The serotonin binding site of human and murine 5-HT _2B receptors. Molecular modeling and site-directed mutagenesis

Bacteriorhodopsin and rhodopsin crystal structures were used as templates to build structural models of the mouse and human serotonin (5-HT)-2B receptors (5-HT _2BRs). Serotonin was docked to the receptors, and the amino acids predicted to participate to its binding were subjected to mutagenesis. 5-HT binding affinity and 5-HT-induced inositol triphosphate production were measured in LMTK ^- cells transfected with either wild-type or mutated receptor genes. According to these measurements, the bacteriorhodopsin-based models of the 5-HT _2BRs appear more confident than the rhodopsin-based ones. Residues belonging to the transmembrane domains 3 and 6, i.e. Asp ^3.32, Ser ^3.36, Phe ^6.52, and Asn ^6-55, make direct contacts with 5-HT. In addition, Trp ^3.28, Phe ^3.35, Phe ^6.52, and Phe ^7.38 form an aromatic box surrounding 5-HT. The specificity of human and mouse 5-HT _2BRs may be reflected by different rearrangements of the aromatic network upon 5-HT binding. Two amino acids close to Pro ^5.50 in the human transmembrane domain 5 sequence were permuted to introduce a "mouse-like" sequence. This change was enough to confer the human 5-HT _2BR properties similar to those of the mouse. Taken together, the computed models and the site-directed mutagenesis experiments give a structural explanation to (i) the different 5-HT pK _D values measured with the human and mouse 5-HT _2BRs (7.9 and 5.8, respectively) and (ii) the specificity of 5-HT binding to 5-HT _2BRs as compared with other serotonergic G-protein coupled receptors.