This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This proposal concerns three projects with a common theme in G protein-mediated signaling. (1)We propose to determine new structures of two members of the RGS-RhoGEF family, which are guanine nucleotide exchange factors (GEFs) that activate the small G protein Rho. RGS-RhoGEFs are themselves stimulated by G?13, a heterotrimeric G protein alpha subunit. We shall measure monochromatic (and if necessary SAD or MAD) data for complexes of two members of the RGS-RhoGEF family bound to G?13. One member of the RGS-RhoGEF family is a GTPase activating protein (GAP) for G?13, and the other is not. We have generated chimeras within the GAP domin of the two RhoGEFs and plan to determine the structures of these proteins to determine the basis of GAP activity. (2) mammalian membrane adenylyl cyclases (mAC)are activated by the G?s, the prototypical member of the family of heterotrimeric G protein alpha subunits. The structure of the catalytic domain of mAC bound to G?s has been determined. We will measure monochromatic data from crystals of the catalytic domain in the absence of G?s to better understand the mechanism of its activation. mAC is also activated by the diterpene forskolin. We shall determine the structure of mAC bound to a forskolin antagonist and therefore a potential mAC inhibitor. Human soluble adenylyl cyclase (sAC) is not regulated by G?s, but rather by calcium and bicarbonate. We will measure monochromatic diffraction data for crystals of the catalytic domain of sAC. (3) Par6 is a regulator of cell division that binds simultaneously to two small G proteins, cdc42 and Rin or Rit. The structures of Rit and Rin are not known, but we have crystals of both in GTP and GDP-bound states for which we shall measure monochromatic data. Small crystals of a Par6 fragment bound to Rit?GDP have been prepared for monochromatic data collection.
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