Molecular and Cellular Analysis of G Protein Function

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DESCRIPTION (provided by applicant): More than a thousand G protein-coupled receptors (GPCRs) play roles in a vast range of biological processes and their importance in health and disease is underscored by the fact that they are the targets of hundreds of drugs, including antihypertensives, neuroleptics, antihistamines, and antidepressants. An important, but poorly understood issue is how signaling specificity is maintained in vivo. Knockout experiments have shown that particular components often have indispensable roles in vivo, but many pathways and interactions can be reconstituted in vitro, suggesting a large amount of redundancy. The experiments in this proposal will test the hypothesis that the cellular localization and organization of signaling proteins play an important role in the specificity. Functional fluorescent wild-type and dominant negative G protein subunits will be used to probe the cellular regulation of signaling in living cells in order to answer the following questions: Aim I. How is targeting of G protein subunits regulated? In HEK-293 cells, the formation and localization of different G protein betagamma complexes and their abilities to target Galphas to the plasma membrane will be examined. Aim II. How are hormone-dependent trafficking of G protein subunits and their receptors regulated? In HEK-293 cells, the mechanisms that regulate activation-dependent trafficking of the subunits of Gs will be determined, the spatial and temporal aspects of G protein heterotrimer dissociation and reassociation will be visualized, and the importance of specific alphabetagamma combinations for receptor signaling will be investigated. Aim III. How are expression, localization, and complex formation of G protein subunits and their receptors regulated during differentiation of a specialized cell? Gs localization and signaling will be examined during differentiation of PC12 cells, a model system for growth-factor stimulated differentiation. The localization patterns of betagamma complexes that form and interact with Galphas and the Gs heterotrimers that are activated by the D1 dopamine receptor will be determined. These proposed studies will produce information and reagents that can be applied to elucidate the molecular and cellular basis for G protein signaling specificity in a variety of cell types and animal models. Ultimately, these approaches will facilitate the design of strategies to manipulate aberrant signaling pathways responsible for disease.