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INTRODUCTION |
ADP-ribosylation factors (ARF)1 are Ras-related
GTPases that regulate vesicular
trafficking pathways in eukaryotic cells (1, 2). Activated ARF
stimulates phospholipase D (PLD) in several cellular systems as well as
in isolated membranes (Refs. 3-5; reviewed in Refs. 6 and 7).
Stimulation of PLD and formation of its product, phosphatidic acid,
appears to be a crucial signal in intracellular vesicle formation and
trafficking (8-10). PLD is stimulated by receptors with intrinsic
tyrosine kinase activity as well as by G protein-coupled receptors
through ARF, Rho, and/or protein kinase C (PKC)-dependent
pathways (reviewed in Refs. 6 and 7). In HEK-293 cells expressing the
muscarinic acetylcholine receptor-3 (M3 receptor),
M3 receptor-induced PLD stimulation is mediated by ARF and
Rho proteins, but is independent of PKC activation (11-13). So far,
two mammalian PLD isoforms with different subcellular localization and
regulatory characteristics have been identified. PLD1 (with the two
splice variants PLD1a and PLD1b) was found in the perinuclear region,
and is markedly stimulated by phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2), PKC, ARF, and Rho GTPases (14, 15). In
contrast, PLD2 is associated with the plasma membrane, and appears to
be less responsive to ARF than PLD1 (16-18).
ARF-related protein (ARP) is a 25-kDa GTPase with remote similarity to
the ARF family (33% and 39% identical amino acids with ARF1 and
ARF-like 3, respectively). Its cDNA was isolated in a PCR-based
cloning approach (19), which led to the identification of four other
ARF-like GTPases (20-22). Based on the presence of conserved residues
which are characteristic for the ARF family, in particular Trp-86,
Asp-101, Asp-104, and Arg-107, the GTPase was assigned to the extended
ARF family. However, ARP exhibits features that are unusual in the ARF
family, i.e. its rather high intrinsic GTPase activity (19).
Compared with other GTPases, guanine nucleotide exchange of ARP is
slow, suggesting that this function is catalyzed by a guanine
nucleotide exchange factor.
In the present study, we have employed the yeast two-hybrid system in
order to identify proteins interacting with ARP, and have isolated a
cDNA clone encoding a partial sequence of cytohesin, a mammalian
homologue of the yeast protein Sec7. Cytohesin belongs to a small
family of cytosolic adapter proteins (here referred to as
mSec7-1/cytohesin, mSec7-2/ARNO, and mSec7-3/GRP1; Ref. 23) that harbor
an N-terminal coiled-coil domain, a Sec7 domain, and a C-terminal
pleckstrin homology domain (24-26). Their Sec7 domain accelerates
GDP/GTP exchange of ARF proteins (Refs. 24, 27, and 28; reviewed in
Ref. 29). Thus, we assumed that the interaction of ARP with a mammalian
Sec7 isoform might modulate the ARF-mediated signal transduction, and
have employed the assay of M3 receptor- or ARF-stimulated
PLD activity (11, 30) as a detection system in order to characterize
the potential role of ARP in this pathway. Evidence is presented here
suggesting that ARP-GTP inhibits the activation of PLD by ARF in
isolated membranes, inhibits the M3 receptor-mediated
stimulation of PLD in intact cells, and also produces a subcellular
re-distribution of co-expressed mSec7-1/cytohesin or mSec7-2/ARNO.
Thus, ARP may be involved in a signaling pathway inhibiting the
ARF-controlled activity of PLD.
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MATERIALS AND METHODS |
Plasmids
For subcloning of cDNAs, suitable restriction sites were
used or introduced by PCR with primers containing the desired site. All
PCR-generated constructs were controlled by sequencing.
ARP and ARL4 Constructs--
ARP-T31N, ARP-Q79L, ARL4-T34N, and
ARL4-Q79L mutants were generated by oligonucleotide-directed
mutagenesis (Ref. 31; Muta-Gene phagemid in vitro
mutagenesis kit, Bio-Rad) from the rat ARP or ARL4 cDNA (Refs. 19
and 20; accession nos. X78603 and X77235). Wild-type and mutant ARP
cDNAs were subcloned as described (19) into pGEX-2TK (Pharmacia,
Freiburg, Germany) for preparation of recombinant proteins, or into
pCMV for expression in mammalian cells.
