Site-directed mutagenesis of the serotonin 5-hydroxytryptamine2C receptor:: Identification of amino acids responsible for sarpogrelate binding

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 [H-3]mesulergine did not show any binding to Asp3.32A1a mutant of 5-HT2C receptor. Therefore, it was not possible to find any sarpogrelate affinity to the mutant using [H-3]mesulergine. The mutation also abolished agonist-stimulated IP formation of [H-3]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 [H-3] 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-HTstimulated IP formation of [H-3]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 [H-3] mesulergine as well as sarpogrelate. More interestingly, Ser7.46Ala increases the receptor expression (20-fold vs. wild-type) of the mutant receptors and basal [H-3]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.