RRC ID 6510
著者 Muntasir HA, Takahashi J, Rashid M, Ahmed M, Komiyama T, Hossain M, Kawakami J, Nashimoto M, Nagatomo T.
タイトル Site-directed mutagenesis of the serotonin 5-Hydroxytryptamine2c receptor: identification of amino acids responsible for sarpogrelate binding.
ジャーナル Biol Pharm Bull
Abstract Site-directed mutagenesis was used to investigate the molecular interactions involved in sarpogrelate binding to the human 5-Hydroxytryptamine(5-HT)2C receptor. Based on molecular modeling studies, Aspartic acid (Asp)155[3.32] in transmembrane region III and Serine(Ser)361[7.46] in transmembrane region VII of the 5-HT2C receptor were found to interact with sarpogrelate. Asp3.32 and Ser7.46 were mutated to alanine (Ala) and expressed in COS-7 cells. The radioligand [3H]mesulergine did not show any binding to Asp3.32Ala mutant of 5-HT2C receptor. Therefore, it was not possible to find any sarpogrelate affinity to the mutant using [3H]mesulergine. The mutation also abolished agonist-stimulated IP formation of [3H]myo-inositol. Introduction of dual mutation at position Ser7.46 (Asp3.32Ala-Ser7.46Ala) could not restore the function disrupted by the first mutation (Asp3.32Ala). On the other hand, the Ser7.46Ala mutant showed reduced binding affinity for [3H]mesulergine (Kd 3557 pM versus 573 pM for the wild-type receptor) and had reduced affinity for sarpogrelate. Moreover, the Ser7.46Ala mutant receptor also showed a great loss of potency for sarpogrelate in inhibiting 5-HT-stimulated IP formation of [3H]myo-inositol. The results provide direct evidence that Asp3.32 and less importantly, Ser7.46 are responsible for the interaction between 5-HT2C receptor and [3H]mesulergine as well as sarpogrelate. More interestingly, Ser7.46Ala increases the receptor expression (20-fold vs. wild-type) of the mutant receptors and basal [3H]myo-inositol formation (2.5-fold vs. wild-type), which indicates that the 5-HT2C receptor could be rendered constitutively active by mutating the amino acid serine at position 7.46 to alanine.
巻・号 29(8)
ページ 1645-50
公開日 2006-8-1
DOI 10.1248/bpb.29.1645
PII JST.JSTAGE/bpb/29.1645
PMID 16880620
MeSH Amino Acids / genetics* Animals Binding Sites COS Cells Chlorocebus aethiops Humans Inositol Phosphates / metabolism Models, Molecular Mutagenesis, Site-Directed Radioligand Assay Receptor, Serotonin, 5-HT2C / chemistry Receptor, Serotonin, 5-HT2C / genetics Receptor, Serotonin, 5-HT2C / metabolism* Succinates / metabolism* Tritium
IF 1.863
引用数 12
WOS 分野 PHARMACOLOGY & PHARMACY
リソース情報
ヒト・動物細胞