Two-hybrid Vectors--
For assay of the yeast two-hybrid
interaction, the plasmids pBTM116 and pVP16 were used (32). The
plasmids pLexA-ARP, pLexA-ARP-T31N, pLexA-ARP-Q79L, pLexA-ARL4,
pLexA-ARL4-T34N, and pLexA-ARL4-Q79L were constructed by
inserting a PCR-generated fragment of the ARP, ARP-T31N, ARP-Q79L,
ARL4, ARL4-T34N, and ARL4-Q79L cDNA into the EcoRI site
of the pBTM116 plasmid. The resulting plasmids express ARP as a fusion
protein with the DNA binding domain of LexA connected by a short linker
(amino acid sequence: EFRSGRSSSST). The plasmids pLexA-Rab6 and
pLexA-lamin (33) were gifts of Dr. B. Goud (Institut Pasteur,
Unité de Génétique Somatique, Paris, France).
pVP16-mSec7-(1-52) and pVP16-mSec7-(1-150) were generated by
subcloning of the respective cDNA fragments (23) into the BamHI/NotI sites of pVP16.
mSec7 Constructs--
Plasmids of mSec7-1/cytohesin and
mSec7-2/ARNO cDNA subcloned into pGEX-KG or the GFP expression
vector pEGFP-C1 (CLONTECH) were gifts from Dr. N. Brose (Max Planck Institute of Biophysical Chemistry, Göttingen,
Germany). mSec7-2/ARNO cDNA subcloned into pGEX-2T was
obtained from Dr. P. Chardin (CNRS, Institute de Pharmacologie Moléculaire et Cellulaire, Valbonne, France).
ARF1 Constructs--
For preparation of recombinant ARF1,
cDNA was subcloned into the baculovirus transfer vector pAcGHLT
(PharMingen, Hamburg, Germany).
Yeast Strains and Media
Yeast strains, plasmids, and library for the yeast two-hybrid
screen were obtained from Dr. B. Goud. The genotype of the
Saccharomyces cerevisiae reporter strain L40 is
MATa trp1 leu2 his3
LYS2::lexA-HIS3 URA3::lexA-lacZ (32). Yeast strains were grown at
30 °C in rich medium (1% yeast extract, 2% Bacto-peptone, 2%
glucose) or in synthetic minimal medium with appropriate supplements.
Two-hybrid Screen
The yeast reporter strain L40, which contains the reporter genes
lacZ and HIS3 downstream of the binding sequences
for LexA, was transformed with pLexA-ARP-T31N or pLexA-ARP-Q79L and a
mouse embryo pVP16 cDNA library (34) with the lithium acetate
method (35), and was subsequently treated as described (32). Double transformants were plated to synthetic medium lacking histidine, leucine, tryptophan, uracil, and lysine. The plates were incubated at
30 °C for 3 days. His+ colonies were patched on selective plates and
assayed for
-galactosidase activity by a filter assay (36). Plasmid
DNA was prepared from colonies displaying a
HIS+/lacZ+ phenotype by
electrotransformation of HB101 cells and used to re-transform the L40
strain containing pLexA-ARP, pLexA-ARP-T31N, pLexA-ARP-Q79L, pLexARL4,
pLexA-Rab6, and pLexA-lamin, respectively, to test for specificity. For
assay of
-galactosidase activity, transformants were grown in
histidine-containing medium, and were lysed and assayed as described
(37).
Preparation of Recombinant Proteins
Escherichia coli DH5
was transformed with
cDNAs of ARP, ARP-T31N, ARP-Q79L, mSec7-1/cytohesin, or
mSec7-2/ARNO subcloned in the pGEX vector (Pharmacia), and an
exponentially growing culture was induced with 0.1 mM
isopropyl-D-thiogalactoside. After incubation at room
temperature for 4-6 h, cells were lysed and centrifuged at 12,000 × g for 10 min. GST fusion proteins were isolated by adsorption to glutathione-Sepharose beads (Pharmacia) for 2 h at
4 °C. In some experiments (Fig. 4), recombinant ARP fusion proteins
were loaded with GTP (ARP, ARP-Q79L) or GDP (ARP-T31N) by incubation
for 2 h at 4 °C. Thereafter, the beads were washed with
phosphate buffer (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.7 mM
KH2PO4 (pH 7.4)), and fusion proteins were
eluted with buffer containing 30 mM glutathione in 100 mM Tris buffer (pH 8.0), or by treatment of the Sepharose
beads with thrombin (1 unit/100 µg of fusion protein). Recombinant
ARF1 was prepared by transfection of Sf9 cells with cDNA
subcloned in the vector pAcGHLT, and isolated by adsorption of the GST
fusion protein to glutathione-Sepharose.
