The transduction of biological signals such as light, hormones and neurotransmitters starts by a specific interaction of the ligand or stimulus with a receptor protein. The ultimate cell-specific responses to most biological signals are produced via G protein coupled receptor (GPCR) activation of specific GTP-binding proteins. Our lab has elucidated mechanisms of G protein interaction with cognate receptors and effectors as well as the structure-function relationships of G proteins. In the last grant period, detailed hypotheses of G protein function based upon crystallographic three-dimensional structural data were tested in biochemical and cellular assays, using a variety of model systems with chimeric constructs and site-directed mutagenesis of G protein alpha and beta/gamma subunits expression in heterologous systems. In this renewal, we will dissect out the interactions of Gbetagamma subunits with their effectors, and determine the mechanisms underlying the cellular specificity of Gbetagamma signaling. Although activation of G proteins by GPCRs is always thought to lead to G protein subunit dissociation, Gbetagamma signaling is known to play an important role in cellular responses especially when the Gi/o family of G proteins is activated. We will systematically determine whether Gbetagamma signaling is not an important part of the response when Gq, Gs, G12 and 13 are activated, and the basis for this specificity. There are a number of well-characterized Gbetagamma effectors, however, the number of Gbetagamma interacting proteins continues to grow. We have recently shown another function: the ability of Gbetagamma released from Gi-coupled receptors to interact with syntaxin and SNAP25A, components of the vesicular fusion or SNARE complex, and inhibit vesicular fusion. We will determine the molecular basis for this effect. In addition, we have discovered yet another Gbetagamma binding protein in a yeast-two-hybrid screen: the receptor for activated C kinase or RACK 1. The fact that Gbetagamma has so many different effectors (Hamm review, 1999) poses a problem for cells, which normally express a number of different Gbetagamma effectors. When a G protein-coupled receptor is activated, both Galpha and Gbetagamma-dependent signaling will take place, and if multiple Gbetagamma effectors are activated, they will have multiple effects in the cell. The implications for cellular physiology have never been systematically examined.
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