HeterotrimericG 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 alpha subunit of G13 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/AKAP110 protein ensemble contribute to cellular functions is the major goal of this proposal. Galpha13 is expressed ubiquitously, whereas AKAP 110 expression is restricted to particular locations, including cerebellum. As the physiological role of Galpha13 and AKAP110 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 AKAP110 can serve as Galpha13-interacting protein that connects Galpha13 with regulatory subunit of protein kinase A and thus regulates cell functions. We will test this hypothesis by determining functional interactions between Galpha13 and AKAP110 in in vitro and in vivo experiments.
Aim 1. Define mechanisms of biochemical interaction of AKAP110 with Galpha13. We will first express AKAP110 and Galpha13 as well as recombinant proteins containing various combinations of their individual functional domains. The Galpha regions necessary for binding to AKAP110 will be localized using chimeric G13/Gi2alpha-subunits. Galpha mutants with substitutions within identified Galpha regions will be analyzed for their ability to interact with AKAP110.
Aim 2. Analyze the regulation of Galpha13-AKAP110 signaling pathway in neuronal cell lines and primary cultured neurons. We will investigate the physiological relevance of the Galpha13-AKAP110 interaction in primary cultured neurons. We also will analyze signaling pathways regulated by Galpha13-AKAP110 and their downstream targets.
Aim 3. Define the role of Galpha13 and AKAP110 in brain. To further understand the Galpha13-AKAP110 function in vivo, we will generate transgenic mice with constitutive activation of Galpha13-AKAP110 pathway. This study will provide new information about signaling and cellular responses regulated by heterotrimeric G proteins.
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