Binding of mSec7-1/Cytohesin to Glutathione-Sepharose Loaded
with GST-ARP
[35S]Methionine-labeled mSec7-1/cytohesin was
prepared by in vitro translation with a kit (TNT T7 quick
coupled transcription/translation system) from Promega (Madison, WI)
according to instructions of the manufacturer. Samples of 4 µl of the
reticulocyte lysate were added to 10 µl of glutathione-Sepharose
beads loaded with approximately 50 µg of recombinant GST-ARP,
GST-ARP-Q79L, or GST-ARP-T31N, and were incubated for 2 h at
4 °C in a total volume of 100 µl. The beads were washed four times
with Tris buffer (50 mM, pH 7.5) containing 1% Nonidet
P-40, 1 mM EDTA, and 500 mM NaCl, and were boiled with Laemmli buffer. Eluted proteins were separated by SDS-polyacrylamide gel electrophoresis (12% gels) and detected by autoradiography.
Deconvolution Microscopy
COS-7 cells were seeded at 7 × 104
cells/coverslip and transfected with 2.5 µg of the plasmids harboring
the GFP-mSec7 and ARP constructs with the aid of LipofectAMINE (Life
Technologies, Inc.). After 30 h of culture, cells were fixed with
3.7% formaldehyde for 20 min, washed, and mounted in Fluoromount-G
(Southern Biotechnology Associates, Birmingham, AL). Images were
obtained with a cooled CCD camera (Photometrics, Tucson, AZ) with
exposures of 0.2 and 0.4 s and HQ fluorescein filters from Chroma
(Brattleboro, VT). Microscope automation and image analysis were
performed with ISEE Inovision (Durham, NC) and Adobe Photoshop software
(Mountain View, CA). For each cell, a series of images spaced 4 µm
apart through the vertical axis was obtained. Out of focus light was removed using the nearest neighbor deconvolution algorithm (38). Background was subtracted and contrast was stretched with the Adobe
Photoshop software.
Assay of GTP Binding
Guanine nucleotide binding to recombinant GTPases was assayed by
a previously described procedure (39, 24) with minor modifications.
Samples of 15 pmol of GTPase were incubated at 37 °C in a buffer
containing 4 µM [35S]GTP
S (4 × 105 cpm/sample), 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM imidazole, 3.1 mM
MgCl2, 10 mM dithiothreitol, 40 µg of bovine
serum albumin, 3 mM dimyristoylphosphatidylcholine, and
0.1% (w/v) sodium cholate in a total volume of 100 µl. The
incubation was terminated by addition of 1 ml of ice-cold buffer
containing 20 mM Tris-HCl (pH 8.0), 100 mM
NaCl, and 25 mM MgCl2. The samples were
filtered through nitrocellulose membranes (Sartorius GmbH,
Göttingen, Germany; pore size 0.2 µm) and washed four times
with 1 ml of fresh buffer. Radioactivity on the filters was determined
by scintillation counting in a water-compatible scintillation mixture
(Ready Protein, Beckman, Fullerton, CA).
Cell Culture and Transfection
HEK-293 cells overexpressing M3 receptors were grown
in Dulbecco's modified Eagle's medium/F-12 medium as described
previously (11) and were transfected by incubation with co-precipitates of calcium phosphate and DNA from the different plasmids. The transfection efficiency (50-80%) was controlled by co-transfection with pSV
-gal (Promega) and histochemical detection of
-galactosidase activity. All assays were performed 48 h after
transfection. Overexpression of ARP was confirmed by immunoblotting of
whole cell lysates as described (19).
