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.
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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 |
|