Fraser C M, Wang C D, Robinson D A, Gocayne J D, Venter J C
Section of Receptor Biochemistry and Molecular Biology, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892.
Mol Pharmacol. 1989 Dec;36(6):840-7.
Muscarinic acetylcholine receptors contain a region encompassing the second and third transmembrane domains that is rich in conserved aspartic acid residues. To investigate the role of four conserved aspartic acids at positions 71, 99, 105, and 122 in muscarinic receptor function, point mutations in the rat m1 muscarinic receptor gene were made that converted each Asp to Asn, and wild type or mutant genes were stably expressed in Chinese hamster ovary cells that normally lack muscarinic receptors. Substitution of Asp71 or Asp122 with Asn produced mutant receptors that displayed high affinity for carbachol but decreased efficacy and potency, respectively, in agonist-induced activation of phosphoinositide hydrolysis, suggesting that these residues may mediate receptor-GTP binding protein interactions. Substitution of Asp99 or Asp105 with Asn produced marked decreases in ligand binding affinities and/or covalent incorporation of [3H] propylbenzilylcholine mustard, suggesting that these residues may be involved in receptor-ligand interactions.
毒蕈碱型乙酰胆碱受体包含一个区域，该区域涵盖富含保守天冬氨酸残基的第二和第三跨膜结构域。为了研究位于71、99、105和122位的四个保守天冬氨酸在毒蕈碱受体功能中的作用，对大鼠m1毒蕈碱受体基因进行了点突变，将每个天冬氨酸转换为天冬酰胺，并且野生型或突变型基因在通常缺乏毒蕈碱受体的中国仓鼠卵巢细胞中稳定表达。用天冬酰胺取代71位或122位的天冬氨酸产生了突变受体，这些受体对卡巴胆碱显示出高亲和力，但在激动剂诱导的磷酸肌醇水解激活中分别降低了效能和效力，这表明这些残基可能介导受体-GTP结合蛋白相互作用。用天冬酰胺取代99位或105位的天冬氨酸导致配体结合亲和力和/或[3H]丙基苯甲酰胆碱氮芥的共价掺入显著降低，这表明这些残基可能参与受体-配体相互作用。
