Exploration of the ligand binding site of the human 5-HT4 receptor by site-directed mutagenesis and molecular modeling

1 Among the five human 5-HT4 (h5-HT4) receptor isoforms, the hS-HT4(a) rece ptor was studied with a particular emphasis on the molecular interactions i nvolved in ligand binding. For this purpose, we used site-directed mutagene sis of the transmembrane domain. Twelve mutants were constructed with a spe cial focus on the residue P4.53 of helix IV which substitutes in h5-HT4 rec eptors the highly conserved S residue among the rhodopsin family receptors. The mutated receptors were transiently expressed in COS-7 cells. 2 Ligand binding or competition studies with two h5-HT4 receptor agonists, serotonin and ML10302 and two h5-HT4 receptor antagonists, [H-3]-GR113808 a nd ML10375 were performed on wild type and mutant receptors. Functional act ivity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase. 3 Ligand binding experiments revealed that [H-3]-GR113808 did not bind to m utants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. O n the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5-HT4(a) receptor functional activity. From these stu dies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagoni st [H-3]-GR113808 and to serotonin. 4 According to these results, we propose ligand-receptor complex models wit h serotonin and [H-3]-GR113808. For serotonin, three interaction points wer e selected including ionic interaction with D3.32, a stabilizing interactio n of this ion pair by Y7.43 and a hydrogen bond with S5.43. [H-3]-GR113808 was also docked, based on the same type of interactions with S5.43 and D3.3 2: the proposed model suggested a possible role of P4.53 in helix IV struct ure allowing the involvement of a close hydrophobic residue, W4.50, in a hy drophobic pocket for hydrophobic interactions with the indole ring of [H-3] -GR113808.