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

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 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.

Amylin Receptor Signaling in the Ventral Tegmental Area is Physiologically Relevant for the Control of Food Intake

“…It is worth noting that the binding association between sCT and amylin receptors is reported to be more longlasting than binding of native amylin to the amylin receptor (Christopoulos et al, 1999;Lutz et al, 2000). However, a major discovery from the current studies is the finding that VTA amylin receptors are also a physiologically relevant site of action for food intake control by endogenous amylin, as blockade of VTA amylin receptors by the antagonist AC187 increased food intake.…”

“…Different combinations of RAMP and CTR subtypes create receptor complexes that all bind amylin but also have differing affinities for other calcitonin family ligands such as calcitonin generelated peptide and adrenomedullin. Worth noting is the fact that RAMP2 and RAMP3 may confer additional specificity for amylin binding (Christopoulos et al, 1999). Therefore, given that all RAMP subtypes are equally expressed in the VTA, compared with a more selective expression of RAMP1 in the NAc, amylin receptor complexes within the VTA may respond more preferentially and specifically to amylin analogs than do NAc amylin receptors.…”

“…The amylin receptor complex consists of a CTR (subtypes: A (CTR-A) and B (CTR-B)) heterodimerized with a RAMP (subtypes: RAMP1, RAMP2, and RAMP3; Christopoulos et al, 1999). The different combinations of CTR and RAMP subtypes allow for six possible amylin receptor complexes.…”

Autonomic regulation of islet hormone secretion - Implications for health and disease

“…Depending on which RAMP is transporting the receptor to the surface, affinity for either CGRP, IAPP, calcitonin or adrenomedullin evolves. It has thus been proposed that it is the RAMP1-transported CRLR which results in a receptor form with high affinity for CGRP [101,102]. Similar regulation has been proposed for the calcitonin receptor [103].…”

Amylin Modulates the Mesolimbic Dopamine System to Control Energy Balance

“…The AmyR is a complex consisting of a dimer of the CTR and a receptor activity-modifying protein (Christopoulos et al, 1999;Young, 2005). Therefore, to analyze expression and colocalization of the AmyR on VTA DA neurons, we performed double-label IHC for CTR and the DA marker TH in the VTA.…”