Assay of PLD in Membranes
Membranes of HEK-293 cells were prepared by nitrogen cavitation
and differential centrifugation as described previously (30). PLD
activity in membranes was assayed as described (3) with the following
modifications. For generation of a micellar lipid preparation,
[3H]phosphatidylcholine was mixed with
PtdIns(4,5)P2 in a molar ratio of 8:1, dried, and
resuspended in a buffer containing 50 mM HEPES (pH 7.5), 3 mM EGTA, 80 mM KCl, and 1 mM
dithiothreitol. The suspension was sonicated on ice, and samples
containing the membranes with the indicated agents and/or proteins were
added. The reaction was allowed to proceed for 60 min at 37 °C in a
total volume of 100 µl containing (final concentrations) 200 µM [3H]phosphatidylcholine (approximately
0.5 × 106 cpm/sample), 25 µM
PtdIns(4,5)P2, 50 mM HEPES (pH 7.5), 80 mM KCl, 3 mM MgCl2, 3 mM EGTA, 2 mM CaCl2, 1 mM dithiothreitol, and 2% (v/v) ethanol. The reaction was
stopped with 2 ml of chloroform/methanol (1:1), and
[3H]phosphatidylethanol ([3H]PtdEtOH)
formed was isolated and analyzed as described (11, 30).
Assay of PLD in Intact Cells
Cellular phospholipids were labeled by culture of cells in the
presence of [3H]oleic acid (2 µCi/ml) for 20-24 h.
Thereafter, cells were washed twice with a buffer containing 118 mM NaCl, 5 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, 5 mM
glucose, and 15 mM HEPES (pH 7.4). The cells were treated
as indicated with 1 mM carbachol or 100 nM
phorbol 12-myristate 13-acetate (PMA) in the presence or absence of
ethanol (400 mM) for 30 min at 37 °C. Total
phospholipids and [3H]PtdEtOH were isolated as described
previously (11-13). Formation of [3H]PtdEtOH is
expressed as percentage of total labeled phospholipids.
Assay of ARF Translocation
Translocation of ARF in HEK-293 cells was assayed with a
previously published procedure (12). Cells were stimulated with carbachol for 10 min, and were permeabilized with 10 µM
digitonin for 15 min. After centrifugation of cells (15,000 × g, 10 min), the ARF content of the supernatant was assayed
as described by immunoblotting. Antiserum against recombinant ARF1 was
provided by Dr. J. B. Helms (University of Heidelberg, Heidelberg,
Germany). Densitometry of bands corresponding to ARF was performed with ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
Specific Binding of ARP-GTP to mSec7-1/Cytohesin--
In a yeast
two-hybrid screen designed to identify proteins interacting with ARP,
we isolated a cDNA clone that induced growth of
lacZ-positive colonies on histidine-free agar plates. The
clone harbored 626 nucleotides of the mouse mSec7-1/cytohesin cDNA
(accession no. AF051337) fused to the VP16 cDNA; the translate of
this cDNA contained the N-terminal coiled-coil domain and the Sec7 domain of cytohesin (codons 1-200) fused in-frame with the VP16 activation domain. Cytohesin belongs to a small family of cytosolic adapter proteins with nucleotide exchange-accelerating activity for ARF
(24, 27-29). As is illustrated in Fig.
1A, the clone induced growth
of colonies on histidine-free agar plates when co-transfected with
pLex-ARP or with pLex-ARP-Q79L (GTPase-negative mutant); growth of
colonies co-transfected with pLex-ARP-T31N (exchange-defective mutant)
was much slower. When transferred to nitrocellulose filters, these
colonies tested positive for the lacZ reporter (data not
shown). Similarly, in cells grown in suspension in histidine-containing
medium (Fig. 1B), pLex-ARP and pLex-ARP-Q79L induced a
significant
-galactosidase activity that was higher than that of
pLex-ARP-T31N transfectants. Thus, the protein encoded by the partial
mSec7-1/cytohesin cDNA appears to bind ARP-GTP with a higher
affinity than ARP-GDP. mSec7-1/cytohesin (1-200) also exhibited some
interaction with lamin C (Fig. 1, A and B).
However, full (mSec7-(1-52)) or partial (mSec7-(1-150)) deletion of
the Sec7 domain suppressed the interaction with ARP but not with lamin
C (Fig. 1B). Thus, lamin C appears to interact with the
coiled-coil domain of cytohesin, whereas the Sec7 domain seems
necessary for the specific interaction with ARP. Furthermore, no
interaction was observed between mSec7-1-(1-200) and the GTPases ARL4
or Rab6.

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Fig. 1.
Specific interaction of ARP with
mSec7-1/cytohesin as detected in the yeast two-hybrid assay.
A, yeast cells were co-transfected with the indicated LexA
(ARP, ARL4, Rab6, lamin C) and VP16 constructs (mSec7-(1-200) or
mSec7-(1-52)), and were grown on histidine-free agar plates.
B, cells transfected with the indicated constructs
were grown in histidine-containing medium in suspension, lysed,
and -galactosidase activity was assayed and normalized for protein
concentration. The data represent means ± S.D. of three
experiments. -Galactosidase activity from cells co-transfected with
pLex constructs of ARL4, ARL4-T34N, ARL4Q79L, or Rab6, and the
indicated VP16 constructs was undetectable (shown only for
mSec7-(1-200)).
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In order to further demonstrate the interaction of ARP with
mSec7-1/cytohesin, we used glutathione-Sepharose loaded with
recombinant GST-ARP constructs, and assayed the binding of full-length
35S-labeled mSec7-1/cytohesin prepared by in
vitro translation. As is illustrated in Fig.
2A, recombinant GST-ARP-Q79L
adsorbed a significant portion of the tracer, whereas no binding was
detected to GST alone or to an unrelated protein (Bcl2). In addition,
Sepharose loaded with GST-ARP-Q79L failed to adsorb an unrelated
labeled protein, Bcl2 (Fig. 2A), ruling out nonspecific
stickiness of the GTPase. An additional band at approximately 40 kDa,
which was also specifically adsorbed, appears to be incompletely
translated mSec7-1/cytohesin; according to its size, this product
should contain the full Sec7 domain. Fig. 2B illustrates the
quantitative evaluation of a series of experiments comparing the
different ARP constructs. The highest binding was found with the
GTPase-negative mutant ARP-Q79L. The exchange-defective mutant ARP-T31N
bound significant, but much lower, amounts of mSec7-1/cytohesin than ARP-Q79L. It should be noted that the wild-type ARP employed in this
experiment carried GTP and GDP at a ratio of approximately 1:3 (19).
Thus, these data are consistent with the conclusion that
mSec7-1/cytohesin specifically binds the GTP-loaded ARP with a higher
affinity than the GDP-loaded form.

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Fig. 2.
Specific adsorption of mSec7-1/cytohesin on
glutathione-Sepharose loaded with GST-ARP-GTP. Radiolabeled
mSec7-1/cytohesin or Bcl2 was prepared by in vitro
translation in the presence of [35S]methionine, and was
incubated with glutathione-Sepharose loaded with the indicated
recombinant GST-ARP preparations (ARP; ARP-Q79L; ARP-T31N). The
Sepharose beads were washed four times and were eluted with Laemmli's
sample buffer. Co, Sepharose loaded with GST;
Bcl2, Sepharose with GST-Bcl2; St, standards of
radiolabeled mSec7 or Bcl2 preparations.
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Subcellular Localization of mSec7 Isoforms in Cells Co-transfected
with ARP--
In order to demonstrate the interaction of ARP with Sec7
domain proteins in a system of intact mammalian cells, COS-7-cells were
co-transfected with ARP and GFP-tagged mSec7-1/cytohesin or
mSec7-2/ARNO. Since we assumed that the specific interaction of the
proteins might produce a re-distribution of the mSec7 isoforms, the
subcellular localization of the GFP fluorescence was analyzed by
deconvolution microscopy. Deconvolution microscopy eliminates most
light from portions of the specimen not in the focal plane, thereby
allowing a more precise localization of proteins in cells. When cells
were co-transfected with GFP-mSec7-1/cytohesin or GFP-mSec7-2/ARNO plus
the bland pCMV vector, an essentially continuous labeling of the
cellular periphery, i.e. of the plasma membrane, with the GFP fluorescence was observed, in addition to labeling of intracellular compartments (panels a and d). In
contrast, when cells were co-transfected with ARP-Q79L
(panels b and e), the GFP fluorescence
disappeared almost completely from the plasma membrane
(GFP-mSec7-1/cytohesin, panel b) or from large
portions of it (GFP-mSec7-2/ARNO, panel e). As is
illustrated in panels c and f of Fig.
