From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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ADP-ribosylation factors (ARFs) are ~20-kDa guanine nucleotide-binding proteins recognized as critical components in intracellular vesicular transport and phospholipase D activation. Both guanine nucleotide-exchange proteins and GTPase-activating proteins (GAPs) for ARFs have been cloned recently. A zinc finger motif near the amino terminus of the ARF1 GAP was required for stimulation of GTP hydrolysis. ARD1 is an ARF family member that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We had reported that the ARF domain of ARD1 binds specifically GDP and GTP and that the amino-terminal extension acts as a GAP for the ARF domain of ARD1 but not for ARF proteins. The GAP domain of ARD1, synthesized in Escherichia coli, stimulated hydrolysis of GTP bound to the ARF domain of ARD1. Using ARD1 truncations, it appears that amino acids 101-190 are critical for GAP activity, whereas residues 190-333 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis. The GAP function of the amino-terminal extension of ARD1 required two arginines, an intact zinc finger motif, and a group of residues which resembles a sequence present in Rho/Rac GAPs. Interaction between the two domains of ARD1 required two negatively charged residues (Asp427 and Glu428) located in the effector region of the ARF domain and two basic amino acids (Arg249 and Lys250) found in the amino-terminal extension. The GAP domain of ARD1 thus is similar to ARF GAPs but differs from other GAPs in its covalent association with the GTP-binding domain.
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INTRODUCTION |
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ARD1 (1) is a 64-kDa protein that contains a 18-kDa carboxyl-terminal ADP-ribosylation factor (ARF)1 domain (p3) and a 46-kDa amino-terminal domain (p5) (see Fig. 1). ARFs are ~20-kDa guanine nucleotide-binding proteins (G proteins), initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity (CTA) and later recognized as critical components in vesicular trafficking involving endoplasmic reticulum, Golgi, endosomes, and nuclear envelope (for review, see Ref. 2). ARF1 acts as a key regulator of the interactions of non-clathrin coat protein (coatomer) with Golgi stacks (3) and of clathrin adaptor particles with the trans-Golgi network (4). ARF proteins also activate phospholipase D (5, 6). Guanine nucleotide binding to ARFs, like that to other monomeric G proteins, appears to be governed by guanine nucleotide-exchange proteins (GEPs) and GTPase-activating proteins (GAPs) (2). ARF GEPs (7, 8; for review, see Ref. 2) and GAPs (9-12) have been purified and cloned. The deduced amino acid sequence of ARF1 GAP from rat liver has a zinc finger motif near the amino terminus, which was required for GAP activity (11). The GAP appeared to be recruited to the Golgi by an ARF1-dependent mechanism (11).
Although the roles of G proteins are extremely diverse, they all operate by a fundamentally similar mechanism (13). When GTP occupies the guanine nucleotide-binding site, the G protein can interact with and modify the activity of a downstream target protein. Hydrolysis of GTP causes dissociation of the G protein-target complex and terminates the "active state" of the G protein. Cells regulate the ratio of active and inactive G proteins by modulating the rates of GDP release and GTP hydrolysis (GTPase activity).
It was reported that dissociation of GDP from the ARF domain of ARD1 was faster than from ARD1 itself (14). Using ARD1 truncations, the 15 amino acids immediately preceding the ARF domain were shown to be responsible for decreasing the rate of GDP, but not GTP, dissociation (15). By site-specific mutagenesis it was shown that hydrophobic residues in this region were particularly important in stabilizing the GDP-bound form of ARD1. Therefore, it was suggested that, like the amino-terminal segment of ARF, the equivalent region of ARD1 may act as a GDP dissociation inhibitor.
Until recently, it was believed that GTP hydrolysis by monomeric G
proteins was stimulated by separate GAPs, whereas the presence of an
intrinsic GAP-like domain in G was responsible for GTPase activity
in the heterotrimeric G proteins (16). Some effector proteins that are
regulated by heterotrimeric G proteins also act as GAPs for their G
protein regulators. The GAP activities of the effectors, such as
phospholipase C-
(17) and the cGMP phosphodiesterase
subunit
(18), may allow effector-specific modulation of responses. A relatively
new class of GAPs for heterotrimeric G proteins includes the RGS
(regulator of G protein signaling) family (19). They can contribute to
desensitization induced by a prolonged signal or act as long term
attenuators of signal amplitude, presumably by stimulating GTP
hydrolysis (for review, see Ref. 20).
ARFs are ~20-kDa proteins that exhibit no detectable GTPase activity (21). Like ARFs, the 18-kDa ARF domain (p3) of the 64-kDa ARD1 binds specifically GDP and GTP and lacks detectable GTPase activity (22). Using recombinant proteins, it was shown that the 46-kDa amino-terminal domain of ARD1 (p5) stimulates hydrolysis of GTP bound to p3, and consequently it appears to be the GAP component of this bifunctional protein (14). The stimulatory effect of the p5 domain on the GTPase activity of p3 was specific, as GTP hydrolysis by other members of the ARF family was not increased (12). Based on these and prior data on ARD1 (14, 15), it appeared that p5 may control GTP hydrolysis as well as GDP dissociation.
We reported that functional and physical interactions between p3 and p5 required two negatively charged amino acids in the "effector" region of p3 (22). We report here that these residues probably interact with two positively charged amino acids in the amino-terminal extension (p5). Using affinity-purified antibodies and truncated mutants of ARD1, we show here that the amino terminus of ARD1 is not required for these interactions. By site-specific mutagenesis, we demonstrate further that in p5 an intact zinc finger motif, two arginines, and a sequence that resembles a consensus motif present in Rho/Rac GAPs are required for GAP activity.
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EXPERIMENTAL PROCEDURES |
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Materials-- Bovine thrombin was purchased from Sigma, TLC plates from VWR Scientific, and GSH-Sepharose beads from Pharmacia Biotech Inc. Polymerase chain reaction reagents and restriction enzymes, unless otherwise indicated, were from Boehringer Mannheim. Sources of other materials have been published (7, 14, 15, 22, 23).
Preparation of Recombinant Fusion Proteins (p3, p5, and
p8)--
For large scale production of fusion proteins (14), 10 ml of
overnight culture of transformed bacteria were added to a flask with 1 liter of LB broth and ampicillin, 100 µg/ml, followed by incubation
at 37 °C with shaking. When the culture reached an A600 of 0.6, 500 µl of 1 M
isopropyl--D-thiogalactopyranoside were added (0.5 mM final concentration). After incubation for an additional
3 h, bacteria were collected by centrifugation (Sorvall GSA, 6,000 rpm, 4 °C, 10 min) and stored at
20 °C. Bacterial pellets were
dispersed in 10 ml of cold phosphate-buffered saline, pH 7.4, with
trypsin inhibitor, 20 mg/ml, leupeptin and aprotinin, each 5 mg/ml, and
0.5 mM phenylmethylsulfonyl fluoride. Lysozyme (20 mg in 10 ml) was added. After 30 min at 4 °C, cells were disrupted by
sonication and centrifuged (Sorvall SS34, 16,000 rpm, 4 °C, 20 min).
Fusion proteins, purified on glutathione-Sepharose, were ~90% pure
as estimated by silver staining after SDS-PAGE (12). After cleavage by
bovine thrombin, GST was removed with glutathione-Sepharose beads and
thrombin with benzamidine-Sepharose 6B (24). Proteins were purified
further by gel filtration through Ultrogel AcA 54 and then Ultrogel AcA
34 before storage in small portions at
20 °C. Purity, estimated by
silver staining after SDS-PAGE, was >98%. Amounts of purified
proteins were estimated by a dye-binding assay (25) and by SDS-PAGE
using bovine serum albumin as standard. ARF1(39-45p3) was synthesized
as published (22).
Construction and Expression of Mutated Forms of
ARD1--
Fragments of ARD1 with deletions at the 5-end (EMBL Data
Bank 1993, Accession Number L04510) from pGEX5G/LIC (1) were amplified by polymerase chain reaction using the forward primers 5
-GATTCAGGTGTCCCGCGGATGAAA-3
, 5
-TGTCAAACTAGCCCGCGGATGTCG-3
,
5
-GAAACTCTGTGTCGTCAACCGCGGATGGCT-3
, 5
-AATCAGTTGGATGCCCCGCGGATGGTCACTTTTACAAAG-3
(differences from the original clones are underlined), and the reverse
primer 5
-GAATTCCCGGGGATCCAACTGCG-3
(italicized sequence is
a BamHI restriction site). The forward primers
introduced a SacII restriction site (italicized
sequences) and/or an initiation codon in-frame (bold sequences) in the
deleted fragments yielding, respectively, N
88p8, N
200p8,
N
304p8, and N
387p8. The polymerase chain reaction fragments were
digested with SacII and BamHI for 1 h at
37 °C, extracted from LM-agarose gels, and purified by phenol/chloroform precipitation. The resulting fragments were then
ligated in-frame to the SacII- and BamHI-digested
pGEX5G/LIC expression vector. Ultra competent cells (Stratagene) were
transformed with the plasmids pGEX5G/LIC/N
88p8,
pGEX5G/LIC/N
200p8, pGEX5G/LIC/N
304p8, and
pGEX5G/LIC/N
387p8. Entire sequences of the ARD1 deletion constructs were confirmed by automated sequencing (Applied Biosystems, 373 DNA Sequencer) using the primers 5
-TTATACGACTCACTATAGGG-3
, 5
-AGCTGCAGAAGAATCCATT-3
, 5
-ATCAATTTTAGATATGGCT-3
,
5
-ATGATTGTAGAGTTGTCTT-3
, 5
-TTATTACCTCAATACTCAA-3
, and
5
-GCTAGTTATTGCTCAGCGG-3
. Mutant ARD1 fusion proteins were
expressed and purified as described for the non-mutant ARD1
proteins.
Construction and Expression of Mutated p5--
For site-directed
mutagenesis of p5, a modification of the unique site-elimination
mutagenesis procedure of Deng and Nickoloff (26) was used. 25 pmol of a
5-phosphorylated selection primer and 25 pmol of a 5
-phosphorylated
mutagenic primer were annealed simultaneously to 750 ng of
p5-pGEX5G/LIC in 20 ml of 10 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 50 mM potassium acetate by heating for 5 min at 100 °C and cooling 5 min on ice, followed by
incubation at room temperature for 30 min. The selection primer 5
-CTGTGACTGGTGACGCGTCAACCAAGTC-3
changed a
ScaI restriction site in the Ampr gene of pT7
into a MluI restriction site (underlined). Mutagenic primers
(see Fig. 6 and Tables I-III) introduced the desired mutations. Primers were extended with T7 DNA polymerase, and the new strands were
ligated with T4 DNA ligase for 1 h at 37 °C (final volume 30 µl). Plasmids were then digested for 2 h at 37 °C with 20 units of ScaI (final volume 60 µl). 4-µl samples were
used to transform 90 ml of Epicurian Coli XL1-Bluecompetent cells
(Stratagene). Plasmids were purified with Miniprep Wizard (Promega)
from bacteria grown overnight in 2 ml of 2 × YT broth with
ampicillin, 100 mg/ml. Samples (500 ng) of the plasmids were digested
with 20 units of ScaI for 3 h at 37 °C. 4-µl
samples were used to transform 40 µl of XLmutS-competent cells
(Stratagene). Colonies were screened selectively by digestion with
MluI, and the presence of the mutations was confirmed by
automated sequencing (Applied Biosystems, 373 DNA Sequencer) using the
primers 5
-TTATACGACTCACTATAGGG-3
, 5
-ATGATTGTAGAGTTGTCTT-3
, and
5
-GCTAGTTATTGCTCAGCGG-3
. Large scale production of mutated p5
proteins was carried out as described for ARD1.
