Dong Maoqing, Cox Richard F, Miller Laurence J
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259.
GlaxoSmithKline, Research Triangle Park, North Carolina 27709.
J Biol Chem. 2009 Aug 14;284(33):21839-21847. doi: 10.1074/jbc.M109.011924. Epub 2009 May 15.
The Family B G protein-coupled calcitonin receptor is an important drug target. The aim of this work was to elucidate the molecular mechanism of action of small-molecule agonist ligands acting at this receptor, comparing it with the action mechanism of the receptor's natural peptide ligand. cAMP responses to four non-peptidyl ligands and calcitonin were studied in COS-1 cells expressing wild-type and chimeric calcitonin-secretin receptors. All compounds were full agonists at the calcitonin receptor with no activity at the secretin receptor. Only chimeric constructs including the calcitonin receptor amino terminus exhibited responses to any of these ligands. We progressively truncated this domain and tested constructs for cAMP responses. Although calcitonin was able to activate the calcitonin receptor fully with the first 58 residues absent, its potency was 3 orders of magnitude lower than that at the wild-type receptor. After truncation of 114 residues, there was no response to calcitonin. In contrast, small-molecule ligands were fully active at receptors having up to 149 amino-terminal residues absent. Those compounds finally became inactive after truncation of 153 residues. Deletion and/or alanine replacement of the region of the calcitonin receptor between residues 150 and 153 resulted in marked reduction in cAMP responses to these compounds, with some compound-specific differences observed, supporting a critical role for this region. Binding studies further supported distinct sites of action of small molecules relative to that of calcitonin. These findings focus attention on the potential importance of the juxtamembranous region of the amino terminus of the Family B calcitonin receptor for agonist drug action.
B族G蛋白偶联降钙素受体是一个重要的药物靶点。这项工作的目的是阐明作用于该受体的小分子激动剂配体的分子作用机制，并将其与该受体天然肽配体的作用机制进行比较。在表达野生型和嵌合降钙素-促胰液素受体的COS-1细胞中研究了对四种非肽基配体和降钙素的cAMP反应。所有化合物在降钙素受体上都是完全激动剂，在促胰液素受体上无活性。只有包含降钙素受体氨基末端的嵌合构建体对这些配体中的任何一种有反应。我们逐步截短该结构域并测试构建体的cAMP反应。尽管降钙素在前58个残基缺失的情况下仍能完全激活降钙素受体，但其效力比野生型受体低3个数量级。在截短114个残基后，对降钙素无反应。相反，小分子配体在氨基末端缺失多达149个残基的受体上仍具有完全活性。这些化合物在截短153个残基后最终失去活性。对降钙素受体150至153位残基之间区域进行缺失和/或丙氨酸替换导致对这些化合物的cAMP反应显著降低，观察到一些化合物特异性差异，支持该区域的关键作用。结合研究进一步支持了小分子相对于降钙素的不同作用位点。这些发现将注意力集中在B族降钙素受体氨基末端近膜区域对激动剂药物作用的潜在重要性上。