3, the nucleotide exchange-defective
mutant ARP-T31N failed to remove the GFP fluorescence from the plasma
membrane. The transfections were repeated three times, and in each
experiment 5-7 cells per treatment were examined. In all cells
examined, ARP-Q79L produced a marked reduction of the GFP fluorescence
associated with the plasma membrane as compared with cells transfected
with bland vector or ARP-T31N.

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Fig. 3.
Effects of ARP on the subcellular
distribution of mSec7 proteins in transfected COS-7 cells. COS-7
cells were co-transfected with GFP-mSec7-1/cytohesin (a-c)
or GFP-mSec7-2/ARNO (d-f) and bland vector (a
and d), ARP-Q79L (b and e) or ARP-T31N
(c and f). Cells were fixed with formaldehyde,
and images of the GFP fluorescence were obtained by automated
deconvolution microscopy and image analysis as described under
"Materials and Methods."
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Lack of Effect of mSec7-1/Cytohesin and mSec7-2/ARNO on Guanine
Nucleotide Exchange of ARP--
In order to test the possibility that
mSec7 isoforms alters the guanine nucleotide exchange rate of ARP,
binding of [35S]GTP
S to recombinant ARP was assayed in
the presence of mSec7-1/cytohesin or mSec7-2/ARNO. Both mSec7 isoforms
failed to alter the rate of binding of [35S]GTP
S to
ARP preloaded with either GDP or unlabeled GTP
S
(t1/2 = 20 min, data not shown). In contrast,
mSec7-1/cytohesin and mSec7-2/ARNO produced the expected acceleration
of guanine nucleotide exchange on (
17)ARF
(t1/2 = 2.5 min in the presence
versus >30 min in the absence of the mSec7 isoforms).
Addition of recombinant ARP failed to affect the stimulatory effect of
mSec7-1/cytohesin on nucleotide exchange of (
17)ARF (data not shown).
Inhibitory Effect of ARP on the mSec7-2/ARNO-stimulated PLD
Activity in Isolated Membranes--
It has previously been shown that
ARF stimulates the activity of PLD (3, 4). This effect can be
demonstrated in an in vitro system of isolated membranes by
addition of mSec7-2/ARNO, ARF, and GTP
S, resulting in stimulated
PtdEtOH production (30). As is illustrated in Fig.
4, the stimulatory effect of mSec7-2/ARNO and ARF on PLD was specifically inhibited by the recombinant ARP-Q79L and to a somewhat lesser extent by ARP, but not by ARP-T31N. In these
experiments, the recombinant proteins were preloaded with GTP (ARP,
ARP-Q79L) or GDP (ARP-T31N). Boiling of ARP-Q79L prevented its
inhibitory effect on PLD activity (data not shown). These data are
consistent with the conclusion that ARP-GTP may interfere with the
ARF-controlled activity of PLD.

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Fig. 4.
ARP inhibits the ARF-dependent
stimulation of phospholipase D in membranes of HEK-293 cells.
Membranes from HEK-293 cells were incubated as indicated with GTP S
(100 µM), ARF1 (4 µM), mSec7-2/ARNO (2 µM), ARP (6 µM), ARP-Q79L (7 µM), or ARP-T31N (8 µM) for 60 min at
37 °C, and PLD activity was assayed as described under "Materials
and Methods." Data represent means ± S.D. of triplicate samples
from a representative experiment, which was repeated four times. PLD
activity in the presence of GTP S, ARF, ARNO, and boiled ARP-Q79L was
1.95 ± 0.2 nmol/(mg × h).
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ARP Inhibits the M3 Receptor-mediated Stimulation of
PLD Activity in HEK Cells--
In order to define the involvement of
ARP in PLD regulation in a system of intact cells, we employed HEK-293
cells transiently transfected with the various ARP constructs. As was
shown previously (11, 12), PLD is activated by M3 receptor
stimulation with carbachol; this effect can be enhanced by
overexpression of mSec7-2/ARNO or ARF (data not shown). The effects of
ARP appeared antagonistic to those of the M3 receptor
agonist, carbachol. As is illustrated in Fig.
