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Vol. 18, Issue 6, 2244-2253, June 2007

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Active Arf6 Recruits ARNO/Cytohesin GEFs to the PM by Binding Their PH Domains

Lee Ann Cohen^*, Akira Honda^*, Peter Varnai, Fraser D. Brown^*, Tamas Balla, and Julie G. Donaldson^*

*Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, and Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892

Submitted November 8, 2006; Revised March 20, 2007; Accepted March 26, 2007
Monitoring Editor: Martin A. Schwartz

ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GEF. Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Julie G. Donaldson (jdonalds{at}helix.nih.gov)

Abbreviations used: GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; PH, pleckstrin homology; PIP₂, phosphatidylinositol 4,5 bisphosphate; PIP_3,, phosphatidylinositol 3,4,5 trisphosphate; PIP5-kinase, phosphatidylinositol 4-phosphate 5-kinase; PM, plasma membrane.

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