The approximately 1 ,000 G protein-coupled receptors (GPCRs) of humans mediate key signals - triggered by photons, odorants, hormones, and neurotransmitters - in brain, heart, blood vessels, white blood cells, and virtually every organ and endocrine gland. The fact that most of these signals represent potential targets for drug therapy justifies the broad-based strategy of this proposal, which aims to understand the conserved molecular mechanisms responsible for transmitting G protein-mediated signals between signaling molecules, in vitro and in the context of the cell. The first two aims test relations between structure and function of the GPCR and the G protein trimer at the level of individual molecules. Experiments with GPCRs aim to: a. use molecular probes to determine how the extracellular surface of a GPCR actually binds the activating ligand; b. engineer metal binding sites that activate a GPCR by inducing coordinated movement of its transmembrane helices, allowing us to infer how the natural ligand induces similar movements; c. identify sites on the GPCR's intracellular surface that interact specifically with peptides representing different parts of the trimeric target. To understand the conformational changes in a G protein trimer that mediate its activation by the GPCR, a second set of experiments will: a. test the hypothesis that the GPCR uses the beta-gamma subunit of the G protein trimer as a lever to open a route for bound GDP to exit from its binding pocket in the alpha subunit and thereby activate the trimer; b. determine how key structural elements of the G protein alpha subunit cooperate during the activation process, by constructing metal binding sites that restrict movements of these elements, relative to one another. The third set of experiments uses fluorescent probes and fluorescence energy transfer (FRET) to determine the locations of G protein alpha and beta-gamma subunits in intact cells and ask how hormonal activation affects their interaction. Biochemical experiments with pure G protein subunits indicate that activation in the test tube causes the alpha subunit to dissociate from the beta-gamma heterodimer; it is not known, however, whether such a dissociation accompanies activation in an intact cell responding to a hormone. Investigation of this question begins by constructing functioning G protein alpha and beta-gamma subunits attached to fluorescent tags; the fluorescent subunits are used to assess their subcellular distributions in intact cultured cells, and FRET between co-expressed tagged beta-gamma and alpha subunits will reveal whether hormones cause them to dissociate.
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