The ADP-ribosylation factor (ARF)-related GTPase ARF-related protein binds to the ARF-specific guanine nucleotide exchange factor cytohesin and inhibits the ARF-dependent activation of phospholipase D.
ADP-ribosylation factor-related protein (ARP) is a membrane-associated GTPase with remote similarity to the family of ADP-ribosylation factors (ARF). In a yeast two-hybrid screen designed to identify proteins interacting with ARP, we isolated a partial cDNA of the ARF-specific guanine nucleotide exchange factor mSec7-1/cytohesin encoding its N terminus and most of ... the Sec7 domain (codons 1-200). ARP and ARP-Q79L (GTPase-negative ARP) exhibited a higher affinity to mSec7-1-(1-200) than ARP-T31N (nucleotide exchange-defective ARP) in the two-hybrid assay. Similarly, full-length [35S]mSec7-1/cytohesin was specifically adsorbed to glutathione-Sepharose loaded with glutathione S-transferase (GST)-ARP-Q79L, GST-ARP, or GST-ARP-T31N, the latter exhibiting the lowest binding affinity. Overexpression of ARP-Q79L, but not of ARP-T31N, in COS-7 cells reduced the fluorescence from co-expressed green fluorescent protein fused with mSec7-1/cytohesin or mSec7-2/ARNO in plasma membranes as detected by deconvolution microscopy. Recombinant ARP and ARP-Q79L, but not ARP-T31N, inhibited the phospholipase D (PLD) activity stimulated by mSec7-2/ARNO and ARF in a system of isolated membranes. Furthermore, transfection of HEK-293 cells with ARP or ARP-Q79L, but not ARP-T31N, inhibited the muscarinic acetylcholine receptor-3 induced PLD stimulation and translocation of ARF from cytosol to membranes. These data suggest that the GTP-bound form of ARP specifically binds mSec7-1/cytohesin, and that ARP may be involved in a pathway inhibiting the ARF-controlled activity of PLD.
Mesh Terms:
ADP-Ribosylation Factors, Carbachol, Cell Adhesion Molecules, Cell Line, Enzyme Activation, GTP Phosphohydrolases, GTP-Binding Proteins, GTPase-Activating Proteins, Guanine Nucleotide Exchange Factors, Guanosine Triphosphate, Humans, Membrane Proteins, Phospholipase D, Proteins, Receptor, Muscarinic M3, Receptors, Muscarinic, Sequence Homology, Amino Acid, Signal Transduction, Yeasts
ADP-Ribosylation Factors, Carbachol, Cell Adhesion Molecules, Cell Line, Enzyme Activation, GTP Phosphohydrolases, GTP-Binding Proteins, GTPase-Activating Proteins, Guanine Nucleotide Exchange Factors, Guanosine Triphosphate, Humans, Membrane Proteins, Phospholipase D, Proteins, Receptor, Muscarinic M3, Receptors, Muscarinic, Sequence Homology, Amino Acid, Signal Transduction, Yeasts
J. Biol. Chem.
Date: Apr. 02, 1999
PubMed ID: 10092663
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