Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product

George Christopoulos, Katie J. Perry, Maria Morfis, Nanda Tilakaratne, Yongyi Gao, Neil J. Fraser, Martin J. Main, Steven M. Foord, Patrick M. Sexton

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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 (hCTR(I1-)) into COS-7 cells induced specific 125 I-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 (IC 50 , 3.01 ± 1.44 x 10 -10 M), a high to moderate affinity for rat amylin (IC 50 , 7.86 ± 4.49 x 10 -9 M) and human CGRPα (IC 50 , 2.09 ± 1.63 x 10 -8 M), and a low affinity for human CT (IC 50 , 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 CGRPα was markedly reduced (IC 50 , 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 hCTR(I1-) 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 125 I-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.

Original languageEnglish
Pages (from-to)235-242
Number of pages8
JournalMolecular Pharmacology
Issue number1
Publication statusPublished - 1 Jan 1999
Externally publishedYes

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