Rho GTPases are members of the Ras superfamily which regulate multiple cell functions. Rho GTPases cycle between active, GTP-bound and inactive, GDP-bound states and are regulated by guanine exchange factors (GEF), GTPase activating proteins (GAPs) and guanine dissociation inhibitors (GDIs). Rac (including the related Rac1, Rac2 and Rac3 proteins), Cdc42 and RhoA represent the best studied members of the Rho family of small GTPases. Previous studies have relied primarily on transfection and expression of dominant negative and constitutive active mutants in cell lines, most notably fibroblasts and these approaches, while informative, suffer from experimental deficiencies. In the previous funding period, we have utilized knock-out and conditional knock-out mouse lines generated by gene targeting to study the role of Rac1 and Rac2 in blood cell development and function. We have determined that Rac is activated via stimulation of CXCR4 by SDF-1, by adhesion via beta1 integrins and via binding of c-kit by SCF-all pathways involved in stem cell engraftment. We have defined the Rac proteins as key regulators of hematopoietic stem and progenitor (HSC/P) cell function, delineated key unique and overlapping functions of these two highly related GTPases in a variety of primary hematopoietic cell lineages in vitro and in vivo. In this continuation application, we seek to expand our studies to further define the role of individual Rac proteins, Rac3 in particular, in blood cell function. These studies will utilize gene-targeted mice deficient in Rac3 and Rac3 in combination with Rac1/Rac2. Utilizing insights gained in the previous funding period we also seek to define key upstream regulators and downstream effectors of the Rac-dependent hematopoietic cell functions, particularly engraftment and retention in the bone marrow niche. These studies will utilize 'candidate gene'approaches focusing on the role of downstream Rac targets Pak, POR-1 and WAVE and Stat5 in engraftment and marrow retention. Finally, again utilizing knockout mice deficient in the Vav family of activators of Rac (Vav1, Vav2, and Vav3) (all in hand), we will determine the role of these GEFs in stem cell functions focusing on the hematopoietic-specific Vav1 GEG. Thus, overall the goal of these studies is to build on the recent identification of Rac GTPases as major regulators of HSC localization and retention in the hematopoietic microenvironment to determine the 'biochemical footprint'of stem cell engraftment and marrow retention.
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