Nehring R B, Meyerhof W, Richter D
Institut für Zellbiochemie und klinische Neurobiologie, UKE, Universität Hamburg, Germany.
DNA Cell Biol. 1995 Nov;14(11):939-44. doi: 10.1089/dna.1995.14.939.
A highly conserved aspartic acid residue present in the third membrane-spanning region of adrenergic and muscarinic receptors is directly involved in ligand binding. The five cloned somatostatin receptor subtypes also contain this residue at the same relative position. To test whether Asp-124 of the rat somatostatin receptor subtype 3 (SSTR3) is responsible for the binding of somatostain-14 (SST-14), this amino acid residue was replaced by an asparagine or a glutamic acid by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Expression and binding activity of the wild-type and mutant receptor constructs were studied in COS and HEK cells by ligand binding, UV cross-linking, Western blot, and immunocytochemical analysis. The Asn or Glu mutations result in a significant loss of SST-14 binding, although the mutant receptors are correctly transferred to the cell surface, demonstrating that Asp-124 is directly involved in binding of SST-14.
肾上腺素能受体和毒蕈碱受体第三个跨膜区域中存在的一个高度保守的天冬氨酸残基直接参与配体结合。五个克隆的生长抑素受体亚型在相同的相对位置也含有此残基。为了检测大鼠生长抑素受体亚型3（SSTR3）的天冬氨酸-124是否负责生长抑素-14（SST-14）的结合，通过聚合酶链反应（PCR）介导的定点诱变将这个氨基酸残基替换为天冬酰胺或谷氨酸。通过配体结合、紫外线交联、蛋白质印迹和免疫细胞化学分析，在COS和HEK细胞中研究野生型和突变型受体构建体的表达和结合活性。尽管突变型受体能正确转运至细胞表面，但天冬酰胺或谷氨酸突变导致SST-14结合显著丧失，表明天冬氨酸-124直接参与SST-14的结合。
