The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.
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Adrenomedullin, a potently hypotensive peptide isolated from human pheochromocytoma, is known to elicit a rise in cAMP levels within mammalian endothelial and smooth muscle cells. Until now, however, little has been known about the adrenomedullin receptor. Recently, a group called receptor activity-modifying proteins that complex with the calcitonin receptor-like receptor, and thereby regulate its transport and ligand specificity, were identified. Here we show that mRNA for both the calcitonin receptor-like receptor and the receptor activity-modifying protein 2, but not the receptor activity-modifying protein 1 or receptor activity-modifying protein 3, are expressed in human endothelial and vascular smooth muscle cells. We also found that adrenomedullin increased cAMP levels in HeLa EBNA and 293 EBNA cells, expressing both the receptor activity-modifying protein 2 and calcitonin receptor-like receptor proteins. Thus, the receptor activity-modifying protein 2/calcitonin receptor-like receptor complex apparently serves as a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells. (+info)
The amino terminus of receptor activity modifying proteins is a critical determinant of glycosylation state and ligand binding of calcitonin receptor-like receptor.
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The calcitonin receptor-like receptor (CRLR) can function as either a receptor for calcitonin gene-related peptide (CGRP) or for adrenomedullin (ADM), depending upon the coexpression of a novel family of single transmembrane proteins, which we have called receptor activity modifying proteins or RAMPs. RAMPs 1, 2, and 3 transport CRLR to the plasma membrane with similar efficiencies, however RAMP1 presents CRLR as a terminally glycosylated, mature glycoprotein and a CGRP receptor, whereas RAMPs 2 and 3 present CRLR as an immature, core glycosylated ADM receptor. Characterization of the RAMP2/CRLR and RAMP3/CRLR receptors in HEK293T cells by radioligand binding (125I-ADM as radioligand), functional assay (cAMP measurement), or biochemical analysis (SDS-polyacrylamide gel electrophoresis) revealed them to be indistinguishable, even though RAMPs 2 and 3 share only 30% identity. Chimeric proteins were created with the transmembrane and cytosolic portions of RAMP1 associated with the amino terminus of RAMP2 (RAMP2/1) and vice versa (RAMP1/2). Coexpression of RAMP2/1 with CRLR formed a core glycosylated ADM receptor, whereas the RAMP1/2 chimera generated both core glycosylated and mature forms of CRLR and enabled both ADM and CGRP receptor binding. Hence, the glycosylation state of CRLR appears to correlate with its pharmacology. (+info)
Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product.
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Receptor activity-modifying proteins (RAMPs) are single-transmembrane proteins that transport the calcitonin receptor-like receptor (CRLR) to the cell surface. RAMP 1-transported CRLR is a calcitonin gene-related peptide (CGRP) receptor. RAMP 2- or RAMP 3-transported CRLR is an adrenomedullin receptor. The role of RAMPs beyond their interaction with CRLR, a class II G protein-coupled receptor, is unclear. In this study, we have examined the role of RAMPs in generating amylin receptor phenotypes from the calcitonin (CT) receptor gene product. Cotransfection of RAMP 1 or RAMP 3 with the human CT receptor lacking the 16-amino acid insert in intracellular domain 1 (hCTRI1-) into COS-7 cells induced specific 125I-labeled rat amylin binding. RAMP 2 or vector cotransfection did not cause significant increases in specific amylin binding. Competition-binding characterization of the RAMP-induced amylin receptors revealed two distinct phenotypes. The RAMP 1-derived amylin receptor demonstrated the highest affinity for salmon CT (IC50, 3.01 +/- 1.44 x 10(-10) M), a high to moderate affinity for rat amylin (IC50, 7.86 +/- 4.49 x 10(-9) M) and human CGRPalpha (IC50, 2.09 +/- 1.63 x 10(-8) M), and a low affinity for human CT (IC50, 4.47 +/- 0.78 x 10(-7) M). In contrast, whereas affinities for amylin and the CTs were similar for the RAMP 3-derived receptor, the efficacy of human CGRPalpha was markedly reduced (IC50, 1.12 +/- 0.45 x 10(-7) M; P <.05 versus RAMP 1). Functional cyclic AMP responses in COS-7 cells cotransfected with individual RAMPs and hCTRI1- were reflective of the phenotypes seen in competition for amylin binding. Confocal microscopic localization of c-myc-tagged RAMP 1 indicated that, when transfected alone, RAMP 1 almost exclusively was located intracellularly. Cotransfection with calcitonin receptor (CTR)I1- induced cell surface expression of RAMP 1. The results of experiments cross-linking 125I-labeled amylin to RAMP 1/hCTR-transfected cells with bis succidimidyl suberate were suggestive of a cell-surface association of RAMP 1 and the receptors. Our data suggest that in the CT family of receptors, and potentially in other class II G protein-coupled receptors, the cellular phenotype is likely to be dynamic in regard to the level and combination of both the receptor and the RAMP proteins. (+info)
Induction of a myocardial adrenomedullin signaling system during ischemic heart failure in rats.
