We have discovered a protein family termed RGSs that impair signal transduction through signaling pathways that use seven transmembrane receptors and heterotrimeric G proteins. Such receptors, when activated following the binding of a ligand such as a hormone or chemokine, trigger G-alpha subunit to exchange GTP for GDP; this causes the dissociation of G-alpha and G-beta- gamma subunits and downstream signaling. RGS proteins bind G- alpha subunits and function as GTPase activating proteins (GAPs), thereby deactivating the G-alpha subunit and facilitating their re-association with G-beta-gamma. We have created a series of mutant RGS mutant proteins, which has revealed several key residues required for Gi-alpha binding and provided several important reagents for studying RGS protein function. Recently, we have shown that several RGS proteins also interfere with G-beta-gamma signaling likely by behaving as effector antagonists for the free G-beta-gamma subunits. Since chemokine receptors utilize Beta-gamma subunits to trigger chemotaxis, RGS proteins may be particularly important in regulating chemokine signaling. We have shown in a transient transfection assay that RGS1 and RGS3 are potent inhibitors of chemokine signaling and chemotaxis. By examining signaling pathways that utilize different G-alpha subunits, we have found some specificity of individual RGS proteins for certain G-alpha subunits. RGS2 and RGS3 are very effective Gq-inhibitors, while RGS14 inhibits G13 signaling. In addition, RGS3 is an effective inhibitor of signaling by the Kaposi Sarcoma G protein coupled receptor. Since this receptor has transforming properties, RGS3 may prove to be a potent inhibitor of cellular transformation by G protein coupled receptors. We have also shown that RGS proteins can be recruited to the cell membrane from the cytosol by different stimuli. This provides a mechanism by which signaling through G protein coupled receptors can be attenuated. Yeast 2-hybrid studies revealed several proteins that interact with RGS proteins including 14-3-3e and t, proteins that regulate intracellular signaling, and beta'COP, a protein that participates in coating secretory vesicles. Based on these results we have shown a role for 14-3-3 proteins in regulating RGS protein function and that RGS proteins may regulate certain intracellular secretory pathways. RGS proteins have proved to be effective inhibitors of chemokine- induced chemotaxis and the analysis of mice deficient in RGS1 has revealed an enhanced chemotaxis response to SDF-1. Gene targeting experiments for RGS4 and RGS14 are in progress. In the past year we have accumulated increasing evidence that RGS proteins are important regulators of signaling through G protein coupled receptors and have broadened our understanding of the mechanisms by which they accomplish this regulation.
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