The serotonin binding site of human and murine 5-HT2B 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(2B)Rs). 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(2B)Rs 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(2B)Rs 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(2B)R 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(2B)Rs (7.9 and 5.8, respectively) and (ii) the specificity of 5-HT binding to 5-HT(2B)Rs as compared with other serotonergic G-protein coupled receptors.
Manivet
Philippe
P
Centre de Recherche Claude Bernard Pathologie Expérimentale et Communications Cellulaires, IFR 6, Service de Biochimie, Hôpital Lariboisière Assistance Publique-Hopitaux de Paris (AP-HP), 75475 Paris Cedex 10, France.
Schneider
Benoit
B
Smith
Jeremy Christopher
JC
Choi
Doo-Sup
DS
Maroteaux
Luc
L
Kellermann
Odile
O
Launay
Jean-Marie
JM
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044403:Receptor, Serotonin, 5-HT2B; D011985:Receptors, Serotonin / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry
D008024:Ligands; D044403:Receptor, Serotonin, 5-HT2B; D011985:Receptors, Serotonin; D001224:Aspartic Acid; D012694:Serine; D010649:Phenylalanine; D014364:Tryptophan