 +/- 0.2 mmol/mole of GAPDH and 0.57 +/- 0.03 mmol/mole of GAPDH (mean +/- SE, n = 6), respectively. RAMP3 mRNA levels decreased significantly to about 50% and about 70% of control (sham-operated rats) 4 days and 14 days after 5/6 nephrectomy, respectively. CRLR mRNA levels also decreased significantly to about 30% and about 43% of control. Sodium intake restriction had no significant effects on the RAMP3 and CRLR gene expression. On the other hand, RAMP2 mRNA expression in the kidney was suppressed by sodium intake restriction regardless of nephrectomy, while RAMP2 levels in the remnant kidney were not significantly changed by 5/6 nephrectomy. Neither 5/6 nephrectomy or sodium intake restriction had any significant effects on the AM gene expression in the kidney. The present study showed that expression of mRNAs encoding AM, RAMP2, RAMP3 and CRLR were differentially regulated in remnant kidneys of rats with renal mass ablation.

Adrenomedullin (AM) is a multifunctional peptide with a range of cardiovascular functions including direct effects on cardiomyocytes. Despite the demonstration of high numbers of AM binding sites in rat heart, the receptor responsible for cardiac AM action has not been characterized. We show here that the hearts of adult and neonatal rats, and neonatal cardiomyocyte cultures coexpress mRNA transcripts for the calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs). RAMP2 mRNA predominated over RAMP1 in adult (20:1 ratio) and neonatal heart (10:1 ratio), and in cardiomyocyte cultures (1.7:1 ratio), though the relative abundance of these transcripts appears to be developmentally regulated. Transient transfection of cardiomyocytes using a cAMP-responsive element (CRE)-luciferase reporter gene as an indirect measure of cAMP activation, demonstrated that overexpression of either CRLR or RAMP2 potentiated the AM signalling response. Overexpression of CRLR and RAMP2 together led to an additive effect on AM signalling that was approximately 4-fold greater than control cardiomyocytes stimulated with AM. Furthermore, the AM-mediated induction of CRE-luciferase activity was abolished by coincubation with the receptor antagonist CGRP(8-37) or by overexpression of a CRLR antisense construct. These data demonstrate that AM action in the rat cardiomyocyte requires CRLR and RAMP2 to constitute functional AM receptors.

Adrenomedullin (AM) is a potent vasodilator. Pregnancy-induced hypertension (PIH) is a common cause of maternal or fetal mortality. We measured the changes of adrenomedullin receptor components gene expression, receptor activity-modifying protein 2 (RAMP2) and calcitonin receptor-like receptor (CRLR), at feto-maternal tissues in human normotensive pregnant women and pregnancy-induced hypertensive women by Northern blot analysis. Samples of the placenta, uterine muscle, umbilical artery, and fetal membranes were obtained from each patient under informed consent. RAMP2 mRNA significantly decreased in the umbilical artery (54%, P < 0.01) and uterus (53%, P < 0.01) of the patients with PIH. CRLR mRNA also significantly decreased in both tissues of the patients with PIH. On the other hand, the RAMP2 mRNA was significantly increased in the fetal membrane of the patients with PIH. In addition, there was a significant negative correlation between the RAMP2 mRNA levels in the umbilical artery (systolic; r = -0.623, P < 0.01, diastolic; r = -0.552, P < 0.01) and uterine muscle (systolic; r = -0.563, P < 0.01, diastolic; r = -0.553, P< 0.01) and blood pressure. However, there was no correlation between the mRNA level and blood pressure in fetal membrane and placenta, suggesting that there is no close relationship to the pathogenesis in PIH. These findings suggested that the reduced expression of adrenomedullin receptor component in umbilical artery and uterus may have some role in PIH.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM), two closely related peptides, initiate their biological responses through their interaction with calcitonin receptor-like receptor (CRLR). The CRLR receptor phenotype can be determined by coexpression of CRLR with one of the three-receptor activity modifying proteins (RAMPs). In this report, we characterized the pharmacological properties of the human or porcine CRLR with individual RAMPs transiently expressed in human embryonic kidney cell line (HEK-293). Characterization of RAMP1/human or porcine CRLR combination by radioligand binding ([125I] halphaCGRP) and functional assay (activation of adenylyl cyclase) revealed the properties of CGRP receptor. Similarly characterization of RAMP2/human or porcine CRLR and RAMP3/human or porcine CRLR combination by radioligand binding ([125I] rADM) and functional assay (activation of adenylyl cyclase) revealed the properties of ADM (22-52) sensitive-ADM receptor. In addition, porcine CRLR/RAMP2 or 3 combination displayed specific high affinity [125I] halphaCGRP binding also. Also, co-transfection of porcine CRLR with RAMPs provided higher expression level of the receptor than the human counterpart. Thus the present study along with earlier studies strongly support the role of RAMPs in the functional expression of specific CRLRs.