title: Site-directed mutagenesis of the serotonin 5-Hydroxytryptamine2c receptor: identification of amino acids responsible for sarpogrelate binding.
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.
authors.affiliation: Department of Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Japan.
authors.forename: Habib Abul
authors.initials: HA
authors.lastname: Muntasir
authors.forename: Jyuniti
authors.initials: J
authors.lastname: Takahashi
authors.forename: Mamunur
authors.initials: M
authors.lastname: Rashid
authors.forename: Maruf
authors.initials: M
authors.lastname: Ahmed
authors.forename: Tadazumi
authors.initials: T
authors.lastname: Komiyama
authors.forename: Murad
authors.initials: M
authors.lastname: Hossain
authors.forename: Jun
authors.initials: J
authors.lastname: Kawakami
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nashimoto
authors.forename: Takafumi
authors.initials: T
authors.lastname: Nagatomo
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D044405:Receptor, Serotonin, 5-HT2C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013386:Succinates / Q000378:metabolism*; D014316:Tritium
chemical_list: D000596:Amino Acids; D007295:Inositol Phosphates; D044405:Receptor, Serotonin, 5-HT2C; D013386:Succinates; D014316:Tritium; C064294:sarpogrelate