Chow Billy K C, Moon Thomas W, Hoo Ruby L C, Yeung Chung-Man, Müller Miklós, Christos Paul J, Mojsov Svetlana
Department of Zoology, University of Hong Kong, Hong Kong.
Endocrinology. 2004 Jul;145(7):3273-88. doi: 10.1210/en.2003-0597. Epub 2004 Mar 19.
The structural basis of ligand selectivity of G protein-coupled receptors for metabolic hormones has been an area of intense investigation, and yet it remains unresolved. One approach to delineating the mechanism of ligand-receptor interactions is to compare the ligand specificities of receptors expressed in species that emerged at different times within vertebrate evolution. In this paper we describe the isolation, functional, and phylogenetic characterization of the glucagon receptor from the goldfish Carassius auratus (Teleostei, order Cypriniformes), and compare its ligand specificity with that of the homologous rat receptor. Goldfish (gf) glucagon stimulated glucose production in a dose-dependent manner from isolated goldfish hepatocytes, resulting in 5-fold increase at 1 microm. The goldfish glucagon receptor (gfGlucR) shares 56, 51, 50, and 52% amino acid identities with frog Rana tigrina regulosa, mouse, rat, and human glucagon receptors, respectively. In competitive binding experiments, the recombinant gfGlucR displays high affinity toward goldfish, zebrafish, and human glucagons (IC(50) = 0.6, 9, and 13 nm, respectively) but not toward goldfish glucagon-like peptide-1 or human glucagon-like peptide-1 (7-36) amide. Whereas both goldfish and human glucagons stimulated dose-dependent increases in intracellular cAMP through the recombinant gfGlucR, the recombinant rat GlucR interacted only with human glucagon, analogous to the specificity of the previously characterized glucagon receptor from the frog R. tigrina regulosa. Our results demonstrate that the binding pocket of gfGlucR can accommodate a broad range of glucagon structures and that in the frogs and mammals, there is a structural switch to a more restrictive conformation of glucagon receptors.
G蛋白偶联代谢激素受体的配体选择性的结构基础一直是深入研究的领域，但仍未得到解决。一种描述配体-受体相互作用机制的方法是比较在脊椎动物进化过程中不同时期出现的物种中表达的受体的配体特异性。在本文中，我们描述了金鱼（鲤形目鲤科鲫属）胰高血糖素受体的分离、功能及系统发育特征，并将其配体特异性与同源大鼠受体的配体特异性进行了比较。金鱼胰高血糖素以剂量依赖的方式刺激分离的金鱼肝细胞产生葡萄糖，在1微摩尔时增加了5倍。金鱼胰高血糖素受体（gfGlucR）与虎纹蛙、小鼠、大鼠和人类胰高血糖素受体的氨基酸同源性分别为56%、51%、50%和52%。在竞争性结合实验中，重组gfGlucR对金鱼、斑马鱼和人类胰高血糖素显示出高亲和力（IC(50)分别为0.6、9和13纳米），但对金鱼胰高血糖素样肽-1或人类胰高血糖素样肽-1（7-36）酰胺没有亲和力。虽然金鱼和人类胰高血糖素都通过重组gfGlucR刺激细胞内cAMP剂量依赖性增加，但重组大鼠GlucR仅与人胰高血糖素相互作用，类似于先前表征的虎纹蛙胰高血糖素受体的特异性。我们的结果表明，gfGlucR的结合口袋可以容纳广泛的胰高血糖素结构，并且在青蛙和哺乳动物中，胰高血糖素受体的构象发生了结构转变，变得更加受限。