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BACKGROUND: Increased plasma adrenomedullin (ADM) levels have been reported in congestive heart failure (HF). The present study was designed to investigate myocardial regulation of the different components of the ADM signaling system (ADM, ADM receptor, and receptor-activity-modifying protein-2, RAMP-2) during ischemic HF in rats and to identify the cells in the myocardium displaying ADM-like immunoreactivity (ADM-ir). Furthermore, the effects of endothelin (ET) receptor antagonism on expression of the myocardial ADM system during HF were investigated. METHODS AND RESULTS: Northern blot analysis revealed increased ADM mRNA expression in the nonischemic left ventricle, with maximal levels 28 days after induction of myocardial infarction (1.5-fold, P<0.05) compared with the sham group. Parallel elevations of myocardial ADM receptor and RAMP-2 mRNA levels were also observed (2.3- and 1.5-fold increase, respectively; P<0.05). In addition, high levels of ADM mRNA were seen in the ischemic region. Immunohistochemical analysis revealed a substantial increase of ADM-ir in microvascular endothelium and perivascular interstitial cells of myocardial tissue contiguous to the ischemic region. In addition, radioligand binding studies demonstrated a 1.6-fold increase of specific ADM binding sites in the failing left ventricle (P<0.05). Intervention with the mixed ET(A)/ET(B) receptor antagonist bosentan (100 mg. kg(-1). day(-1) PO) for 15 days prevented the increase of RAMP-2 mRNA. CONCLUSIONS: The study demonstrates a concerted induction of several components of the myocardial ADM signaling system during postinfarction failure and that the vessels are the main source of myocardial ADM. Our observations indicate a role for ADM as an autocrine/paracrine factor during ventricular remodeling after myocardial infarction. (+info)
Mammalian calcitonin receptor-like receptor/receptor activity modifying protein complexes define calcitonin gene-related peptide and adrenomedullin receptors in Drosophila Schneider 2 cells.
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Differential glycosylation of human and rat (r) calcitonin (CT) receptor-like receptors (CRLR) as a result of interactions with accessory receptor activity-modifying proteins (RAMP)1 or -2 was considered to define CT gene-related peptide (CGRP) or adrenomedullin (ADM) receptors in mammalian cells. Here, Drosophila Schneider (S2) cells stably co-expressed rCRLR and RAMP1 or -2 as functional CGRP or ADM receptors. Different from mammalian cells, rCRLR expressed in S2 cells are uniformly glycosylated proteins independent of RAMP1 or RAMP2. Bis(sulfosuccinimidyl)suberate cross-linking revealed receptor components with the size of rCRLR, increased by the molecular weights of the corresponding RAMPs and [(125)I]CGRP or [(125)I]ADM. In conclusion, [(125)I]CGRP/rCRLR/RAMP1 and [(125)I]ADM/rCRLR/RAMP2 complexes have been recognized in Drosophila S2 cells. (+info)
CGRP and adrenomedullin binding correlates with transcript levels for calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) in rat tissues.
