0.01M PBS pH7.4, 15 mM sodium azide
recombinant, human ARNO (ARFnucleotide-binding-site opener).
For In vitro laboratory use only. Not for any clinical, therapeutic, or diagnostic use in humans or animals. Not for animal or human consumption.
ARFs (ADP ribosylation factors) are GTP-binding proteins (20 kDa), which catalyze ADP-ribosylation of the alpha-subunit of the adenylyl cyclase-stimulatory G protein. ARFs have been shown to regulate various aspects of vesicular trafficking pathways in mammalian cells, including endocytosis, phagocytosis, secretion, and endoplasmic reticulum protein transport and budding of transport vesicles from Golgi in both anterograde and retrograde directions. Mammalian ARFs are grouped into three classes (class I: ARFs 1, 2, and 3, class II: ARFs 4 and 5, and class III: ARF 6), based on size, gene structure, and sequence identity. ARFs are active when GTP, but not GDP or ATP, is bound. Hydrolysis of bound GTP to GDP with assistance of GTPase-activating protein, results in inactive ARF-GDP. Conversion of ARF-GDP to ARFGTP is promoted by GEP (guanine-exchange protein). The ARF-GEP family of guanine nucleotide-exchange proteins (also referred to as cytohesins) includes cytohesin 1, ARNO (ARF nucleotide binding site opener, also called cytohesin 2 or Sec7p), and GRP1 (general receptor for phosphoinositides-1, also known as cytohesin 3 or ARNO3). ARF-GEP family members are characterized by an N-terminal coiled-coil domain of 40 amino acids, a PtdIns(3,4,5)P3-binding C-terminal PH (pleckstrin homology) domain, and a central Sec7 domain. Sec7 is a conserved catalytic domain of approximately 200 amino acids, which stimulates the exchange of GDP to GTP on members of the ARF family of GTPases. The PH domain, by interacting with phospholipids, is believed to be responsible for association of cytoplasm with membranes. ARNO (47 kDa) appears as a dimer of approximately 90 kDa in gel filteration, but has an apparent molecular weight of approximately 40 kDa in SDS-PAGE. It is 83% homologous to cytohesin 1. However, while cytohesin 1 expression appears to be limited to hematopoietic cells, ARNO is more ubiquitously expressed. The catalytic activity of ARNO has been localized to the region of the Sec7 domain, and it appears to be positively regulated by interaction of the PH domain with inositol phospholipids. ARNO is localized to the plasma membrane in most mammlian cells, where it functions as an exchange factor for the plasma membrane-located ARF6 rather than ARF1. In HeLa cells, ARNO is mostly cytosolic, and its recruitment from cytosol to endosomes in the epithelial cells of the kidney, is pH-dependent.