Defining the functions, structure and cell biology of G-protein-mediated transmembrane signaling devices is the long-term objective of the proposal. Information transduction across the lipid bilayer by G-proteins represents a major transmembrane signaling pathway integral to hormone action, neurotransmission, and modes of sensory physiology. Spectacular advances in the description of the biochemical and molecular biology of G-proteins have occurred, and new additions continue to be made to this family of heterotrimeric, GTP-binding, regulatory proteins. Integral specific questions are proposed that focus upon the function, localization, and organization of G-protein-mediated transmembrane signaling devices. First, to which receptor(s) and effector(s) are specific G-proteins coupled in vivo? To address this fundamental question we propose a new strategy, i.e., sequence-specific antisense-mRNA to suppress expression of G-protein subunits. Antisense-mRNA will be introduced (i) indirectly by a CMV- promoter-driven antisense construct introduced by retroviral infection of cells, thereby generating stable subclones deficient in the expression of a specific subunit, and (ii) directly as oligodeoxynucleotides into cells in culture, providing a reversible blockade of subunit expression. Stimulatory and inhibitory adenylyl cyclase, stimulatory phospholipase C, and ligand-dependent K+ currents and Ca++ fluxes will be analyzed in subunit-deficient subclones. The role of G-proteins in complex responses like differentiation will be explored using the oligomer antisense strategy. The second question is, how are the signals that are transduced by different G-protein-mediated pathways integrated? We propose to probe cross-regulation among G-protein-mediated pathways. Cross-regulation will be explored by activating a specific pathway and defining modulation of parallel G-protein-mediated pathways, establishing both acute and longer- term regulation. Transcriptional, post-transcriptional, and post- translational controls will be investigated to define the mechanism(s) regulating expression or function of system elements by intracellular networking among G-protein-mediated responses. Finally, how are G-protein- mediated signaling devices organized in the cell and at the lipid bilayer? We propose to probe the intracellular localization of G-proteins and their macromolecular organization by direct and indirect immunofluorescence in tandem with specific antibodies to each element. Chemical crosslinking and nearest-neighbor analysis with site-directed cleavable probes will be used to dissect the macromolecular organization in both basal and activated (information transducing) states. Sensitivity is amplified by strategies that make use of metabolic labeling of elements and analysis following immunoprecipitation. In combination, this effort provides an integrated, multifaceted approach to the study of the cell biology and regulation of G- protein-mediated transmembrane signaling.
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