The proposed research will elucidate molecular mechanisms by which G proteins regulate effectors and are themselves regulated. Because G proteins are central to regulation of multiple cellular pathways, a clear understanding of their molecular function is ultimately critical for recognition of many disease processes and their treatment.
Aim 1 will define the dynamic components of G alpha-i1 that are coupled to GTP hydrolysis and conformational changes and that result in the termination of its signaling activity. Proline or Glycine mutations shall be introduced to constrain or relax backbone conformation at permissive sites throughout the molecule. Rates of nucleotide dissociation, conformational change and GTP hydrolysis shall be measured, and crystal structures determined of mutants with altered properties. Molecular dynamics calculations will be used to find correlations between changes in function and structure and alterations in essential dynamic components.
Aim 2 will address the mechanism by which p115Rho GEF is stimulated by G alpha-13, leading to the activation of the small G protein Rho, which induces stress fiber formation required for cell motility. The crystal structure of p115Rho GEF shall be determined, together with those of its complexes with G alpha-13/i chimeras and RhoA, singly and in combination. To accomplish this goal, well-expressed chimeric proteins containing structural elements of G alpha-i1 and G alpha-13 will be used, in their GDP-magnesium fluoroaluminate form, together with negative-dominant mutants of RhoA to stabilize the desired complexes.
The third aim explores a newly discovered G protein-coupled receptor independent pathway in which G alpha-i controls asymmetric cell division, and Ric-8 serves as the GEF for Galpha-i1. Complexes of G alpha-i1 with Ric-8, or subdomains of Ric-8 identified by sequence analysis and proteolytic mapping of Ric-8, shall be formed in the absence of nucleotide. These, together with Ric-8 alone will be crystallized and structures of Ric-8 and its complexes with G alpha-i1 determined. ? ?
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