Peptides and Antibodies--
High performance liquid
chromatography-purified (>95% pure) synthetic peptides (rARF1p,
KLKLGEIVTTIPT; p3p, KLKQDEFMQPIPT; p5p, SDYSRKLVGIVQ; p5 random (p5Rp),
LSKIGVRDVYSQ; Nt-ARD1, ATLVVNKLGAG; Ct-ARD1, QLVAAGVLDVA) were
purchased from Bio-Synthesis, Inc. (Lewisville, TX). Mass spectral
analysis, amino acid analysis, and peptide sequencing were performed on
each peptide. Peptides were dissolved in water at a final concentration
of 10 mg/ml. Antibodies against the NH2-terminal and the
COOH-terminal sequence of ARD1 were prepared by injecting into rabbits
either ATLVVNKLGAG (Nt-ARD1) or QLVAAGVLDVA (Ct-ARD1) coupled to
hemocyanin (27). After (NH4)2SO4
precipitation, dialyzed IgG from serum of rabbits immunized with the
amino- and carboxyl-terminal ARD1 peptides was purified on protein
A-Sepharose G (Pierce) and then affinity purified on Affi-Gel 15 or
Affi-Gel 10 (Bio-Rad) coupled to Nt-ARD1 and Ct-ARD1, respectively
(28). Specific antibodies were eluted in 12 ml of 0.2 M
glycine, pH 2.7, 10% ethylene glycol, and the pH was adjusted
immediately to 7.5 with 1 N NaOH. After dialysis against
phosphate-buffered saline, antibodies were concentrated (Centricon 50)
and stored at 80 °C in 30% glycerol. Antibodies against p3 and p5
were prepared by injecting purified recombinant fusion proteins (GST-p3
or GST-p5) into rabbits. After
(NH4)2SO4 precipitation, dialyzed
IgG was purified on protein A-Sepharose G, and antibodies against GST
were removed on a GST-glutathione-Sepharose column. The p3 and p5
antibodies were affinity purified on GST-p3- and
GST-p5-glutathione-Sepharose columns, respectively, and stored as
described above. Preimmune sera (diluted at 1/1,000) did not react
detectably with 1 µg of ARD1 by Western blotting. Typically, the
affinity-purified Nt-ARD1, Ct-ARD1, p3, and p5 polyclonal antibodies,
at dilutions of 1/200,000 (~3.3 ng), 1/200,000 (2.4 ng), 1/5,000 (82 ng), and 1/10,000 (78 ng), respectively, detected ~0.2 µg of ARD1
after transfer to nitrocellulose membrane. Nt-ARD1 and Ct-ARD1
antibodies did not react with any of the recombinant ARF proteins
tested (rARF1, 2, 3, 4, 5, 6), and cross-reactivity between the amino-
or carboxyl-terminal ARD1 and p3 or p5 antibodies was not detected.
Assay of GTPase Activity--
Samples were incubated for 30 min
at 30 °C in 20 mM Tris, pH 8.0, 10 mM
dithiothreitol, 2.5 mM EDTA with bovine serum albumin, 0.3 mg/ml, and cardiolipin, 1 mg/ml, then for 40 min at 30 °C in the
same medium with 0.5 µM [-32P]GTP (3,000 Ci/mmol) and 10 mM MgCl2 (total volume 120 µl). After addition of p5 or mutant proteins (40 µl), incubation at
room temperature was continued for 1 h (final volume 160 µl)
before proteins with bound nucleotides were collected on nitrocellulose (23). Bound nucleotides were eluted in 250 µl of 2 M
formic acid, of which 3-4-µl samples were analyzed by TLC on
polyethyleneimine-cellulose plates (14), and 240 µl was used for
radioassay to quantify total 32P-nucleotide. TLC plates
were subjected to autoradiography at
80 °C for 18-28 h. Total
amounts of labeled nucleotides (GTP + GDP) bound to p3, p8, or p3 after
incubation with p5, whether quantified by radioassay of the formic acid
solution, by counting total radioactivity on the filter, or by
PhosphorImaging (Molecular Dynamics) after TLC, were not significantly
different under any condition (14), except as mentioned. An increase in
bound GDP was always correlated with a decrease in bound GTP (22).
Assay of Cholera Toxin-catalyzed ADP-ribosylagmatine
Formation--
p3 or ARD1 was incubated for 30 min at 30 °C in 40 µl of 20 mM Tris, pH 8.0, 10 mM
dithiothreitol, 2.5 mM EDTA with bovine serum albumin, 0.3 mg/ml, and cardiolipin, 1 mg/ml, before addition of 20 µl of solution
to yield final concentrations of 100 µM GTPS or GTP
and 10 mM MgCl2. Where indicated, p5 or mutant
protein was then added for 30 min. Components needed to quantify ARD
stimulation of cholera toxin-catalyzed ADP-ribosylagmatine formation
were then added in 70 µl to yield final concentrations of 50 mM potassium phosphate, pH 7.5, 6 mM
MgCl2, 20 mM dithiothreitol, ovalbumin, 0.3 mg/ml, 0.2 mM [adenine-14C]NAD
(0.05 µCi), 20 mM agmatine, cardiolipin, 1 mg/ml, and 100 µM GTP
S or GTP with 0.5 µg of cholera toxin (29).
