G protein—coupled receptors (GPCRs) represent a class of important therapeutic targets for drug discovery. The integration of GPCRs into contemporary high-throughput functional assays is critically dependent on the presence of appropriate G proteins. Given that different GPCRs can discriminate against distinct G proteins, a universal G protein adapter is extremely desirable. In this report, the authors evaluated two highly promiscuous Gα16/zchimeras, 16z25 and 16z44, for their ability to translate GPCR activation into Ca2+mobilization using the fluorescence imaging plate reader (FLIPR) and aequorin. A panel of 24 Gs- or Gi-coupled receptors was examined for their functional association with the Gα16/zchimeras. Although most of the GPCRs tested were incapable of inducing Ca2+mobilization upon their activation by specific agonists, the introduction of 16z25 or 16z44 allowed all of these GPCRs to mediate agonist-induced Ca2+mobilization. In contrast, only 16 of the GPCRs tested were capable of using Gα16to mobilize intracellular Ca2+. Analysis of dose-response curves obtained with the δ-opioid, dopamine D1, and Xenopus melatonin Mel1c receptors revealed that the Gα16/zchimeras possess better sensitivity than Gα16in both the FLIPR and aequorin assays. Collectively, these studies help to validate the promiscuity of the Gα16/zchimeras as well as their application in contemporary drug-screening assays that are based on ligand-induced Ca2+mobilization. ( Journal of Biomolecular Screening 2003:39-49)
The bovine Gα14 is a member of the Gq subfamily of G proteins that can regulate phospholipase Cβ isoforms but the extent to which Gα14 recognizes different receptor classes is not known.
Gα14 was cotransfected with a variety of receptors in COS‐7 cells, and agonist‐induced stimulation of phospholipase C was then measured.
Activation of the type 2 but not type 1 somatostatin receptor in cells coexpressing Gα14 stimulated the accumulation of inositol phosphates; functional expression of both subtypes of somatostatin receptors was determined by the ability of somatostatin to inhibit cyclic AMP accumulation.
Among the three opioid receptors (μ, δ, and κ), only the δ receptor was capable of stimulating IP formation when coexpressed with Gα14 in COS‐7 cells.
A panel of Gi‐ and Gs‐linked receptors was screened for their ability to stimulate IP accumulation via Gα14. The adenosine A1, complement C5a, dopamine D1, D2 and D5, formyl peptide, luteinizing hormone, secretin, and the three subtypes of melatonin (mt1, MT2, and Xenopus) receptors were all incapable of activating Gα14, while the α2‐ and β2‐adrenoceptors were able to do so.
Gα14‐mediated stimulation of phospholipase Cβ was agonist dose‐dependent. These data demonstrate that although Gα14 can interact with different classes of receptors, it is much less promiscuous than Gα15 or Gα16.
British Journal of Pharmacology (2001) 132, 1431–1440; doi:
A broad repertory of G-protein-coupled receptors shows effective coupling with the haematopoietic G16 protein. In the present study, individual residues along the C-terminal alpha5 helix of Galpha16 were examined for their contributions in defining receptor-coupling specificity. Residues that are relatively conserved within, but diverse between, the subfamilies of cloned Galpha subunits were mutated into the corresponding Galpha(z) residues. Six G(i)-linked receptors with different coupling efficiencies to Galpha16 were examined for their ability to utilize the various Galpha16 mutants to mediate agonist-induced inositol phosphate accumulation and Ca2+ mobilization. Co-operative enhancements of receptor coupling were observed with chimaeras harbouring multiple mutations at Glu350, Lys357 and Leu364 of Galpha16. Mutation of Leu364 into isoleucine appeared to be more efficient in enhancing receptor recognition compared with mutations at the other two sites. Mutation of a stretch of six consecutive residues (362-367) lying towards the end of the alpha5 helix was found to broaden significantly the receptor-coupling profile of Galpha16, and the effect was mediated partly through interactions with the beta2-beta3 loop. These results suggested that a stretch of six distinctive residues at the alpha5 helix of Galpha16 is particularly important, whereas other discrete residues spreading along the alpha5 helix function co-operatively for determining the specificity of receptor recognition.
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