ARNO Is a Guanine Nucleotide Exchange Factor for ADP-ribosylation Factor 6

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 vitro nucleotide 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 ␣L␤ 2 integrins at the plasma membrane of lymphocytes.

The ADP ribosylation factor nucleotide exchange factor ARNO promotes β-arrestin release necessary for luteinizing hormone/choriogonadotropin receptor desensitization

“…Arf6 in vehicle and hCG-treated granulosa cells exhibited a predominant association with the cell periphery (arrowheads) (Fig. 5A), consistent with previous reports (15,32,41), which was blocked with competing peptide (c), and was associated with the membrane pellet fraction (Fig. 5B).…”

“…Our results point to Arf6 as the small G protein involved in LH͞CGR desensitization based on the insensitivity of LH͞CGR desensitization to brefeldin A (32) and activation by ARNO (15,33). To confirm its identification as Arf6 (and not Arf1), membranes were preincubated with synthetic N-terminal Arf peptides reported to block Arf activity (17,37).…”

“…Because Arf6 is highly expressed in ovaries and uniquely localized to plasma membranes of an ovarian-derived cell line (32), we tested whether ARNO promotes LH͞CGR desensitization. ARNO catalyzes the exchange of GTP for GDP on Arfs 1 and 6 (15,33). Preincubation of follicular membranes with ARNO yielded a dose-dependent reduction in hCG-stimulated AC activity ( Fig.…”

“…Recombinant ␤-arrestin (14), ADP ribosylation factor (Arf) nucleotide-binding site opener (ARNO) and ARNO mutant proteins (15), and Clostridium difficile and Clostridium sordelli toxins (16) were expressed and purified as described. Myristoylated (Myr)-Arf6(2-13), non-Myr-Arf6(2-13), and Myr-Arf1(2-17) N-terminal peptides were synthesized as described previously (17).…”

A Large T Cell Invagination with CD2 Enrichment Resets Receptor Engagement in the Immunological Synapse

“…T cell/APC couples were identified by larger size and increased Indo-1 and SNARF fluorescence. Tat SEC7 domain guanine nucleotide exchange factor activity for Arf6 was determined in vitro using purified tat SEC7 and recombinant myristoylated Arf6 (22) according to standard procedures (23).…”