Similarities in Function and Gene Structure of Cytohesin-4 and Cytohesin-1, Guanine Nucleotide-exchange Proteins for ADP-ribosylation Factors

doi:10.1074/jbc.275.5.3221

Similarities in Function and Gene Structure of Cytohesin-4 and Cytohesin-1, Guanine Nucleotide-exchange Proteins for ADP-ribosylation Factors*

From the Pulmonary-Critical Care Medicine Branch,^§Pathology Section and ^¶Hematology Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1434

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 47-kDa 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 10–20% 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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) – .
↵‡ To whom correspondence should be addressed: 10 Center Dr. MSC 1434, Bldg. 10, Rm. 5N307, National Institutes of Health, Bethesda, MD 20892-1434. Tel.: 301-496-3150; Fax: 301-402-1610; E-mail: ogasawam@gwgate.nhlbi.nih.gov.
Abbreviations:

ARF

ADP-ribosylation factor

rARF

recombinant ARF

BFA

brefeldin A

GEP

guanine nucleotide-exchange protein

RT-PCR

reverse transcriptase-polymerase chain reaction

PtdIns3

4,5P₃, phosphatidylinositol 3,4,5-triphosphate

PtdIns4

5P₂, phosphatidylinositol 4,5-bisphosphate

PtdIns3

4P₂, phosphatidylinositol 3,4-bisphosphate

PI3K

phosphoinositide 3-kinase

GTPγS

guanosine 5′-3-O-(thio)triphosphate

bp

base pair

kb

kilobase

kbp

kilobase pair

PH

pleckstrin homology

PE

phycoerythrin

PBS

phosphate-buffered saline

FITC

fluorescein isothiocyanate

RACE

rapid amplification of cDNA ends

UTR

untranslated region
- Received August 13, 1999.
- Revision received October 5, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.