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.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 Sequence polymorphisms can potentially influence the efficacy of drugs in patient populations and are therefore an important consideration in the drug development process. To identify DNA sequence variants of the mGluR1 receptor, comparative DNA sequencing was performed on DNA samples (n=186) from apparently healthy subjects representing 2 ethnic groups. In total, 8 non synonymous single nucleotide polymorphisms (SNPs) were identified and one SNP (c2977>T) was found to be particularly common, this SNP results in a Proline to Serine substitution at residue 993 (P993S). The WT (P993) and S993 variants were expressed in an inducible system which allowed us to titrate gene expression to equivalent levels and were pharmacologically characterised. We determined the potency and affinity of standard antagonist compounds as well as the potency and efficacy of the endogenous ligand Glutamate and other agonist compounds at both receptor variants. Agonist evoked increases in intracellular Ca 2+ were measured by FLIPR. The potency of mGluR1 antagonists was evaluated by their ability to inhibit quisqualate induced increases in intracellular Ca 2+ , while their affinities were determined by radio-ligand binding studies. This study demonstrates that the Pro993Ser amino acid exchange is highly frequent in the human mGluR1gene. This polymorphism however, does not appear to affect the potency of agonist compounds or the potencies or affinities of small molecule antagonist compounds.

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