Signal transduction via guanine nucleotide binding proteins (G proteins) is crucial in regulation of cardiovascular, neural, and endocrine function. In addition, roles for the classical heterotrimeric G proteins are being defined in intracellular trafficking of proteins and in cell growth regulations. Activating mutations in several heterotrimeric G proteins have been found in human endocrine adenomas and in vitro studies with phospholipase C-coupled G proteins have shown the potential for cellular transformation as well. Drugs to either activate or inhibit G protein function may be useful agents in treatment of hypertensin and vascular disease as well as in certain neoplasias. To assist in the assist in the design of G protein activators or inhibitors and to better understand how certain mutations lead to activation of G proteins, we propose to study the kinetic and structural mechanisms of G protein activation by use of several newly developed biophysical techniques. Specifically, the steps between binding of nucleotide and activation of the G protein will be explored by rapid-kinetic studies of fluorescent- labelled nucleotide analogs and fluorescent-labelled G proteins subunits. Real-time measurements of spectral shifts of the fluorescent nucleotide analog and energy transfer between fluorescent on the alpha and beta gamma subunits provide novel means to dissect G proteins conformational equilibria on the subsecond time scale. Differences between G protein subtypes and normal and mutant G proteins will be determined to better understand the structural function basis of G protein conformational regulation. Also, chimeric G protein alpha subunits will help identify regions of the protein which govern the rates of the steps in the G protein activation on the second and sub-second time scale by use of these spectroscopic techniques. The alpha2 adrenergic receptor and the formyl peptide receptor will provide two systems known to activate inhibitory G proteins. The receptor conformations which accompany G protein coupling and G proteins activation can be examined for the receptors by rapid-mix quench radiochemical and fluorescence spectroscopic techniques, respectively. Finally, G protein structural organization will be examined. The relation of known sites in the alpha subunit to one another will be determined by fluoresences energy transfer. Also, organization of sites in the alphabetagamma hetertrimer and their relation to the membrane will be examine by fluorescence techniques and radio-labelling and proteolytic mapping. Changes in the relative location of these sites upon G protein activation will be examined to provide additional monitors of the steps in G protein activation. These biophysical studies of the mechanisms of G proteins activation will complement the great variety of molecular biological techniques currently being applied to the understanding of G proteins structure and function.
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