The heterotrimeric G proteins G12 and G13 have received considerable attention for their roles in the regulation of gene transcription, Na+/H+ exchange, and changes in cell contractility and shape. Despite the importance of G12 and G13, much regarding the engagement of these G proteins by agonist-activated receptors remains unclear. The identity of receptors unequivocally coupled to G12 and/or G13, the extent to which agonists vary in capacity to activate these G proteins, the extent to which these G proteins contribute to recognition of agonists by receptors, and how signals are allocated among these and other G proteins are important questions. The overall objective of this proposal is to define the properties of receptor coupling to G12 and GI3 and to evaluate the relevance of superimposed forms of regulation. The first specific aim addresses the concept of agonism as it applies to G12 and G13. We will use Sf9 and mammalian cell expression systems to define the coupling of receptors to GI2 and G13 employing ligand-stimulated [35S]GTP?S-binding and radioligand displacement assays, asking whether signals from agonist-activated receptors are interpreted differently by G12 and GI3 than by other G proteins. The second specific aim addresses the relevance of Gs, Gi, and Gq to the functionality of receptors coordinately coupled to G12 and G13. We will test the hypothesis that different G proteins impart to a single receptor different increases in affinities for agonists and in so doing shape the sensitivity of cells uniquely according to coupling profiles. We will also examine the concept of agonist-directed trafficking of receptor stimulus as it applies to Gs, Gi, Gq, G12, and G13 using isolated and intact modes of a three-state model. The third specific aim is to define the underlying basis and impact of G12 and G13 phosphorylation in the intact cell. The alpha subunits of G12 and G13 are phosphorylated in a variety of cells in response to activation of protein kinase C. We propose to examine the sites and extent of phosphorylation in human platelets, test the notion that Galpha3 is phosphorylated by a protein kinase downstream of protein kinase C, and determine the impact of phosphorylation on receptor-G protein coupling.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Pharmacology A Study Section (PHRA)
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Dunsmore, Sarah
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University of Pennsylvania
Schools of Medicine
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Zhang, Li; Brass, Lawrence F; Manning, David R (2009) The Gq and G12 families of heterotrimeric G proteins report functional selectivity. Mol Pharmacol 75:235-41
Riobo, Natalia A; Manning, David R (2007) Pathways of signal transduction employed by vertebrate Hedgehogs. Biochem J 403:369-79
Riobo, Natalia A; Saucy, Berangere; Dilizio, Cherisse et al. (2006) Activation of heterotrimeric G proteins by Smoothened. Proc Natl Acad Sci U S A 103:12607-12
Zhang, Li; DiLizio, Cherisse; Kim, David et al. (2006) The G12 family of G proteins as a reporter of thromboxane A2 receptor activity. Mol Pharmacol 69:1433-40
Riobo, Natalia A; Manning, David R (2005) Receptors coupled to heterotrimeric G proteins of the G12 family. Trends Pharmacol Sci 26:146-54