PT - JOURNAL ARTICLE AU - Nataliya Gorinski AU - Noga Kowalsman AU - Ute Renner AU - Alexander Wirth AU - Michael T. Reinartz AU - Roland Seifert AU - Andre Zeug AU - Evgeni Ponimaskin AU - Masha Y. Niv TI - Computational and Experimental Analysis of the Transmembrane Domain 4/5 Dimerization Interface of the Serotonin 5-HT<sub>1A</sub> Receptor AID - 10.1124/mol.112.079137 DP - 2012 Sep 01 TA - Molecular Pharmacology PG - 448--463 VI - 82 IP - 3 4099 - http://molpharm.aspetjournals.org/content/82/3/448.short 4100 - http://molpharm.aspetjournals.org/content/82/3/448.full SO - Mol Pharmacol2012 Sep 01; 82 AB - Experimental evidence suggests that most members of class A G-protein coupled receptors (GPCRs) can form homomers and heteromers in addition to functioning as single monomers. In particular, serotonin (5-HT) receptors were shown to homodimerize and heterodimerize with other GPCRs, although the details and the physiological role of the oligomerization has not yet been fully elucidated. Here we used computational modeling of the 5-HT1A receptor monomer and dimer to predict residues important for dimerization. Based on these results, we carried out rationally designed site-directed mutagenesis. The ability of the mutants to dimerize was evaluated using different FRET-based approaches. The reduced levels of acceptor photobleaching-Förster resonance energy transfer (FRET) and the lower number of monomers participating in oligomers, as assessed by lux-FRET, confirmed the decreased ability of the mutants to dimerize and the involvement of the predicted contacts (Trp1754.64, Tyr1985.41, Arg1514.40, and Arg1524.41) at the interface. This information was reintroduced as constraints for computational protein-protein docking to obtain a high-quality dimer model. Analysis of the refined model as well as molecular dynamics simulations of wild-type (WT) and mutant dimers revealed compensating interactions in dimers composed of WT and W175A mutant. This provides an explanation for the requirement of mutations of Trp1754.64 in both homomers for disrupting dimerization. Our iterative computational-experimental study demonstrates that transmembrane domains TM4/TM5 can form an interaction interface in 5-HT1A receptor dimers and indicates that specific amino acid interactions maintain this interface. The mutants and the optimized model of the dimer structure may be used in functional studies of serotonin dimers.