Oligomerization of the α and β isoforms of the thromboxane A2 receptor: Relevance to receptor signaling and endocytosis

Abstract Thromboxane A 2 (TXA 2 ) is a potent mediator of inflammation, vasoconstriction and oxidative stress. The TXA 2 receptor (TP) is a G protein-coupled receptor (GPCR) that is expressed as two alternatively spliced isoforms, α (343 residues) and β (407 residues) that share the first 328 residues. For many years GPCRs were assumed to exist and function as monomeric species, but increasing evidence suggests that a dimer is the minimal functional unit of GPCRs. In the present report, using co-immunoprecipitation of differentially tagged TP expressed in HEK293 cells, we demonstrate that TPα and TPβ form homo- and hetero-oligomers. Immunoblotting of lysates from human platelets with an anti-TP specific antibody revealed the presence of endogenously expressed TP oligomers. We show that TP oligomerization is an agonist-independent process highly affected by the reducing agent dithiothreitol suggesting the involvement of disulfide bonds in TP oligomerization. Over-expression of G protein-coupled receptor kinases and arrestins did not modulate the extent of receptor dimerization/oligomerization. Co-expression of two TP signaling-deficient mutants, R60L and E2402R, resulted in rescuing of receptor signal transduction suggesting that dimers/oligomers constitute the functional units of this receptor. Interestingly, TPα which does not undergo constitutive or agonist-induced endocytosis on its own was subjected to both types of endocytosis when co-expressed with TPβ, indicating that TPα can display intracellular trafficking when complexed through hetero-oligomerization with TPβ.