Site-Directed Mutagenesis of the Serotonin 5-Hydroxytryptamine_2C 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-HT_2C 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-HT_2C 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 (K_d 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-HT_2C 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-HT_2C receptor could be rendered constitutively active by mutating the amino acid serine at position 7.46 to alanine.