verbatim): Heterotrimeric GTP-binding (G) proteins transduce external signals that act on G Protein Coupled Receptors (GPCRs). When a signal binds its GPCR, it is thought that the activated G protein splits into its component subunits, the alpha-GTP (Ga-GTP) subunit and the betagamma (Gbg) complex, thus generating two signaling molecules that interact with specific effectors. Ga effector targets have long been recognized and studied extensively. The effector function of the bg complex was recognized more recently and although controversial at first, it is now well accepted. Gbg targets include, ion channels, (e.g. K+ (or GIRK) channels, neuronal type Ca2+ channels and the tetrodotoxin-insensitive Na+ channels), enzymes (e.g. PLA2, PLCb isoforms, adenylyl cyclase isoforms) and kinases (e.g. PI3K, the Bruton and Tsk tyrosine kinases). The list of effector targets continues to grow for each of the two signaling components of G proteins. Multiple isoforms for each of the subunits (20 Ga, 5 Gb and 14 Gg) have been identified. In vitro or heterotogous expression experiments have shown that unlike Ga subunit isoforms (4Gas, 3 Gai, 2 Gao, 4Gaq/11, etc.) which seem to associate with specific GPCRs and specific effectors, most Gbg combinations show little to no specificity towards their effector partners. Yet, in vivo (meaning in native tissues), there is exquisite specificity of Gbg signaling, so that for example the Gbg complexes associated with Gaq/11 do not signal to GIRK channels but those associated with Gai/o subunits do. The mechanism responsible for specificity of Gbg action is poorly understood, although it is basic to our understanding of G protein signaling at large. In this competitive renewal grant, we propose experiments in heterologous expression systems geared to understand the molecular basis for the specificity of Gbg signaling. In our ending current award we have identified functionally important amino acid residues on GIRK channels and on the Gbg subunits. We propose to identify residues in each protein that interact with the other, using a powerful mutant screening assay in yeast. In heterologous expression systems, where we have reconstituted specific Gbg signaling, we will determine the particular G protein subunits involved in the signaling. Ga subunits associated directly with the Gbg subunits seem to affect Gb's interactions with the K+ channel. We propose studies to elucidate the molecular basis for the dependence of the effector function of Gbg on Ga. We will test the hypothesis that distinct Ga subunits may determine specificity of Gbg signaling, using electrophysiology, molecular biology, biochemistry and fluorescence spectroscopy.
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