The Regulator of G protein Signaling (RGS) proteins originally were identified as GTPase-activating proteins (GAPs) for heterotrimeric G protein ? subunits. However, many RGS proteins possess highly- conserved domains in addition to the signature RGS box, which empower a multifunctional character that underlies poorly understood but physiologically important interactions among components of heterotrimeric G protein signaling cascades as well as with other signaling pathways. The R7 subfamily of RGS proteins possess a distinctive G-?-like (GGL) domain that mediates specific and obligate heterodimer formation with the atypical G protein subunit, G?5, suggesting that these signaling proteins exhibit functions similar to conventional G?? dimers. Our recently refined crystal structure of G?5/RGS9 support this idea and provides a framework for testing hypotheses related to various binding interfaces of this dimer that likely subserve the organization and integration of higher-order, multifunctional G protein/GPCR/RGS complexes. Consequently, we propose to: 1) use crystallography and mutational analyses to define the complete functionality and G? specificity of RGS domains within full-length G??5/R7 dimers; 2) extend our understanding of the functional and structural relationships between G??5/R7 dimers and their anchoring proteins, R9AP and R7BP; and 3) quantify the functional capacities of G??5/R7 dimers to interact with GDP- G? subunits and GPCRs using reconstituted systems of purified components. While it is clear that R7 proteins are required for proper signaling mediated by G protein-coupled receptors under a variety of physiological settings, the roles of these proteins in coordinating these signaling events are relatively poorly understood. The work proposed here is designed to place R7 proteins within a detailed structural and functional context with respect to G protein-coupled receptors and heterotrimeric G proteins. It is anticipated that these studies will then be used to guide treatment regimens in cases where coordinated signaling mediated by R7 proteins has failed. ? ? ?
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