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 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.
Christopoulos
G
G
Molecular Pharmacology Laboratory, Department of Pharmacology, The University of Melbourne, Victoria, Australia.
Perry
K J
KJ
Morfis
M
M
Tilakaratne
N
N
Gao
Y
Y
Fraser
N J
NJ
Main
M J
MJ
Foord
S M
SM
Sexton
P M
PM
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000378:metabolism*; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide / Q000378:metabolism*
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide; D000242:Cyclic AMP