ARNO Is a Guanine Nucleotide Exchange Factor for ADP-ribosylation Factor 6*
- From the ‡Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital East, Charlestown, Masschusetts 02129 and the Programs in§Immunology and ‖Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02115
Abstract
ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases. ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis. Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs). ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitronucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi. However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization. We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi. It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of αLβ2integrins at the plasma membrane of lymphocytes.
Footnotes
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↵* 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) .
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↵¶ Supported by a predoctoral training grant to the Harvard University Program in Immunology.
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↵** Supported by National Institutes of Health Grants AI32991 and DK33506 and a gift from the Good Samaritan Foundation. To whom correspondence should be addressed: Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-4173; Fax: 617-726-4172.
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↵1 The abbreviations used are: ARF, ADP-ribosylation factor; GEF, guanine nucleotide exchange factor; PH, pleckstrin homology; GST, glutathione S-transferase; GTPγS, guanosine 5′-(γ-thio)triphosphate; PtdIns-4,5-P2, phosphatidylinositol 4,5-bisphosphate; PtdIns-3,4,5-P2, phosphatidylinositol 3,4,5-trisphosphate; BFA, brefeldin A; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; BHK, baby hamster kidney.
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↵2 S. Frank, unpublished data.
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↵3 P. Melançon, personal communication.
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- Received September 25, 1997.
- Revision received October 31, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
