Cloning and characterization of bovine low molecular weight GTPases (Rac1 and Rac2) and Rho GDP-dissociation inhibitor 2 (D4-GDI)

GTPases of the Rho family play important roles in human leukocyte signal tr ansduction pathways; however, little is known about the function of these p roteins in bovine cells. In the present studies, we isolated molecular clon es of bovine Rac1, Rac2, and the Rac/Rho GTPase regulatory protein D4-GDP d issociation inhibitor (D4-GDI) from a bovine bone marrow cDNA library. Thes e clones contained complete open reading frames, encoding 192, 192, and 200 amino acids, respectively. Comparison of the bovine amino acid sequences w ith those of other species demonstrated a high degree of identity of these proteins across all species, suggesting that these proteins likely play con served functional roles in bovine leukocyte signal transduction pathways. C omparative Western blotting of these proteins in human and bovine neutrophi l cytosol demonstrated that Rac2 was the predominant Rac species and that D 4-GDI was the predominant GDI species in bovine neutrophil cytosol. Despite the high degree of homology between human and bovine Rac2, some of the ant i-peptide antibody probes prepared against human Rac2 failed to recognize t he bovine homologue. We also showed by subcellular fractionation techniques that Rac2 is localized primarily to the cytosolic compartment of resting b ovine neutrophils, but is translocated to the plasma membrane after stimula tion with PMA. These findings suggest that Rac2 does play a role in bovine neutrophil activation. In addition, these data will be helpful in developin g more specific probes for investigating the role of these proteins in bovi ne leukocyte signal transduction pathways and for studying various inflamma tory diseases in cattle. (C) 2000 Elsevier Science B.V. All rights reserved .