Exploration of the ligand binding site of the human 5-HT 4 receptor by site-directed mutagenesis and molecular modeling

Jeanne Mialet (1) , Yamina Dahmoune (2, 3) , Frank Lezoualc'H (1) , Isabelle Berque-Bestel (2, 3) , Pierre Eftekhari (4) , Johan Hoebeke (4) , Sames Sicsic (2, 3) , Michel Langlois (3, 2) , Rodolphe Fischmeister (1)

1 CARPAT - Signalisation et physiopathologie cardiovasculaire
2 ISIT - Institut de signalisation et innovation thérapeutiques
3 BioCIS - Biomolécules : Conception, Isolement, Synthèse
4 ICT - Immunologie et chimie thérapeutiques

Among the ®ve human 5-HT 4 (h5-HT 4) receptor isoforms, the h5-HT 4(a) receptor was studied with a particular emphasis on the molecular interactions involved in ligand binding. For this purpose, we used site-directed mutagenesis of the transmembrane domain. Twelve mutants were constructed with a special focus on the residue P4.53 of helix IV which substitutes in h5-HT 4 receptors 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-HT 4 receptor agonists, serotonin and ML10302 and two h5-HT 4 receptor antagonists, [ 3 H]-GR113808 and ML10375 were performed on wild type and mutant receptors. Functional activity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase. 3 Ligand binding experiments revealed that [ 3 H]-GR113808 did not bind to mutants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. On the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5-HT 4(a) receptor functional activity. From these studies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagonist [ 3 H]-GR113808 and to serotonin. 4 According to these results, we propose ligand-receptor complex models with serotonin and [ 3 H]-GR113808. For serotonin, three interaction points were selected including ionic interaction with D3.32, a stabilizing interaction of this ion pair by Y7.43 and a hydrogen bond with S5.43. [ 3 H]-GR113808 was also docked, based on the same type of interactions with S5.43 and D3.32: the proposed model suggested a possible role of P4.53 in helix IV structure allowing the involvement of a close hydrophobic residue, W4.50, in a hydrophobic pocket for hydrophobic interactions with the indole ring of [ 3 H]-GR113808.

Keywords: Serotonin; 5-HT 4 receptor; human; binding site; directed mutagenesis; molecular modeling

Domains: Molecular biology

Jeanne Mialet, Yamina Dahmoune, Frank Lezoualc'H, Isabelle Berque-Bestel, Pierre Eftekhari, et al.. Exploration of the ligand binding site of the human 5-HT 4 receptor by site-directed mutagenesis and molecular modeling. British Journal of Pharmacology, 2000, 130 (3), pp.527-538. ⟨10.1038/sj.bjp.0703356⟩. ⟨hal-03611626⟩