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 TPα and TPβ 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 TPα and TPβ, epitope-tagged or fused to fluorescent proteins (GFP, CFP, YFP), ectopically expressed in transfected cells. We show that at steady state TPα and TPβ 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 TPα and TPβ mutants unable to dimerize, thus conferming the theoretical predictions. When individually expressed in cells TPβ, not TPα, underwent endocytosis upon agonist stimulation via the clathrin-dependent route. However, TPα was capable of agonist-induced endocytosis when co-expressed with TPβ thus suggesting co-internalization of TPα-TPβ hetero-complexes. However, the intracellular route of TPα-TPβ dimer was different from that of TPβ, 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.