A major signaling paradigm for modulation of hormonal and other extracellular stimuli is the use of heterotrimeric G proteins by cell surface receptors. Besides direct regulation of intracellular enzymes that produce second messengers, these receptor/G protein pathways influence the action of several members of the Ras superfamily of monomeric GTPases. Members of this family, such as Ras and Rho proteins, regulate cellular growth, differentiation, shape, adhesion and programs of gene transcription. Misregulation of these pathways is often found in cancer as well as other diseases. RhoGEFs are guanine nucleotide exchange factors for Rho proteins. Several of these are modulated directly by heterotrimeric G proteins. An example is p115-RhoGEF, which can be specifically regulated by the heterotrimeric G13 protein via interaction with the RH (RGS homology) domain of the GEF. In addition, we have shown that a homolog, PDZRhoGEF, can bind to its activated substrate, RhoA-GTP, to form a putative autoactivation loop. We propose to enhance our understanding of these pathways by a combination of detailed studies on the mechanisms of individual components and broader studies to define the spectrum and specificity of G protein regulation, autoregulation, and crosstalk within a select group of RhoGEFs including the Lbc family. Structural studies utilize a combination of classical crystallography and small-angle x-ray scattering (SAXS) to identify details of regulatory interactions between molecules. Specific mutagenesis to selectively alter function can then be used to determine impact in regulation. Experiments focused on the use of phospholipid vesicles to mimic the plasma membrane will systematically examine the spectrum and mechanism of regulation of a group of targeted RhoGEFs by G protein subunits and activated monomeric GTPases, especially the use of regulated localization to substrates and integration of multiple inputs. Experiments in vitro and i cells will examine paradigms of cross-regulation between Rho and Rac pathways that may be key to coordinated regulation in cellular shape and migration and test the physiological impact of autoregulatory mechanisms. Completion of the proposed experiments will increase our understanding of how these key pathways function in the regulation of growth, differentiation and cell motility through identification of new interactions and increased insight into mechanisms of regulation, both within and across pathways. This will help to better understand the regulation imparted by a variety of hormones and the contribution of these proteins to cellular dysfunction and disease.
Mutations or misregulated expression of RhoGEFs are known to be oncogenic and G proteins that regulate them also contribute to disease. Progress will increase our understanding of these key pathways in regulating growth, differentiation and cell motility. This will help to better understand the regulation imparted by a variety of hormones and the contribution of these proteins to cellular dysfunction and disease.
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