Oligomerization of the alpha and beta isoforms of the thromboxane A2 receptor: relevance to receptor signaling and endocytosis.
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, alpha (343 residues) and beta (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 TPalpha and TPbeta 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, TPalpha which does not undergo constitutive or agonist-induced endocytosis on its own was subjected to both types of endocytosis when co-expressed with TPbeta, indicating that TPalpha can display intracellular trafficking when complexed through hetero-oligomerization with TPbeta.
Laroche
Geneviève
G
Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique, Université de Sherbrooke, Fleurimont, Qc, Canada J1H 5N4.
Lépine
Marie-Claude
MC
Thériault
Caroline
C
Giguère
Patrick
P
Giguère
Vincent
V
Gallant
Maxime A
MA
de Brum-Fernandes
Artur
A
Parent
Jean-Luc
JL
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D019393:Arrestin / Q000378:metabolism; D001704:Biopolymers; D001792:Blood Platelets / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D004705:Endocytosis; D006801:Humans; D047468:Immunoprecipitation; D009154:Mutation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D019393:Arrestin; D001704:Biopolymers; D020033:Protein Isoforms; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid