Mutational analysis of the highly conserved ERY motif of the thromboxane A2 receptor: alternative role in G protein-coupled receptor signaling

The presence of highly conserved amino acid stretches in G protein-coupled receptors (GPCRs) usually predicts an important role in receptor function. Considerable attention has therefore been focused on the involvement of the highly conserved Glu/Asp –Arg –Tyr (E/DRY) motif at the cytoplasmic end of transmembrane domain 3 in the regulation of GPCR conformational states and/or the mediation of G protein activation. In the present work, we have investigated the role of Glu129 and Arg130 in the ERY of thromboxane A2 receptor α (TPα) in transfected HEK293 cells. We show that no conservative or non-conservative substitutions of Glu129 and Arg130 generated a constitutively active TPα mutant, but a non-conservative mutation of Arg130 (R130V) yielded a mutant receptor with significantly impaired U46619-induced accumulation of inositol phosphates (IPs). This loss-of-function phenotype seems to be due to the uncoupling of the TPα receptor from Gq, as demonstrated by the loss of high-affinity agonist binding, and not to receptor internalization, as shown by localization studies with the R130V-GFP fusion protein. Interestingly, U46619-induced activation of the non-conservative E129V mutant stimulated the production of IPs with a ~10-fold lower EC50 and a ~2-fold higher Emax than in the wild type receptor. Collectively, these data demonstrate that, unlike other GPCRs, mutations of Glu129 do not induce constitutive activity, whereas Arg130 is involved in G protein coupling or recognition, and suggest the existence within class A GPCRs of at least two different subclasses that make different uses of the highly conserved E/DRY motif.