5A, transfection of HEK-293
cells with ARP or GTPase-negative ARP-Q79L markedly inhibited the
stimulatory effect of carbachol on PLD activity. In contrast, the
nucleotide exchange-defective mutant ARP-T31N failed to affect the
stimulatory effect of carbachol. Furthermore, neither basal nor
PMA-stimulated PLD activity was altered by any of the ARP constructs
(Fig. 5B).

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Fig. 5.
ARP inhibits the M3
receptor-stimulated PLD activity in HEK-293 cells. Cells
were transfected with blank plasmid (pCMV), ARP, GTPase-negative mutant
ARP-Q79L, or nucleotide exchange-defective mutant ARP-T31N. After
48 h, the cells were stimulated with 1 mM carbachol
(A) or 0.1 µM PMA (B) for 30 min,
and PLD activities were assayed. Data represent means ± S.D. of
triplicate samples from a representative experiment, which was repeated
four times. Equal expression of ARP constructs was ascertained by
immunoblotting of cell lysates from parallel transfections (data not
shown).
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ARP Inhibits the M3 Receptor-mediated Translocation of
ARF in HEK Cells--
In unstimulated HEK-293 cells, the major portion
of ARF (ARF-GDP) is located in the cytosol, and is released from the
cells upon permeabilization with digitonin (Ref. 12; Fig.
6A). When cells are stimulated
with carbachol or GTP
S, the release of ARF from permeabilized cells
is inhibited, reflecting the translocation of ARF-GTP from the cytosol
to the membranes (Fig. 6, A and B). As the
quantitative evaluation of a series of six experiments indicates (Fig.
6B), transfection of cells with ARP or GTPase-negative ARP-Q79L reversed the inhibitory effect of carbachol on ARF release. In
contrast, the exchange-negative mutant ARP-T31N failed to alter the
effect of carbachol. Furthermore, the effect of GTP
S on ARF release
was not reversed by any of the constructs investigated. These data
indicate that ARP-GTP specifically inhibits the M3 receptor-induced translocation of ARF.

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Fig. 6.
Inhibition of the carbachol-induced
translocation of ARF in HEK-293 cells by ARP. HEK-293 cells
overexpressing M3 receptors were transfected with blank
plasmid (pCMV), ARP, GTPase-negative mutant ARP-Q79L, or nucleotide
exchange-defective mutant ARP-T31N. After 48 h, the cells were
stimulated with carbachol (1 mM) for 10 min, and were
subsequently permeabilized with digitonin in the presence or absence of
GTP S as indicated for 15 min at 37 °C. Release of ARF into the
cytosol was assayed by immunoblotting of the supernatant.
Panel A, immunoblots from a representative
experiment. Panel B, quantitative evaluation of
six independent transfections. The data were normalized for ARF release
in the absence of stimuli and are expressed as means ± S.E.
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 |
DISCUSSION |
The present data indicate that the ARF-related GTPase ARP
specifically inhibits the PLD activation by ARF and its nucleotide exchange factor mSec7-2/ARNO in isolated membranes. The effect was
produced by the GTPase-negative mutant ARP-Q79L and not by the
exchange-defective ARP-T31N, consistent with the conclusion that it is
the activated, GTP-bound form of ARP that exerts the effects.
Furthermore, overexpression of ARP or ARP-Q79L inhibited the
stimulatory effect of carbachol on PLD in HEK-293 cells. In contrast,
ARP failed to inhibit the PMA-stimulated PLD, apparently dissociating
PKC and ARF-mediated stimulation of the enzyme. This result is
consistent with our previous finding that inhibition or down-regulation
of PKC suppressed the effect of PMA, but not that of carbachol, on PLD
stimulation in HEK-293 cells, and that PKC-mediated stimulation of PLD
is ARF-independent (11, 30). Thus, it is suggested that ARP may be
involved in a signaling pathway that inhibits the ARF-controlled
activation of PLD by the M3 receptor.
The inhibitory effect of ARP on the ARF-stimulated PLD activity may be
explained by an interaction of ARP with ARF guanine nucleotide exchange
factors, which are required for conversion of ARF to its GTP-bound,
activated form (24, 27, 28). Indeed, we observed specific binding of
GTP-bound ARP to the nucleotide exchange factor mSec7-2/cytohesin in
two independent assays of protein interaction. Furthermore, GTP-bound
ARP altered the subcellular distribution of both mSec7-1/cytohesin and
mSec7-2/ARNO. However, we failed to detect an inhibitory effect of ARP
on the mSec7-1/cytohesin-stimulated guanine nucleotide exchange of ARF
in a solution of recombinant proteins. This failure of ARP to inhibit
nucleotide exchange might be due to lack of a necessary factor that is
not present in the incubation of recombinant, soluble proteins.
