This proposal focuses on two key mechanisms that control signaling through heterotrimeric G protein alpha subunits (G?): exchange of GDP for GTP at the catalytic site of G?, which initiates signaling, and G? stimulation of its effector as modulated by the activity of the effector as a GTPase Activating Protein (GAP) towards G?, which terminates signaling. The mechanism of two classes of molecules that play these respective roles in two different G protein-regulated cellular pathways will by elucidated at the level of molecular structure and dynamics.
The first aim of this proposal focuses on Ric-8A, a recently identified Guanine nucleotide Exchange Factor (GEF) for Gi-class G? subunits (e.g., G?i1) that regulates cell division in model organisms, and may play a similar role in mammals. Control of cell division is fundamental to cellular proliferation and therefore components of Ric-8A-regulated signaling systems may be misregulated in transformed cells and thus relevant to the health mission of the NIH. Ric-8A is significant because, in contrast to plasma membrane-embedded G protein-coupled receptors (GPCRs), it is localized in the cell cytosol or matrix rather than in the plasma membrane. Based on preliminary biophysical data, we propose that Ric-8A induces substantial conformational rearrangements in G?i1 to catalyze nucleotide exchange. Further, there is evidence that Ric-8A interacts with G?i1 in a manner similar to GPCRs. To test these hypotheses, we shall elucidate the interaction between Ric-8A and its perturbation of G?i1 by a variety of approaches including heteronuclear Nuclear Magnetic Resonance Spectroscopy (NMR), Small Angle X-ray Scattering (SAXS) and X-ray crystallography, together with functional analysis of structure-based site directed mutants. In the second aim of the proposal we seek to determine how the RGS-RhoGEF family of Rho activators are regulated by GPCRs that act through G12 and G13 class G? subunits (G?12 and G?13). RGS-RhoGEF proteins catalyze the exchange of GDP for GTP at the active site of Rho;Rho?GTP generated by this reaction acts directly to mobilize the actin cytoskeleton that power cell motility. Misregulation of certain RGS-RhoGEFs are implicated in the etiology of certain leukemias. G?13 and RGS-RhoGEFs interact in two functional modes. First, G?13 stimulates the GEF activity of RGS-RhoGEF. Second, certain RGS-RhoGEFs function as GAPs toward G?13, thereby terminating its stimulatory activity. By crystallographic analysis of a panel of RGS-RhoGEF mutants with different degrees of GAP activity, we seek to identify the biochemical determinants of this function. We propose that GAP and GEF stimulatory activities arise from distinct interactions involving different molecular contact surfaces between G?13 and RGS-RhoGEF. By small angle X-ray scattering (SAXS), we shall determine the molecular envelope of RGS-RhoGEF in solution, and that of its complex with G?13. Higher resolution structural information will be obtained by X-ray crystallography. The structural models obtained from these results, and the functional hypotheses inferred therefrom will be tested by activity assays of structure- based site directed mutants.
We propose to investigate the mechanism of action of two functionally-related regulatory molecules that control cell movement and cell division. Both of these processes are fundamental to life, and their mis-regulation may occur in disease states, including cancer. We will use experimental methods that will investigate the interactions of these molecules with each other at the molecular and atomic levels.
