The RhoH GTPase Interacts with ZAP-70 Kinase and Is Required for T Cell Receptor Signaling and T Cell Development

Engagement of the T cell receptor (TCR) by antigen/major histocompatibility complex initiates a signaling cascade involving activation and recruitment of multiple key intracellular proteins, including the zeta-chain-associated protein 70 (ZAP-70). ZAP-70-deficient mice show thymocyte developmental arrest and mutations of ZAP-70 are associated with severe combined immunodeficiency in humans. However, regulation of these early biochemical processes is not completely understood. RhoH, first identified as a hypermutable gene in non-Hodgkin’s lymphomas, belongs to the family of Rho guanosine triphosphatases (Rho GTPases), which are recognized as critical mediators of signaling pathways regulating cell growth, differentiation and function (Gu et al., Science 302: 445-9). The expression of RhoH is restricted to hematopoietic cells, and using in vitro protein-binding and immunoprecipitation assays, we found that RhoH interacts with ZAP-70 in the TCR complex. Therefore, to understand the role of this highly expressed protein in T cell development and function, we have generated RhoH-deficient mice using homologous recombination. RhoH-/- mice are viable and normal in body size. However, the thymuses of 4–6 week-old RhoH-/- mice are significantly smaller and show a 3-fold decrease in cellularity compared with those from WT littermates. RhoH-/- thymocyte development is severely blocked at the CD4+ CD8+ double positive (DP) stage, resulting in markedly fewer CD4 or CD8 single positive (SP) T cells in RhoH-/- thymus (CD4 SP: 10.5+/− 1.6 vs. 1.1+/−0.2; CD8 SP: 6.8+/−0.7 vs. 0.8+/−0.1, cell number x106, WT vs. RhoH−/−, n≥15 mice, p To determine the potential role of RhoH in TCR signaling, we studied proliferative responses to anti-CD3 antibody cross-linking of RhoH−/− spleen T cells. RhoH−/− cells show significantly defective proliferation (3H-thymidine incorporation: 10.5+/−0.9 vs. 2.1+/−0.2, cpm x103, WT vs. RhoH−/−, n=6, p