G-protein coupled receptors (GPCRs) are a large group of 7 transmembrane helix receptors that require ligand-dependent activation to initiate heterotrimeric (a, B, y) G-protein mediated intracellular signaling cascades. Due to their physiological relevance and pharmacological tractability, GPCRs are the focus of numerous drug discovery efforts. Activation of a G-protein by its agonist stimulated GPCR (R*) requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide-binding pocket of the G-protein. The structural basis for the interaction of a GPCR with its cognate G-protein, and the subsequent activation of the G-protein by R*, are not fully understood. The overall goal of this research is to develop and apply high-resolution nuclear magnetic resonance (NMR) methods to probe the structural basis for the propagation of structural signals from R* to the G-protein, with a specific focus on elucidating structural changes in the a-subunit that lead to guanine nucleotide exchange. For these studies, signaling of the G-protein, transducin (Gt), by the light-activated GPCR, rhodopsin, will be used as a model system. The interaction of Gt with solubilized native and mutant rhodopsins, as well as soluble mimics of R*, will be employed to allow for the observation and trapping of discrete states accompanying signal transfer. Using isotope-labeled G-protein a-subunits, heteronuclear NMR methods will be used to selectively map structural changes in this subunit upon heterotrimer formation with unlabeled By-subunits and when trapped in discrete R* bound states. 3 fundamental structural questions surrounding the mechanism of R* mediated signal transfer will be addressed: 1. What structural changes in the receptor binding interface of the a-subunit occur upon interaction of the G-protein heterotrimer with R*?; 2. What are the structural changes in the guanine nucleotide binding site that occur upon interaction of the G-protein heterotrimer with R*?; 3. How are structural changes that result from the interaction of the binding interface of the a-subunit with R* correlated to changes in the conformation of the guanine nucleotide binding site? We expect that this work will have a fundamental impact on our understanding of the mechanisms governing activation of G-proteins by R* and form a basis for understanding the structural consequences of naturally occurring rhodopsin and Gt mutations associated with visual dysfunction. [unreadable] [unreadable] [unreadable]
NIH ExPorter
NIH ExPorter