Characterization of cytohesin-1 monoclonal antibodies: Expression in neutrophils and during granulocytic maturation of HL-60 cells

ADP-ribosylation factors (Arf) are small GTP-binding proteins involved in v esicular transport and the activation of phospholipase D (PLD). The convers ion of Arf-GDP to Arf-GTP is promoted in vivo by guanine nucleotide exchang e factors such as ARNO or cytohesin-1. In order to examine the expression o f ARNO and cytohesin-1 in human granulocytes, we generated specific polyclo nal and monoclonal antibodies (mAbs). We also overexpressed GFP-ARNO and GF P-cytohesin-1 in RBL-2H3 cells to characterize the specificity and the abil ity of cytohesin-1 mCibs to immunoprecipitate cytohesin-1. Among the hybrid omas secreting cytohesin-1 mAbs, only the clones 2E11, 1E4, 3C8, 6F5, 4C7, 7A3 and 8F7 were found to be specific for cytohesin-l. Furthermore, mAb 2E1 1 immunoprecipitated GFP-cytohesin-1 but not GFP-ARNO under native conditio ns. In contrast, mAbs 5D8, 4C3, 2G8, 6G11, 4C3, 6D4, 7B4 and 6F8 detected b oth cytohesin-1 and ARNO as monitored by immunoblotting. Although mAb 6G11 detected both proteins, this antibody immunoprecipitated GFP-ARNO but not G FP-cytohesin-1 under native conditions. Another antibody, mAb 10A17, also s electively immunoprecipitated GFP-ARNO under native conditions, but the epi tope recognized by this mAb is unlikely to be linear as no signal was obtai ned by immunoblotting. Immunoprecipitation with a cytohesin-1 polyclonal an tibody and blotting with cytohesin-1 specific mAbs revealed that cytohesin- 1 is highly expressed in neutrophils. Cytohesin-1 can be detected in HL-60 cells but the endogenous protein levels were low in undifferentiated cells. Using the specific cytohesin-1 mAb 2E11 we observed a marked increase in l evels of cytohesin-1 expression during dibutyryl-cyclic AMP-induced granulo cytic differentiation of HL-60 cells. These data suggest that cytohesin-1, which may have important functions in neutrophil physiology, can be: useful as a potential marker for granulocytic differentiation. (C) 2001 Elsevier Science B.V. All rights reserved.