Similarities in function and gene structure of cytohesin-4 and cytohesin-1, guanine nucleotide-exchange proteins for ADP-ribosylation factors

Abstract

Activation of ADP-ribosylation factors (ARFs), ~20-kDa GTPases that are inactive in the GDP-bound form, depends on guanine nucleotide-exchange proteins (GEPs) to accelerate GTP binding. A novel ARF GEP, designated cytohesin-4, was cloned from a human brain cDNA library. Deduced amino acid sequence of the 47kDa protein contains the same structural components present in cytohesin -1, -2, and -3, including an ~200-amino acid Sec7 domain with an ~100-residue pleckstrin homology domain near the C terminus. The Sec7 domain sequence is 77% identical to those of other cytohesins. Structures of the cytohesin-4 and cytohesin-1 genes were remarkably similar, except for an extra 3-base pair (GAG) exon present in cytohesin-1. Two mRNAs with and without the 3-base pair sequence were found in brain in different ratios for cytohesin-1, -2, and -3 but not cytohesin-4. Recombinant cytohesin-4 stimulated guanosine 5'-3-O-(thio)triphosphate binding by human ARF1 and ARF5 but not ARF6. Like other cytohesins and unlike the ~200-kDa ARF GEPs, it was not inhibited by brefeldin A. A cytohesin-4 mRNA of ~3.7 kilobases, abundant in leukocytes, was not detected in most tissues. Among separated populations of blood cells, ~90% of CD33+ (monocytes), 80% of CD2+ (NK/T), and 10-25 of CD19+ (B) cells contained cytohesin-4 mRNA by in situ hybridization. Thus, in gene structure and brefeldin A-insensitive GEP activity, cytohesin-4 resembles other cytohesins, but its tissue distribution differs considerably, consistent with a different specific function.