Biochemical and biological characterization of a human Rac2 GTPase mutant associated with phagocytic immunodeficiency

The Rho GTPase, Rac2, is expressed only in hematopoietic cell lineages, sug gesting a specific cellular function in these cells. Genetic targeting stud ies in mice showed that Rac2 is an essential regulator of neutrophil chemot axis, L-selectin capture and rolling, and superoxide production. Recently, a dominant negative mutation of Rac2, D57N, has been reported to be associa ted with a human phagocytic immunodeficiency. To understand further the cel lular phenotypes associated with this D57N Rac2 mutant we examined its bioc hemical characteristics and functional effects when expressed in primary mu rine bone marrow cells. When compared with wild type (WT) Rac2, D57N Rac2 d isplayed similar to 10% GTP binding ability resulting from a markedly enhan ced rate of GTP dissociation and did not respond to the guanine nucleotide exchange factors. These results suggest that D57N Rac2 may act in a dominan t negative fashion in cells by sequestering endogenous guanine nucleotide e xchange factors, When expressed in hematopoietic cells, D57N Rac2 reduced e ndogenous activities of not only Rac2, but also Rad and decreased cell expa nsion in vitro in the presence of growth factors due to increased cell apop tosis, Unexpectedly, D57N expression had no effect on proliferation. In con trast, expansion of cells transduced with WT Rac2 and a dominant active mut ant, Q61L, was associated with significantly increased proliferation. Trans plantation of transduced bone marrow cells into lethally irradiated recipie nts showed that the percentage of D57N-containing peripheral blood cells de creased markedly from 40% at 1 month to <5% by 3 months postinjection. Neut rophils derived in vitro from the transduced progenitor cells containing D5 7N demonstrated markedly impaired migration and O-2(-) responses to formyl- methionyl-leucyl-phenylalanine, reflecting the same cellular phenotype in t hese differentiated cells as those described previously in patient cells. T hese data suggest that the phenotypic abnormalities associated with D57N Ra c2 may involve not only neutrophil cellular functions, but also abnormal ce ll survival in other hematopoietic cells and that overexpression of Rac lea ds to increased proliferation of normal cells in vitro, whereas deficiency of Rac leads to increased apoptosis.