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.
Schürmann
A
A
Institut für Pharmakologie und Toxikologie, Medizinische Fakultät der Rheinisch-Westfälischen Technischen Hochschule Aachen, D-52074 Aachen, Germany.
Schmidt
M
M
Asmus
M
M
Bayer
S
S
Fliegert
F
F
Koling
S
S
Massmann
S
S
Schilf
C
C
Subauste
M C
MC
Voss
M
M
Jakobs
K H
KH
Joost
H G
HG
D020727:ADP-Ribosylation Factors; D002217:Carbachol / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D015003:Yeasts
D015815:Cell Adhesion Molecules; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D011506:Proteins; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; C066755:Sec7 guanine nucleotide exchange factors; C100723:cytohesin-1; C102474:cytohesin-2; D006160:Guanosine Triphosphate; D002217:Carbachol; D010739:Phospholipase D; C097313:ADP-ribosylation factor related proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors