Oligomerization of thromboxane A2 receptor : from computational modelling to biochemistry

Thromboxane A2 induces platelet aggregation and constriction of vascular and bronchial smooth muscle through the interaction with a G protein-coupled receptor that exists as two alternatively splice variants, termed TPa and TPb with different C-terminal cytoplasmic tails. TPa and TPb are usually co-expressed in native cells and form homo- and hetero-oligomers in transfected cells. Methods: Computational modelling techniques [1, 2] were first used to generate models of TPa monomer in free- and agonist-bound conformations, then subjected to docking simulations for modelling of TPa homo-dimers. Theoretical predictions were challenged by sitedirected mutagenesis of TPa and TPb. Wild type and mutant receptors fused to fluorescent proteins (GFP, CFP, YFP) were transiently expressed in HEK293 cells and their subcellular localization and dimerization studied by a combination of microscopy and fluorescence resonance energy transfer (FRET) techniques. Results: The most supported model of TPa homo-dimer was characterized by contacts between helices 1 of monomers, whereby cysteine35 residues lie at a distance predictive of an inter-helical disulphide bridge. Upon substitution of cysteine35 with alanine (C35A) homo- and hetero- dimerization of TPa and TPb as well as TPa trafficking was greatly impaired. However, C35A mutant TP receptors did not significantly differ from wild type in terms of [3H]-SQ29, 548 antagonist binding and accumulation of inositol phosphates induced by U46619 agonist stimulation. Conclusions: Mutation of cysteine35 greatly impairs dimerization of TP receptors as anticipated by computational modelling but leaves unaffected the binding and signalling properties of monomeric receptors. References: 1. Casciari et al., BMC Bioinformatics 2006; 7: 340, 2006. 2. Fanelli Mol Cell Endocrinol 2007, 260–262 59–64.