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Multiple Amylin Receptors Arise from Receptor Activity-Modifying Protein Interaction with the Calcitonin Receptor Gene Product
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 recept...
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Published in: | Molecular pharmacology 1999-07, Vol.56 (1), p.235-242 |
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Main Authors: | , , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
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Online Access: | Get full text |
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Summary: | 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 I1â ) 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 à 10 â10 M), a high to moderate affinity for rat amylin (IC 50 , 7.86 ± 4.49 à 10 â9 M) and human CGRPα (IC 50 , 2.09 ± 1.63 à 10 â8 M), and a low affinity for human CT (IC 50 , 4.47 ± 0.78 à 10 â7 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 à 10 â7 M; P < .05 versus RAMP 1). Functional cyclic AMP responses in COS-7 cells cotransfected with individual RAMPs and hCTR I1â 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) I1â 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. |
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ISSN: | 0026-895X 1521-0111 |
DOI: | 10.1124/mol.56.1.235 |