Site-directed mutagenesis of the serotonin 5-hydroxytrypamine2 receptor: identification of amino acids necessary for ligand binding and receptor activation.
Site-directed mutagenesis of the serotonin 5-hydroxytrypamine2 receptor: identification of amino acids necessary for ligand binding and receptor activation.
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DOI：
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发表时间：
1993-06
影响因子：
3.6
通讯作者：
C. Wang;T. Gallaher;J. Shih
中科院分区：
文献类型：
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作者：
C. Wang;T. Gallaher;J. Shih
5-羟色胺（5-HT）2受体与精神疾病和抑郁症的病因学有关。与5-HT 2受体相互作用的药物在治疗上用于治疗这些疾病，其作用机制引起了极大的兴趣。在这项研究中，5-HT 2受体配体的相互作用进行了研究，通过定点诱变，其中三个天冬氨酸天冬酰胺突变体（Asn-120，Asn-155，和Asn-172），并在NIH 3 T3细胞中表达。Asp-120突变为天冬酰胺后，对125 I-麦角酸二乙酰胺的亲和力相同（125 I-LSD）与野生型受体一样，对激动剂5-HT和（+/-）-1-HT的结合亲和力降低，且对GTP不敏感。（2，5-二甲氧基-4-碘苯基）-2-氨基-丙烷（约10倍）和拮抗剂酮色林和米安色林（约10倍），但不包括螺哌隆。该突变还消除了激动剂刺激的[3 H]聚磷酸肌醇（PI）的形成。Asn-155突变体对125 I-LSD的结合亲和力降低（Kd，2.8 nM，野生型受体为0.6 nM），对激动剂和拮抗剂的亲和力降低（分别为约30倍和14-75倍）。然而，Asn-155受体保留GTP敏感性和刺激PI形成的能力。Asn-172突变体保留了野生型对125 I-LSD的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，而保留了GTP敏感性激动剂结合，对酮色林的亲和力无变化，米安色林和螺哌隆结合略有降低（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是必需的鸟嘌呤核苷酸结合蛋白的变构激活。Asp-155是高亲和力结合所必需的，可能是通过充当配体的胺基的抗衡剂。三种突变对酮色林、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-HT 2受体结构的治疗位点选择性化合物的设计。
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.