Multiple Amylin Receptors Arise from Receptor Activity-Modifying Protein Interaction with the Calcitonin Receptor Gene Product

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Abstract
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 specific125I-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 × 10−10 M), a high to moderate affinity for rat amylin (IC50, 7.86 ± 4.49 × 10−9 M) and human CGRPα (IC50, 2.09 ± 1.63 × 10−8 M), and a low affinity for human CT (IC50, 4.47 ± 0.78 × 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 (IC50, 1.12 ± 0.45 × 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.