Abstract
Activation of ADP-ribosylation factors (ARFs), approximately 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 47-kDa protein contains the same structural components present in cytohesin -1, -2, and -3, including an approximately 200-amino acid Sec7 domain with an approximately 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 approximately 200-kDa ARF GEPs, it was not inhibited by brefeldin A. A cytohesin-4 mRNA of approximately 3.7 kilobases, abundant in leukocytes, was not detected in most tissues. Among separated populations of blood cells, approximately 90% of CD33(+) (monocytes), 80% of CD2(+) (NK/T), and 10-20% 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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF125349 –AF125362
Activation of ADP-ribosylation factors (ARFs)-GDP depends on the replacement of bound GDP by GTP, which is catalyzed by guanine nucleotide-exchange proteins or GEPs [13]
Part of its deduced amino acid sequence resembles that of the so-called Sec7 domain of the Saccharomyces cerevisiae Sec7 protein (Sec7p), which had been identified as a pivotal component in Golgi protein transport [17]
Summary
3221–3230, 2000 Printed in U.S.A. Similarities in Function and Gene Structure of Cytohesin-4 and Cytohesin-1, Guanine Nucleotide-exchange Proteins for ADP-ribosylation Factors*. Activation of ADP-ribosylation factors (ARFs), ϳ20kDa GTPases that are inactive in the GDP-bound form, depends on guanine nucleotide-exchange proteins (GEPs) to accelerate GTP binding. Deduced amino acid sequence of the 47kDa protein contains the same structural components present in cytohesin -1, -2, and -3, including an ϳ200amino acid Sec domain with an ϳ100-residue pleckstrin homology domain near the C terminus. GEP activity of the Sec domain of yeast Sec7p was inhibited by brefeldin A (BFA), a fungal fatty acid metabolite that reversibly alters Golgi structure and function, suggesting that the 200-amino acid Sec domain contains both the inhibitory and the catalytic sites [24]. Cytohesin-1 and cytohesin-4, which differ most among the cytohesins, have similar gene structures within the coding region, suggesting that the family may have arisen by gene duplication
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