Armour S L, Foord S, Kenakin T, Chen W J
Department of Molecular Sciences, Glaxo Wellcome Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, USA.
J Pharmacol Toxicol Methods. 1999 Dec;42(4):217-24. doi: 10.1016/s1056-8719(00)00074-5.
Receptor-activity-modifying proteins (RAMPs) are a family of single transmembrane domain proteins shown to be important for the transport and ligand specificity of the calcitonin gene-related peptide (CGRP) receptor. In this report, we describe the analysis of pharmacological properties of the human calcitonin receptor (hCTR) coexpressed with different RAMPs with the use of the Xenopus laevis melanophore expression system. We show that coexpression of RAMP3 with human calcitonin receptor changed the relative potency of hCTR to human calcitonin (hCAL) and rat amylin. RAMP1 and RAMP2, in contrast, had little effect on the change of hCTR potency to hCAL or rat amylin. When coexpressed with RAMP3, hCTR reversed the relative potency by a 3.5-fold loss in sensitivity to hCAL and a 19-fold increase in sensitivity to rat amylin. AC66, an inverse agonist, produced apparent simple competitive antagonism of hCAL and rat amylin, as indicated by linear Schild regressions. The potency of AC66 was changed in the blockade of rat amylin but not hCAL responses with RAMP3 coexpression. The mean pK(B) for AC66 to hCAL was 9.4 +/- 0.3 without RAMP3 and 9.45 +/- 0.07 with RAMP3. For the antagonism of AC66 to rat amylin, the pK(B) was 9.25 +/- 0.15 without RAMP3 and 8.2 +/- 0.35 with RAMP3. The finding suggests that RAMP3 might modify the active states of calcitonin receptor in such a way as to create a new receptor phenotype that is "amylin-like." Irrespective of the physiological association of the new receptor species, the finding that a coexpressed membrane protein can completely change agonist and antagonist affinities for a receptor raises implications for screening in recombinant receptor systems.
受体活性修饰蛋白（RAMPs）是一类单跨膜结构域蛋白，已被证明对降钙素基因相关肽（CGRP）受体的转运和配体特异性很重要。在本报告中，我们描述了利用非洲爪蟾黑素细胞表达系统对与不同RAMPs共表达的人降钙素受体（hCTR）的药理学特性进行的分析。我们发现，RAMP3与人降钙素受体共表达改变了hCTR对人降钙素（hCAL）和大鼠胰淀素的相对效力。相比之下，RAMP1和RAMP2对hCTR对hCAL或大鼠胰淀素效力的变化影响很小。当与RAMP3共表达时，hCTR使相对效力发生逆转，对hCAL的敏感性降低了3.5倍，对大鼠胰淀素的敏感性增加了19倍。反向激动剂AC66对hCAL和大鼠胰淀素产生了明显的简单竞争性拮抗作用，线性Schild回归表明了这一点。AC66的效力在阻断大鼠胰淀素反应时发生了变化，但在与RAMP3共表达时对hCAL反应的阻断中未发生变化。AC66对hCAL的平均pK(B)在无RAMP3时为9.4±0.3，在有RAMP3时为9.45±0.07。对于AC66对大鼠胰淀素的拮抗作用，pK(B)在无RAMP3时为9.25±0.15，在有RAMP3时为8.2±0.35。这一发现表明，RAMP3可能以一种创造“胰淀素样”新受体表型的方式改变降钙素受体的活性状态。无论新受体物种的生理关联如何，共表达的膜蛋白能完全改变受体对激动剂和拮抗剂亲和力这一发现对重组受体系统中的筛选具有启示意义。
