Muntasir Habib Abul, Rashid Mamunur, Komiyama Tadazumi, Kawakami Jun, Nagatomo Takafumi
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Japan.
J Pharmacol Sci. 2006 Sep;102(1):55-63. doi: 10.1254/jphs.fp0060171. Epub 2006 Sep 15.
The purpose of the present study was to examine 5-hydroxytryptamine (5-HT)(2A)-receptor sarpogrelate interactions by site-directed mutagenesis. Based on molecular modeling studies, aspartic acid (Asp)155[3.32] and tryptophan (Trp)151[3.28] in transmembrane helix (TMH) III and Trp336[6.48] in TMH VI of the 5-HT(2A) receptor were found to interact with sarpogrelate. All of these residues were mutated to alanine (Ala). The Asp3.32Ala mutant did not exhibit any affinity for [(3)H]ketanserin, whereas the Trp3.28Ala mutant showed a markedly decrease in the binding affinity for [(3)H]ketanserin (K(d) >10,000 nM). Therefore, it was not possible to find any sarpogrelate affinity to the mutants using [(3)H]ketanserin. The mutation also abolished agonist-stimulated formation of [(3)H]inositol phosphates (IP) in both cases. On the other hand, the Trp6.48Ala mutant showed reduced binding affinity for [(3)H]ketanserin (K(d) 2.0 nM vs 0.8 nM for the wild-type receptor) and had reduced affinity for sarpogrelate (pK(i) value of 5.71 vs 9.06 for the wild-type receptor). The Trp6.48Ala mutant also showed the greatest decrease in sensitivity to sarpogrelate (pK(b) value 8.81 for wild-type and 5.11 for mutant) in inhibiting agonist-stimulated IP formation. These results provide direct evidence that Asp3.32, Trp3.28, and less importantly, Trp6.48 are responsible for the interaction between the 5-HT(2A) receptor and sarpogrelate. In addition, these results support the findings obtained from molecular modeling studies.
本研究的目的是通过定点诱变来检测5-羟色胺（5-HT）（2A）受体与沙格雷酯之间的相互作用。基于分子模拟研究，发现5-HT（2A）受体跨膜螺旋（TMH）III中的天冬氨酸（Asp）155[3.32]和色氨酸（Trp）151[3.28]以及TMH VI中的Trp336[6.48]与沙格雷酯相互作用。所有这些残基都被突变为丙氨酸（Ala）。Asp3.32Ala突变体对[（3）H]酮舍林没有任何亲和力，而Trp3.28Ala突变体对[（3）H]酮舍林的结合亲和力显著降低（K（d）>10,000 nM）。因此，使用[（3）H]酮舍林无法发现突变体对沙格雷酯有任何亲和力。在这两种情况下，突变也消除了激动剂刺激的[（3）H]肌醇磷酸（IP）的形成。另一方面，Trp6.48Ala突变体对[（3）H]酮舍林的结合亲和力降低（野生型受体的K（d）为2.0 nM，而突变体为0.8 nM），并且对沙格雷酯的亲和力降低（野生型受体的pK（i）值为9.06，而突变体为5.71）。Trp6.48Ala突变体在抑制激动剂刺激的IP形成方面对沙格雷酯的敏感性也表现出最大程度的降低（野生型的pK（b）值为8.81，突变体为5.11）。这些结果提供了直接证据，表明Asp3.32、Trp3.28以及不太重要的Trp6.48负责5-HT（2A）受体与沙格雷酯之间的相互作用。此外，这些结果支持了从分子模拟研究中获得的发现。
