Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Mechanisms responsible for reversibly targeting peripheral membrane proteins to different cellular membranes are poorly understood. This research grant will address this question in the context of heterotrimeric (alphabetagamma) G proteins. G proteins act as molecular switches to relay information from activated receptors to appropriate effector proteins (e.g. adenylyl cyclase, phospholipase C, cGMP phosphodiesterase, and ion channels). Molecular mechanisms underlying the GTPase cycle of G proteins and its regulation by receptors and effectors are becoming increasingly well understood. Much less well understood are mechanisms responsible for targeting G proteins to their appropriate cellular location, and how a unique cellular environment - and proteins that interact with G proteins to target or retain them there - affect a G protein's function. The major objectives of this proposal are to elucidate mechanisms of cellular palmitoylation, covalent attachment to cysteines of a 16 carbon fatty acid, of G protein alpha subunits (Galpha) and mechanisms of specific targeting of Galpha to intracellular or plasma membranes. The role of G protein betagamma subunits in the palmitoylation of Galpha will be tested by employing strategies to disrupt the alphaybetagamma interaction in vivo. The subcellular site of palmitoylation of Galpha will be addressed by examining the palmitoylation of differentially localized Galpha; alphai2 resides at the plasma membrane, whereas alphai3 and a splice variant of alphai2 (salphai2) are found at Golgi membranes. Inhibitors of membrane vesicle transport will be used to further define the cellular pathway for palmitoylation. To understand mechanisms of specific Golgi localization of Galpha, the role of lipid modifications, both myristoylation and palmitoylation, and betagamma interaction will be tested by expression of mutant Galpha.
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