EXCEED THE SPACE PROVIDED. Heterotrimerie G proteins and protein kinase A (PKA) are two important signal transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses. The established mechanism of PKA activation in response to various hormones involves stimulatory G protein, Gs, which activates adenylyl cyclase resulting in production of cAMP. We have discovered a novel mechanism of PKA stimulation that does not require cAMP. Using yeast two-hybrid screening, we found that the x subunit of GI3 protein interacted with a member of the PKA-anchoring protein family, AKAP110. Our data suggested that AKAP110 may provide a link between heterotrimeric G proteins and cAMP-independent activation of PKA. Understanding the exact molecular mechanism by which members of the G13/AKAPll0 protein ensemble contribute to cellular functions is the major goal of this proposal. GcO3 is expressed ubiquitously, whereas AKAP 110 expression is restricted to particular locations, including cerebellum. As physiological role of G_13 and AKAPll0 in cerebellum is unknown, this proposal is also aimed to understand molecular mechanisms and cellular functions regulated by these proteins in neuronal cell lines, primary cultured neurons, and transgenic mice models. We hypothesize that AKAPll0 can serve as Go_13-interacting protein of that connects Gctl3 with regulatory subunit of protein kinase A and thus regulates cell functions. We will test this hypothesis by determining functional interactions between Gctl3 and AKAPll0 in in vitro and in vivo experiments.
Aim 1. Define mechanisms of biochemical interaction of AKAP110 with G0d3. We will first express AKAPII0 and Gctl3 as well as recombinant proteins containing various combinations of their individual functional domains. The Gtz regions necessary for binding to AKAP1 l0 will be localized using chimeric G13/Gi2 _-subunits. Gc_ mutants with substitutions within identified G0_regions will be analyzed for their ability to interact with AKAPI10.
Aim 2. Analyze the regulation of Gtxl3-AKAPll0 signaling pathway in neuronal cell lines and primary cultured neurons. We will investigate the physiological relevance of the Gtxl3-AKAP110 interaction in primary cultured neurons. We also will analyze signaling pathways regulated by Gctl3-AKAPll0 and their downstream targets.
Aim 3 Define the role of G_13 and AKAP110 in brain. To further understand the Gtxl3-AKAP1 l0 function in vivo, we will generate transgenic mice with constitutive activation of Gctl3-AKAP110 pathway. This study will provide new information about signaling and cellular responses regulated by heterotrimeric G proteins. PERFORMANCE SITE ========================================Section End===========================================
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