Yeung Chung-Man, Mojsov Svetlana, Mok Pui-Yee, Chow Billy K C
Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
Endocrinology. 2002 Dec;143(12):4646-54. doi: 10.1210/en.2002-220694.
A better understanding of the molecular mechanism of ligand-receptor interaction of glucagon-like peptide 1 (GLP-1) receptors (GLP-1Rs) is useful for the design of potent GLP-1 analogs that could potentially be used as a treatment for diabetic patients. Changes in the ligand and receptor sequences during evolution provide invaluable clues to evaluate the functional motifs of the receptor that are responsible for ligand interaction. For these reasons, in the present study, we have isolated and functionally characterized a GLP-1R from goldfish. Its amino acid sequence shows 50.8% and 52.3% identity with the human glucagon (hGLU) and GLP-1Rs, respectively, and 84.1% with the zebrafish GLP-1R (the only other GLP-1R isolated from teleost fish). Peptides that are structurally different from goldfish (gf)GLP-1, such as gfGLU and hGLU and human GLP-1 (7-36)amide, are also capable of stimulating this receptor, albeit with lower potencies than gfGLP-1. gfGLP-1 stimulates the formation of cAMP through the recombinant gfGLP-1R with EC(50) = 0.18 nM, whereas EC(50) values for gfGLU, human GLP-1 (7-36)amide, and hGLU are 0.53 nM, 0.9 nM, and 1.2 nM, respectively. These results indicate that the gfGLP-1R is structurally more flexible than its mammalian counterpart and that its binding pocket can accommodate a wider spectrum of peptide ligands. Previous studies demonstrated that the charged residues in the extracellular domains of mammalian GLP-1R, particularly those found in the N-terminal domain and the first exoloop, are important for ligand binding. We investigated the roles of the conserved charged residues in the function of the gfGLP-1R. Eleven mutant receptors were constructed, and the effects of mutations were determined by functional assays. Our results demonstrated that three charged residues (D(113), R(197), and D(205)) present in the extracellular domains are critical for receptor function.
更好地理解胰高血糖素样肽1（GLP-1）受体（GLP-1Rs）的配体-受体相互作用分子机制，有助于设计出强效的GLP-1类似物，这些类似物有可能用于治疗糖尿病患者。进化过程中配体和受体序列的变化为评估负责配体相互作用的受体功能基序提供了宝贵线索。基于这些原因，在本研究中，我们从金鱼中分离出了一种GLP-1R并对其进行了功能表征。其氨基酸序列与人类胰高血糖素（hGLU）和GLP-1Rs的同一性分别为50.8%和52.3%，与斑马鱼GLP-1R（从硬骨鱼中分离出的唯一另一种GLP-1R）的同一性为84.1%。结构上与金鱼（gf）GLP-1不同的肽，如gfGLU、hGLU和人GLP-1（7-36）酰胺，也能够刺激该受体，尽管其效力低于gfGLP-1。gfGLP-1通过重组的gfGLP-1R刺激cAMP的形成，其半数有效浓度（EC(50)）=0.18 nM，而gfGLU、人GLP-1（7-36）酰胺和hGLU的EC(50)值分别为0.53 nM、0.9 nM和1.2 nM。这些结果表明，gfGLP-1R在结构上比其哺乳动物对应物更灵活，其结合口袋可以容纳更广泛的肽配体。先前的研究表明，哺乳动物GLP-1R细胞外结构域中的带电荷残基，特别是在N端结构域和第一个外环中发现的那些残基，对配体结合很重要。我们研究了gfGLP-1R功能中保守带电荷残基的作用。构建了11种突变受体，并通过功能测定确定了突变的影响。我们的结果表明，细胞外结构域中存在的三个带电荷残基（D(113)、R(197)和D(205)）对受体功能至关重要。
