Identification of amino acid residues important for sarpogrelate binding to the human 5-hydroxytryptamine2A serotonin receptor.
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.
Muntasir
Habib Abul
HA
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Japan.
Rashid
Mamunur
M
Komiyama
Tadazumi
T
Kawakami
Jun
J
Nagatomo
Takafumi
T
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
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