ARF-GEP ₁₀₀ , a guanine nucleotide-exchange protein for ADP-ribosylation factor 6

Abstract

A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP 100, which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a Ϸ8-kb mRNA that hybridized with an ARF-GEP 100 cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP100 accelerated [ 35 S]GTP␥S binding to ARF1 (class I) and ARF5 (class II) 2-to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP 100 Sec7 domain contains Asp 543 and Met 555 , corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe 535 and Ala 536 , associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP 100 activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP 100, a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP 100 in those areas. No similar coincidence of ARF-GEP100 with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

TheDrosophilaARF6-GEF Schizo controls commissure formation by regulating Slit

“…6D). Schizo and its vertebrate homologs exert at least part of the function through the small GTPase Arf6 (Franco et al, 1999;Someya et al, 2001;Chen et al, 2003). Arf6 mRNA is supplied maternally and is expressed ubiquitously during embryonic development (data not shown).…”

“…The Anopheles homolog is about 90% identical. The closest human homologs are EFA6 (Franco et al, 1999;Perletti et al, 1997), being 32% identical to Schizo, lacking the IQ domain, and ARF-GEP 100 (Someya et al, 2001) showing a 40% identity to Schizo (Fig. 4B).…”

“…4B). Both human proteins were shown to act as ADP ribosylation factor 6 (ARF6)-GEFs (Franco et al, 1999;Someya et al, 2001) suggesting that schizo might have a similar function (see below).…”

“…The deduced nature of the Schizo protein suggests that it affects Slit expression by post-transcriptional mechanisms. (Someya et al, 2001) and promotes GDP/GTP exchange on ARF6. The small GTPase ARF6 is a plasma membrane-localized protein and functions in the regulation of membrane ruffling, cell motility, aspects of endocytosis and exocytosis, membrane recycling, reorganization of the cortical actin cytoskeleton and activation of phospholipase D (Kondo et al, 2000;Radhakrishna et al, 1999;Randazzo et al, 2000;Turner and Brown, 2001).…”

Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis

“…Arf6 can be activated by several guanine exchange nucleotide factors (GEFs) [7]. Brag2 (GEP100/IQSEC1) is a GEF activating the small ArfGTPases Arf4, Arf5 and Arf6 [33,48] and is expressed in EC [20,45]. Brag2 was found to promote the invasive activity of breast cancer cells [34] and myoblast fusion [11,37].…”