Imaging the daily life of the G protein-coupled thromboxane A2 receptor : from dimerization to internalization

The thromboxane A2 receptor (TP) is a seven transmembrane protein coded by a single gene that undergoes alternative splicing giving rise to the TP\u3b1 and TP\u3b2 isoforms with identical N-terminal 328 aminoacids and different C-terminal tails of 15 and 79 aminoacids, respectively. Using confocal microscopy or wide-field microscopy combined with FRET analysis we have imaged TP\u3b1 and TP\u3b2, epitope-tagged or fused to fluorescent proteins (GFP, CFP, YFP), ectopically expressed in transfected cells. We show that at steady state TP\u3b1 and TP\u3b2 form homo- and hetero-dimeric complexes at the cell surface. The architecture of homo- and hetero-dimers was predicted by an in silico approach to be essentially characterized by contacts between hydrophobic amino acids I25, W29, C35, V36, L39, L43, L44 and S47 in helix 1 from both monomers. Substitutions of these residues into alanines generated TP\u3b1 and TP\u3b2 mutants unable to dimerize, thus conferming the theoretical predictions. When individually expressed in cells TP\u3b2, not TP\u3b1, underwent endocytosis upon agonist stimulation via the clathrin-dependent route. However, TP\u3b1 was capable of agonist-induced endocytosis when co-expressed with TP\u3b2 thus suggesting co-internalization of TP\u3b1-TP\u3b2 hetero-complexes. However, the intracellular route of TP\u3b1-TP\u3b2 dimer was different from that of TP\u3b2, since it was not affected by co-expression of dominant negative mutants of dynamin and EPS-15, and showed less intense co-localization with transferrin receptor, Rab4, Rab5 vesicle markers