The transduction of biologic signals such as light, hormones and neurotransmttters starts by a specific interaction of the ligand or stimulus with a receptor protein. The ultimate cell-specific responses are produced via receptor activation of specific GTP-binding proteins. While the functional aspects of this process are well-defined, the structural basis of interactions between receptor, G protein and effector, and the activation processes are understood to only a very partial extent. A more profound understanding of these fundamental mechanisms will be achieved once the three-dimensional structures of these molecules are in hand. We have determined some of the sites on heterotrimeric G proteins of interaction with cognate receptors and effectors. In addition, in collaboration with Paul Sigler's laboratory, we have succeeded in obtaining high-quality, diffracting crystals of the alpha-t subunit of a heterotrimeric G protein in the active, GTP-bound form. This structural information, in conjunction with functional studies, suggests detailed hypotheses for the serial activation of G proteins and effectors by activated receptors during signal transduction. In this proposal, site- directed mutagenesis of G protein alpha subunits and protein expression studies will be used to test these hypotheses and determine the critical amino acid residues involved in these processes. Molecular basis of G protein interaction with receptor. The amino acid residues of the alpha subunit involved in binding interactions with receptor and effector will be determined by scanning and site directed mutagenesis of a soluble carboxyl terminal alpha-t fusion protein expressed in E. coli. The interactions will be measured by both binding and functional assays. The mutations that are found to increase or decrease the affinity of a G protein for its cognate receptor and effector will be mapped onto the crystal structure, which should provide structural insight into the interaction mechanisms. Molecular basis of G protein interaction with effector. Determination of the structure of the inactive GDP-bound alpha-t subunit, which is currently underway, and comparison with the structure of the GTP-bound structure, will provide insight into GTP-induced conformational changes in alpha subunits underlying their ability to interact with and activate effector proteins. The molecular basis of effector activation will be determined by site-directed mutagenesis based upon this structural information. Such studies will contribute to our understanding of basic mechanisms of cellular activation by a variety of signals, and may also lead to insights into possible interventions in certain disease states of the cardiovascular and nervous systems.
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