Egan C T, Herrick-Davis K, Teitler M
Department of Pharmacology and Neuroscience, Albany Medical College, New York, USA.
J Pharmacol Exp Ther. 1998 Jul;286(1):85-90.
Single amino acid mutations in the third intracellular loop, as well as other domains of G protein-coupled receptors, have been shown to confer drastic changes in receptor properties and have been postulated to be responsible for various disease states. To determine whether an amino acid mutation can confer dramatic alterations in the 5-hydroxytryptamine2A (5-HT2A) receptor, we mutated amino acid 322 to lysine (C322K), glutamate (C322E) or arginine (C322R). Transient expression of the mutant receptors revealed properties associated with constitutive activity. Radioligand binding studies revealed an increase in 5-HT affinity from 293 nM (native) to 86 nM (C322E), 25 nM (C322K) and 11 nM (C322R). 5-HT potency for stimulation of inositol phosphate production increased from 152 nM (native) to 61 nM (C322E) and 25 nM (C322K). Basal inositol phosphate levels in COS-7 cells expressing C322K and C322E mutant receptors were 8-fold and 4-fold higher, respectively, than cells expressing native 5-HT2A receptors. Basal levels of inositol phosphate stimulated by C322K receptors represented 48% of total inositol phosphate production stimulated by native receptors in the presence of 10 microM 5-HT. Antipsychotic drugs (chlorpromazine, clozapine, haloperidol, loxapine and risperidone) displayed inverse agonist activity by inhibiting C322K constitutive activation of phosphatidylinositol hydrolysis. These data indicate that amino acid 322 in the 5-HT2A receptor plays an important role in maintaining the inactive conformation and provide further evidence that amino acid mutations can produce profound alterations in G protein-coupled receptor activity.
G蛋白偶联受体的第三个细胞内环以及其他结构域中的单氨基酸突变已被证明会导致受体特性发生剧烈变化，并被认为与多种疾病状态有关。为了确定氨基酸突变是否会使5-羟色胺2A（5-HT2A）受体发生显著改变，我们将第322位氨基酸突变为赖氨酸（C322K）、谷氨酸（C322E）或精氨酸（C322R）。突变受体的瞬时表达显示出与组成性活性相关的特性。放射性配体结合研究表明，5-HT亲和力从293 nM（天然型）增加到86 nM（C322E）、25 nM（C322K）和11 nM（C322R）。刺激肌醇磷酸生成的5-HT效力从152 nM（天然型）增加到61 nM（C322E）和25 nM（C322K）。表达C322K和C322E突变受体的COS-7细胞中的基础肌醇磷酸水平分别比表达天然5-HT2A受体的细胞高8倍和4倍。在10 microM 5-HT存在下，C322K受体刺激的基础肌醇磷酸水平占天然受体刺激的总肌醇磷酸生成量的48%。抗精神病药物（氯丙嗪、氯氮平、氟哌啶醇、洛沙平及利培酮）通过抑制C322K介导的磷脂酰肌醇水解的组成性激活而表现出反向激动剂活性。这些数据表明，5-HT2A受体中的第322位氨基酸在维持无活性构象中起重要作用，并进一步证明氨基酸突变可使G蛋白偶联受体活性发生深刻改变。
