Mutational Analysis of the Highly Conserved ERY Motif of the Thromboxane A₂Receptor: Alternative Role in G Protein-Coupled Receptor Signaling

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 Glu 129 and Arg 130 in the ERY of thromboxane A 2 receptor ␣ (TP␣) 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 TP␣ mutant, but a nonconservative mutation of Arg 130 (R130V) yielded a mutant receptor with significantly impaired 9,11-dideoxy-9␣,11␣-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 TP␣ 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 ϳ10-fold lower EC 50 and a ϳ2-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.

Light on the structure of thromboxane A2 receptor heterodimers

“…Receptor expression was monitored by equilibrium mixed type binding curves of [ 3 H]SQ 29,548 (48 Ci/mmol) performed as previously described [37,38]. Briefly, confluent adherent cells in 250 ll of serum-free DMEM, containing 0.2% (w/v) BSA were assayed in the presence of 0.1-1 nM serial dilutions of [ 3 H]SQ 29,548 and 3 nM-10 lM of the homologous cold ligand.…”

“…Total inositol phosphate production Signaling of TPs was assessed by measuring the accumulation of total IPs as previously described [37,38]. Briefly, cells labeled overnight with 0.5 lCi of myo-[2-3 H]inositol (18 Ci/mmol) were pre-incubated with 25 mM LiCl for 10 min followed by incubation with increasing concentrations of the TP selective U46619 agonist for 30 min at 37°C.…”

“…IL1 and EL1 are lime, IL2 and EL2 are slate, IL3 and EL3 are salmon and the N-and C-terms are red. In the bottom image, the side chains of the interface amino acids subjected to alanine substitutions are shown in gray computer analysis, with binding parameters as previously reported [37,41] It has been previously reported that TPb undergoes agonistinduced endocytosis with a kinetics similar to many GPCRs, whereas TPa does so only after prolonged (2-4 h) agonist stimulation, or when co-expressed in the same cells with TPb [4], or prostacyclin receptor (IP) [7]. These observations suggested that TPa associates with TPb or IP, thus forming hetero-dimeric receptor complexes that undergo agonist-promoted intracellular trafficking.…”

TBXA2R gene variants associated with bleeding

“…Molecular modeling indicates that Arg60 interacts via hydrogen bonds with Met126 and Arg130 in transmembrane domain (TM)3 and that this interaction is lost when the Arg is substituted for Leu (11). Arg130 is part of the highly conserved D/ERY motif (Figure 1) critical for TPα receptor activation (12). Therefore, in line with previous mutagenesis studies of the ERY motif (12), the Arg60Leu substituted TPα receptor is predicted to be unable to undergo the conformational changes required to promote efficient G protein coupling.…”

“…Arg130 is part of the highly conserved D/ERY motif (Figure 1) critical for TPα receptor activation (12). Therefore, in line with previous mutagenesis studies of the ERY motif (12), the Arg60Leu substituted TPα receptor is predicted to be unable to undergo the conformational changes required to promote efficient G protein coupling.…”