title: Identification of amino acid residues important for sarpogrelate binding to the human 5-hydroxytryptamine2A serotonin receptor.
abstract: 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.
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: Mamunur
authors.initials: M
authors.lastname: Rashid
authors.forename: Tadazumi
authors.initials: T
authors.lastname: Komiyama
authors.forename: Jun
authors.initials: J
authors.lastname: Kawakami
authors.forename: Takafumi
authors.initials: T
authors.lastname: Nagatomo
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007650:Ketanserin / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D044402:Receptor, Serotonin, 5-HT2A / Q000235:genetics / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000378:metabolism*; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D013386:Succinates / Q000378:metabolism*; D014162:Transfection
chemical_list: D000596:Amino Acids; D007295:Inositol Phosphates; D008024:Ligands; D044402:Receptor, Serotonin, 5-HT2A; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D013386:Succinates; C064294:sarpogrelate; D004247:DNA; D007650:Ketanserin