Heterotrimeric G proteins transmit hormonal and sensory signals received by seven-transmembrane-helix receptors to effector proteins that mediate a wide variety of physiological processes. The importance of G protein signaling pathways in health and disease is underscored by the fact that G protein-coupled receptors are the targets of hundreds of drugs, including antihypertensives, neuroleptics, antihistamines, and antidepressants. An important challenge is to elucidate regulation at the level of how interactions between receptors and G proteins affect their activities and how these interactions are regulated in vivo. An increased understanding of these processes will provide a wider range of targets and strategies for therapeutic interventions for aberrant signaling pathways. The goal of this proposal is to elucidate the mechanism of G protein signaling at both the molecular and cellular levels.
The specific aims of this proposal are to answer these questions: (I) What regulates association/dissociation of G proteins and receptors during the activation cycle? The role of a surface- exposed subunit region of alphas in which substitutions increase receptor affinity will be investigated. The critical residues will be localized and the functional effects of mutating them will be determined. Alphas residues in this region will be tested to see if they can confer specificity for the Beta2- adrenergic receptor. Peptides based on this region will be tested to see if they mimic or block interactions between Gs and the Beta2-adrenergic receptor. (II) Can functionally distinct receptor populations be selectively inactivated by dominant negative alpha subunits? Mutations that both increase receptor affinity and decrease effector affinity will be combined in an effort to produce strong dominant negative alpha subunits. Dominant negative alpha subunits will be used to clarify the physiological roles of receptors that couple to multiple G proteins. (III) How does the cellular organization of receptors and G proteins regulate signaling? Much of what is known about G protein-mediated signal transduction is based on biochemical experiments using purified components and static images of immunohistochemical localization of the proteins in fixed cells. To determine the extent to which these conclusions can be extended to in vivo situations, pairs of proteins (alpha/beta, alpha/receptor, b/receptor) fused to distinguishable GFP mutants will be followed in living cells using time lapse fluorescence microscopy, fluorescence recover after photobleaching, fluorescence resonance energy transfer, and fluroescence correlation spectroscopy.
