Wang C D, Gallaher T K, Shih J C
Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles 90033.
Mol Pharmacol. 1993 Jun;43(6):931-40.
Serotonin 5-hydroxytryptamine (5-HT)2 receptors are implicated in the etiology of mental disease and depression. Drugs that interact with the 5-HT2 receptor are used therapeutically to treat such illnesses, and their mechanisms of action are of great interest. In this study 5-HT2 receptor-ligand interactions were examined by site-directed mutagenesis in which three aspartic acid to asparagine mutants (Asn-120, Asn-155, and Asn-172) were created and expressed in NIH3T3 cells. The Asp-120 to asparagine mutant exhibited the same affinity for 125I-lysergic acid diethylamide (125I-LSD) as did the wild-type receptor and showed a decreased and GTP-insensitive binding affinity for the agonists 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (approximately 10-fold) and the antagonists ketanserin and mianserin (approximately 10-fold) but not spiperone. The mutation also abolished agonist-stimulated formation of [3H]polyphosphoinositides (PI). The Asn-155 mutant showed reduced binding affinity for 125I-LSD (Kd, 2.8 nM versus 0.6 nM for the wild-type receptor) and had reduced affinity for agonists (approximately 30-fold) and for antagonists (14-75-fold). However, the Asn-155 receptor retained GTP sensitivity and the ability to stimulate PI formation. The Asn-172 mutant retained the wild-type Kd value for 125I-LSD, exhibited only approximately 5-fold reduced affinity for 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane while retaining GTP-sensitive agonist binding showed no change in affinity for ketanserin, and had a small decrease in mianserin and spiperone binding (approximately 6-fold). The Asn-172 receptor also retained the ability to form PI. These results indicate that Asp-120 is necessary for allosteric activation of the guanine nucleotide-binding protein. Asp-155 is necessary for high affinity binding, probably by acting as a counterion for the amine group of the ligand. The different effects of the three mutations on ketanserin, mianserin, and spiperone binding affinity suggest that these antagonists may share overlapping but different binding domains. The information provided by this study may facilitate the design of therapeutic site-selective compound based on the structure of the 5-HT2 receptor.
血清素5-羟色胺（5-HT）2受体与精神疾病和抑郁症的病因有关。与5-HT2受体相互作用的药物被用于治疗此类疾病，其作用机制备受关注。在本研究中，通过定点诱变研究了5-HT2受体与配体的相互作用，构建了三个天冬氨酸突变为天冬酰胺的突变体（Asn-120、Asn-155和Asn-172）并在NIH3T3细胞中表达。Asp-120突变为天冬酰胺的突变体对125I-麦角酰二乙胺（125I-LSD）的亲和力与野生型受体相同，对激动剂5-HT和（±）-1-（2,5-二甲氧基-4-碘苯基）-2-氨基丙烷（约10倍）以及拮抗剂酮色林和米安色林（约10倍）的结合亲和力降低且对GTP不敏感，但对螺哌隆不敏感。该突变还消除了激动剂刺激的[3H]多磷酸肌醇（PI）的形成。Asn-155突变体对I-LSD的结合亲和力降低（Kd，2.8 nM，野生型受体为0.6 nM），对激动剂（约30倍）和拮抗剂（14 - 75倍）的亲和力也降低。然而，Asn-155受体保留了对GTP的敏感性以及刺激PI形成的能力。Asn-突变体保留了I-LSD的野生型Kd值，对5-HT和（±）-1-（2,5-二甲氧基-4-碘苯基）-2-氨基丙烷的亲和力仅降低约5倍，同时保留了对GTP敏感的激动剂结合能力，对酮色林的亲和力无变化，对米安色林和螺哌隆的结合亲和力略有降低（约6倍）。Asn-172受体也保留了形成PI的能力。这些结果表明，Asp-120对于鸟嘌呤核苷酸结合蛋白的变构激活是必需的。Asp-155对于高亲和力结合是必需的，可能是作为配体胺基的抗衡离子。这三个突变对酮色林、米安色林和螺哌隆结合亲和力的不同影响表明，这些拮抗剂可能共享重叠但不同的结合结构域。本研究提供的信息可能有助于基于5-HT2受体的结构设计治疗性位点选择性化合物。
