Abstract

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 Glu¹²⁹ and Arg¹³⁰ in the ERY of thromboxane A₂ receptor α (TPα) in transfected human embryonic kidney 293 cells. We show that no conservative or nonconservative substitutions of Glu¹²⁹ and Arg¹³⁰ generated a constitutively active TPα mutant, but a nonconservative mutation of Arg¹³⁰ (R130V) yielded a mutant receptor with significantly impaired 9,11-dideoxy-9α,11α-methanoepoxy-prosta-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 TPα 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 ∼10-fold lower EC₅₀ and a ∼2-fold higher E_max 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 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.