R7 RGS Protein-Mediated Regulation of GPCR Signaling

DESCRIPTION (provided by applicant): RGS proteins were identified originally as GTPase activating proteins (GAPs) for heterotrimeric G protein alpha-subunits. However, multiple members of this protein superfamily possess highly conserved domains in addition to the hallmark RGS box. These additional domains empower RGS proteins with a multifunctional character that may underlie poorly understood but physiologically important interactions among heterotrimeric G proteins as well as cross-talk with other signaling pathways. The R7 family of RGS proteins exists as heterodimers in association with G-beta5. Recent C. elegans genetic studies have led to hypotheses that place an R7-family member (EAT-16) in complex with a G-beta5 homologue (GPB2) as an inhibitory signal between G-alpha-o (GOA-1) and G-alpha-q (EGL-30) in a C. elegans neuronal signaling pathway. Our preliminary results with mammalian beta5-RGS9 suggest that this RGS heterodimer, and possibly other R7 family members, interact with GDP-bound G-alpha and support G protein-coupled receptor (GPCR) activation of G-alpha in the absence of conventional G-beta-gamma-subunits. Project I will directly address the mechanisms by which beta5-R7 dimers regulate GPCR signaling and delineate key regulatory domains in these proteins. In Aim 1 we will use purified proteins reconstituted in phospholipid vesicles to test the hypotheses that mammalian beta5-R7 dimers interact with GDP-bound G-alpha and promote receptor/G-alpha coupling in a GPCR- and G-alpha-subunit-selective manner while concomitantly serving as GAPs for the GPCR-coupled G-alpha or for another G-alpha. Similar studies will be carried out in Aim 2 with purified C. elegans proteins assessing the multifunctional character of the R7 proteins EAT-16 and EGL-10 in complex with GPB2. We will test the hypotheses that GPB2-EAT-16 directly associates with the GDP-bound GOA-1, is released by GPCR-promoted activation of GOA-1, and stimulates GTP hydrolysis by the C. elegans protein, EGL-30 (G-alpha-q). We also will test the hypothesis that a second C. elegans R7 protein, EGL-10, in heterodimeric complex with GPB2, acts in a reciprocal fashion to GPB2-EAT-16. In Aim 3 we will delineate the molecular determinants of interaction of beta5-R7 dimers with GDP-bound G-alpha and GPCRs. These studies will apply directed mutational analyses in combination with biochemical assays of beta5-R7 dimer action. The experiments in this aim will dovetail with work in Project IV of the PPG ultimately aimed at determining the three-dimensional structure of G-beta5-R7 proteins. Taken together our research will resolve mechanisms underlying the complexities of GPCR-signaling and has significance in identification of new drug targets.