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 study, we investigated the role of Glu(129) and Arg(130) in the ERY of thromboxane A(2) receptor alpha (TPalpha) in transfected human embryonic kidney 293 cells. We show that no conservative or nonconservative substitutions of Glu(129) and Arg(130) generated a constitutively active TPalpha mutant, but a nonconservative mutation of Arg(130) (R130V) yielded a mutant receptor with significantly impaired 9,11-dideoxy-9alpha, 11alpha-methanoepoxyprosta-5Z, 13E-dien-1-oic acid (U46619)-induced accumulation of inositol phosphates (IPs). This loss-of-function phenotype seems to be caused by the uncoupling of the TPalpha receptor from G(q), as demonstrated by the loss of high-affinity agonist binding, and not by receptor internalization, as shown by localization studies with the R130V-green fluorescent protein fusion protein. It is interesting to note that U46619-induced activation of the nonconservative E129V mutant stimulated the production of IPs with a similar to10- fold lower EC50 and a similar to2-fold higher E-max than in the wild-type receptor. Collectively, these data demonstrate that, unlike other GPCRs, mutations of Glu(129) do not induce constitutive activity, whereas Arg(130) is involved in G protein coupling or recognition, and they suggest the existence within class A GPCRs of at least two different subclasses that make different uses of the highly conserved E/DRY motif.