Alternatively, ARP may inhibit activation of ARF by mSec7 proteins
indirectly, i.e. by a spatial separation of ARF and its
nucleotide exchange factors, an effect that cannot be produced in a
system of isolated proteins in solution.
Binding of ARP to mSec7-1/cytohesin appears to depend on a portion of
the Sec7 domain (amino acids 150-200), since a truncated construct
(mSec7-(1-150)) failed to induce
-galactosidase in the two-hybrid
assay. According to the crystal structure of the Sec7 domain (40), this
portion of the domain harbors the helices G and H, and two conserved
motifs considered to mediate the contact with ARF (41). However, the
molecular basis of the interaction between ARP and mSec7 proteins as
observed here appears to be quite different from that of the
interaction between ARF and mSec7. ARF has recently been shown to bind
to mSec7-2/ARNO by hydrophobic residues in its switch I region (also
designated effector loop) and by a lysine (Lys-73) in the switch II
region (41, 42). In ARP, the following residues are placed by serine
(Val-43), threonine (Ile-46), or glutamate (Lys-73). Furthermore, the
affinity of ARP and ARP-Q79L to mSec7-1/cytohesin was markedly higher
than that of exchange-defective ARP-T31N, distinct from what was found for the interaction of ARF with mSec7. The failure of mSec7-1/cytohesin to alter the nucleotide exchange of ARP is consistent with these differences between the mSec7/ARP and the mSec7/ARF interaction.
From the present results, a hypothesis might be deduced suggesting that
ARP prevents the translocation of mSec7 proteins and consequently of
ARF to cellular compartments harboring PLD. Two isoforms of PLD with
different regulatory characteristics and subcellular localization have
been identified so far (14-18). Because of its considerably higher
sensitivity to ARF, PLD1 appears to be the isoform responsible for the
effects described here. PLD1 is believed to be located predominantly in
intracellular compartments (16), but has recently been shown to be
translocated to the plasma membrane in response to stimulation of
RBL-2H3 cells (43). Furthermore, ARF-sensitive PLD is abundant in
plasma membranes from liver (44). Thus, a scenario in which ARP removes
mSec7 proteins from the plasma membrane (Fig. 3), thereby preventing activation of PLD1, or of a yet unidentified isoform, seems possible. Alternatively, the possibility cannot be excluded that removal of mSec7
proteins from the plasma membrane of cells overexpressing ARP is a
phenomenon unrelated to the inhibition of PLD activity.
It has to be noted that the above suggested role of ARP in regulating
PLD activity is based on experiments with isolated proteins and
membranes, and with cells overexpressing ARP. This experimental strategy is required because, in the absence of knowledge on the signaling mechanism "upstream" of ARP, it is impossible to activate the endogenous ARP. Thus, it still remains to be shown that the endogenous ARP affects PLD in a "wild-type" cell. Furthermore, it
should be noted that ARP, in addition to the effect on PLD, may exert
additional, cellular effects through interaction with mSec7 proteins.
Since mSec7-1/cytohesin was also identified as a protein interacting
with the intracellular domain of integrins (25), it seems that its
functions are pleiotropic.
ARP is expressed in most tissues, with somewhat higher protein levels
in testis, kidney, and liver. Thus, its pattern of expression does not
suggest a tissue-specific function. Furthermore, a data base search
revealed the existence of a Drosophila homolog (expressed sequence tag DM1327692 encoding a partial sequence of 112 codons; 74%
of amino acids identical with human ARP), and an open reading frame in
the S. cerevisiae genome (YPL051, GenBank accession no. U39205) encoding a homologous sequence (47% identical amino acids).
Both sequences exhibit the characteristics of the mammalian ARP, in
particular the insertion between PM1 and PM2 and an aromatic amino acid
(tyrosine or phenylalanine) instead of glycine in position 2. The
existence of a yeast homolog suggests that ARP is essential for a
monocellular organism, and that it exerts a basic cellular function.