Han G, Hampson D R
Faculty of Pharmacy and Department of Pharmacology University of Toronto, Toronto, Ontario M5S 252, Canada.
J Biol Chem. 1999 Apr 9;274(15):10008-13. doi: 10.1074/jbc.274.15.10008.
The metabotropic glutamate receptor (mGluR) 4 subtype of metabotropic glutamate receptor is a presynaptic receptor that modulates neurotransmitter release. We have characterized the properties of a truncated, epitope-tagged construct containing part of the extracellular amino-terminal domain of mGluR4. The truncated receptor was secreted into the cell culture medium of transfected human embryonic kidney cells. The oligomeric structure of the soluble truncated receptor was assessed by gel electrophoresis. In the presence of high concentrations of a reducing agent, the truncated receptor migrated as a monomer; at lower concentrations of the reducing agent, only higher molecular weight oligomers were observed. Competition binding experiments using the radiolabeled agonist [3H]L-2-amino-4-phosphonobutyric acid revealed that the rank order of potency of metabotropic ligands at the truncated receptor was similar to that of the full-length membrane-bound receptor. However, the truncated receptor displayed higher affinities for agonists and lower affinities for antagonists compared with the full-length receptor. Deglycosylation produced a shift in the relative molecular weight of the soluble protein from Mr = 71,000 to Mr = 63,000; deglycosylation had no effect on the binding of [3H]L-2-amino-4-phosphonobutyric acid, indicating that the asparagine-linked carbohydrates are not necessary for agonist binding. These results demonstrate that although the primary determinants of ligand binding to mGluR4 are contained within the first 548 amino acids of the receptor, additional amino acids located downstream of this region may influence the affinity of ligands for the binding site.
代谢型谷氨酸受体的代谢型谷氨酸受体（mGluR）4亚型是一种调节神经递质释放的突触前受体。我们已对一种截短的、带有表位标签的构建体的特性进行了表征，该构建体包含mGluR4细胞外氨基末端结构域的一部分。截短的受体被分泌到转染的人胚肾细胞的细胞培养基中。通过凝胶电泳评估可溶性截短受体的寡聚结构。在高浓度还原剂存在下，截短的受体以单体形式迁移；在较低浓度的还原剂下，仅观察到较高分子量的寡聚物。使用放射性标记激动剂[3H]L-2-氨基-4-膦酰丁酸进行的竞争结合实验表明，代谢型配体在截短受体上的效价顺序与全长膜结合受体相似。然而，与全长受体相比，截短的受体对激动剂显示出更高的亲和力，对拮抗剂显示出更低的亲和力。去糖基化使可溶性蛋白的相对分子量从Mr = 71,000转变为Mr = 63,000；去糖基化对[3H]L-2-氨基-4-膦酰丁酸的结合没有影响，表明天冬酰胺连接的碳水化合物对于激动剂结合不是必需的。这些结果表明，尽管与mGluR4配体结合的主要决定因素包含在受体的前548个氨基酸内，但该区域下游位置的其他氨基酸可能会影响配体对结合位点的亲和力。
