R-Ras and Rac GTPase Crosstalk Regulates Hematopoietic Progenitor Cell Migration, Homing and Mobilization Running title: R-Ras in hematopoietic progenitor migration

Adult hematopoietic progenitor cells (HPCs) are maintained by highly coordinated signals in the bone marrow. The molecular mechanisms linking intracellular signaling network of HPCs with their microenvironment remain poorly defined. The Rho family GTPase Rac1/Rac2 has previously been implicated in cell functions involved in HPC maintenance, including adhesion, migration, homing, and mobilization. In the present studies we have identified R-Ras, a member of the Ras family, as a key signal mediator required for Rac1/Rac2 activation. We found that while Rac1 activity is upregulated upon SCF, integrin or CXCL12 stimulation, RRas activity is inversely upregulated. Expression of a constitutively active R-Ras mutant resulted in downregulaton of Rac1-activity while deletion of R-Ras led to an increase in Rac1/Rac2 activity and signaling. R-Ras -/HPCs displayed a constitutively assembled cortical actin structure and showed increased directional migration. Rac1/Rac2 inhibition reversed the migration phenotype of R-Ras -/HPCs, similar to that by expressing an R-Ras active mutant. Furthermore, R-Ras -/mice showed enhanced responsiveness to G-CSF for HPC mobilization, and exhibited decreased bone marrow homing. Transplantation experiments indicate that the RRas deficiency-induced HPC mobilization is a HPC intrinsic property. These results indicate that R-Ras is a critical regulator of Rac signaling required for HPC migration, homing, and mobilization.