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1. Putative receptors for CGRP and adrenomedullin have been investigated in the rat. Calcitonin Receptor-Like Receptor (CRLR), in combination with Receptor Activity Modifying Proteins (RAMPs) is hypothesized to bind either CGRP or adrenomedullin. The receptors known as RDC1 and L1 have also been shown to bind CGRP and adrenomedullin respectively. 2. In this study it is shown that rat CRLR cDNA specifies a CGRP receptor when co-transfected with RAMP-1 cDNA and an adrenomedullin receptor when co-transfected with either RAMP-2 or RAMP-3 cDNA in human embryonic kidney 293 cells. 3. CRLR, RAMP, RCD1 and L1 mRNA levels and CGRP and adrenomedullin receptor densities have been measured and correlated with each other in eight rat tissues selected for their distinctive patterns of CGRP and adrenomedullin binding. 4. The data are consistent with the predictions of the CRLR/RAMP model. CGRP binding correlates well with RAMP-1 mRNA levels (R=1.0, P=0.007), adrenomedullin binding shows a tendency to vary with RAMP-2 mRNA levels (R=0.85, P=0.14) and total binding is correlated with CRLR mRNA levels (R=0.94, P=0.03). The data do not support the hypothesis that RDC1 and L1 account for the majority of CGRP and adrenomedullin binding respectively. (+info)
Amylin receptor phenotypes derived from human calcitonin receptor/RAMP coexpression exhibit pharmacological differences dependent on receptor isoform and host cell environment.
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Receptor activity modifying proteins (RAMPs) constitute a group of three proteins, designated as RAMP1, 2, and 3, which are able to effect functional changes in some members of the G protein-coupled receptor family. Thus, RAMP1 or RAMP3 can modify the calcitonin receptor (CTR) to also function as a high-affinity amylin receptor-like phenotype. To examine the RAMP/CTR interaction, individual RAMPs were coexpressed with either of the two human CTR (hCTR) isoforms, the insert negative (hCTR(I1-)) or the insert positive (hCTR(I1+)), in Chinese hamster ovary (CHO-P) or African monkey kidney (COS-7) cells. CHO-P cells provide an environment conducive to a low, but significant, level of amylin binding with either hCTR isoform alone, unlike in COS-7, where RAMP coexpression is imperative for amylin binding. Also, in CHO-P, hCTR(I1-) induced amylin binding with all three RAMPs, in contrast to COS-7, where only RAMP1 or RAMP3 generate an amylin receptor phenotype. hCTR(I1+) induced high-affinity amylin binding with any RAMP in either cell line. In COS-7 cells, hCTR(I1+)/RAMP-generated receptor displayed high- and low-affinity states, in contrast with the single-state binding seen with hCTR(I1-)/RAMP-generated receptor, whereas in CHO-P cells a two-affinity state receptor phenotype was evident with both hCTR isoforms. Endogenous RAMP expression is low and similar between cell lines. The results suggest that CTR/RAMP interaction in these cells is complex with other cellular factors such as the levels of different G proteins and/or receptor/RAMP stoichiometry following heterologous coexpression contributing to the ultimate receptor phenotype. (+info)
Visualization of the calcitonin receptor-like receptor and its receptor activity-modifying proteins during internalization and recycling.
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Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3). A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without co-transfection of RAMPs. CRLR-GFP failed to generate responses to CGRP or AM without RAMPs. Furthermore, CRLR-GFP was not found in the plasma membrane and its localization was unchanged after agonist exposure. When stably coexpressed with RAMPs, CRLR-GFP appeared on the cell surface and was fully active in intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs. This internalization was strongly inhibited by hypertonic medium (0.45 m sucrose) and paralleled localization of rhodamine-labeled transferrin, suggesting that CRLR endocytosis occurred predominantly through a clathrin-dependent pathway. A significant proportion of CRLR was targeted to lysosomes upon binding of the ligands, and recycling of the internalized CRLR was not efficient. In HEK 293 cells stably expressing CRLR-GFP and Myc-RAMPs, these rhodamine-labeled RAMPs were co-localized with CRLR-GFP in the presence and absence of the ligands. Thus, the CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway. (+info)