Christopoulos G, Perry K J, Morfis M, Tilakaratne N, Gao Y, Fraser N J, Main M J, Foord S M, Sexton P M
Molecular Pharmacology Laboratory, Department of Pharmacology, The University of Melbourne, Victoria, Australia.
Mol Pharmacol. 1999 Jul;56(1):235-42. doi: 10.1124/mol.56.1.235.
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.
受体活性修饰蛋白（RAMPs）是单跨膜蛋白，可将降钙素受体样受体（CRLR）转运至细胞表面。RAMP 1转运的CRLR是降钙素基因相关肽（CGRP）受体。RAMP 2或RAMP 3转运的CRLR是肾上腺髓质素受体。除了与II类G蛋白偶联受体CRLR相互作用外，RAMPs的作用尚不清楚。在本研究中，我们研究了RAMPs在从降钙素（CT）受体基因产物产生胰淀素受体表型中的作用。将RAMP 1或RAMP 3与细胞内结构域1中缺乏16个氨基酸插入片段的人CT受体（hCTRI1-）共转染到COS-7细胞中，可诱导特异性的125I标记的大鼠胰淀素结合。RAMP 2或载体共转染不会导致特异性胰淀素结合显著增加。RAMP诱导的胰淀素受体的竞争结合特征揭示了两种不同的表型。RAMP 1衍生的胰淀素受体对鲑鱼CT表现出最高亲和力（IC50，3.01 +/- 1.44 x 10(-10) M），对大鼠胰淀素（IC50，7.86 +/- 4.49 x 10(-9) M）和人CGRPα（IC50，2.09 +/- 1.63 x 10(-8) M）具有高至中等亲和力，对人CT具有低亲和力（IC50，4.47 +/- .78 x 10(-7) M）。相比之下，虽然RAMP 3衍生的受体对胰淀素和CTs的亲和力相似，但人CGRPα的效力明显降低（IC50，1.12 +/- 0.45 x 10(-7) M；与RAMP 1相比，P <.05）。与单个RAMPs和hCTRI1-共转染的COS-7细胞中的功能性环磷酸腺苷反应反映了在竞争胰淀素结合中观察到的表型。c-myc标记的RAMP 1的共聚焦显微镜定位表明，单独转染时，RAMP 1几乎完全位于细胞内。与降钙素受体（CTR）I1-共转染可诱导RAMP 1在细胞表面表达。用双琥珀酰亚胺基辛二酸酯将125I标记的胰淀素与RAMP 1/hCTR转染细胞交联的实验结果提示RAMP 1与受体在细胞表面存在关联。我们的数据表明，在CT受体家族中，以及可能在其他II类G蛋白偶联受体中，细胞表型可能在受体和RAMP蛋白的水平及组合方面是动态的。