After incubation at 30 °C for 1 h, samples (70 µl) were
transferred to columns of AG 1-X2 equilibrated with water and eluted
with five 1-ml volumes of water (29). The eluate, containing
[14C]ADP-ribosylagmatine, was collected for
radioassay.
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RESULTS |
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Identification of the GAP Domain of ARD1-- Incubation of p3 or p5 with affinity-purified polyclonal antibodies raised against recombinant p3 or p5, respectively, markedly reduced, in a concentration-dependent manner, the ability of p5 to stimulate hydrolysis of GTP bound to p3 (Fig. 1), whereas the antibodies did not affect GTP binding (data not shown). 30 µg of either antibody completely blocked p5-stimulated GTPase activity (Fig. 1), whereas up to 50 µg of an anti-GST antibody had no effect (data not shown). On the other hand, 30 µg of anti-p3 or anti-p5 antibodies reduced the intrinsic GTPase activity of ARD1 only 8.8 ± 1.2% and 9.6 ± 0.9%, respectively (data not shown). 30 µg of anti-p3 or anti-p5 antibodies reduced hydrolysis of GTP bound to p3 by only 26.5 ± 2.3% and 32.3 ± 1.9%, respectively, when added to p3 simultaneously with p5 (data not shown). These results indicated that anti-p3 or anti-p5 antibodies inhibited GTP hydrolysis more effectively when the two domains of ARD1 were present in separate proteins than when covalently linked in recombinant ARD1. Based on these data, the two antibodies may decrease GTP hydrolysis by decreasing the ability of the two domains to interact.
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Identification of the Interaction Domain between p3 and
p5--
Physical interaction between p3 and p5 mutant proteins was
evaluated using recombinant GST fusion proteins with p5 or mutant p5
bound to GSH-Sepharose beads that were then incubated with the ARF
domain (p3) of ARD1. Proteins associated with the beads or interacting
with them were eluted with GSH and separated by SDS-PAGE. We reported
earlier that under those conditions p3 interacted with GST-p5 but not
with GST (14). N88GST-p5 and N
200GST-p5 both clearly interacted
physically with p3, whereas N
304GST-p5 did not (Fig.
5). C
69GST-p5, but not C
191GST-p5
and C
293GST-p5, also associated with p3 (Fig. 5). Therefore, removal
of the first 200 or the last 69 amino acids of p5 did not prevent
physical interaction with p3, suggesting that the interaction domain
may be located between residues 200 and 333. Since the mutant
N
200GST-p5 was able to interact with p3 (Fig. 5) but did not
stimulate hydrolysis of GTP bound to p3 (Fig. 4, B and
C), it appears that residues critical for GAP activity may
be located between amino acids 101 and 200 in p5.
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Identification of Critical Residues in the GAP Domain of
ARD1--
Deletion of amino acids can have subtle but adverse effects
on overall protein structure, sometimes with structural changes in
domains of the protein which are (in the linear sequence) far from the
deletion. Although we could not observe any difference in overall
protein structure of the p8/p5 deletion mutants from that of the wild
type p8/p5 proteins, subtle adverse changes in structure cannot be
ruled out completely. Therefore, to reduce the possibility that the
observed differences among N101p8, N
124p8, and N
146p8 in
GTPase activity resulted from subtle perturbation of their
three-dimensional organization, mutant proteins were constructed with
single amino acid replacements, which should cause minimal disturbance
of global protein structure.
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DISCUSSION |
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Crystal structures of Ras (36, 37) and G (38-41) proteins in
their GTP- and GDP-bound forms have been solved. Ras and G
likely
hydrolyze GTP by similar catalytic mechanisms. Nonetheless, by
themselves, monomeric G proteins hydrolyze GTP at a rate about 100-fold
lower than heterotrimeric G proteins (13). In the presence of Ras GAP,
however, Ras hydrolyzes GTP at least 100-fold faster than
G
s (43). One explanation of this difference is that Ras GAP resembles the so-called "helical domain" that is present in G
s but absent in Ras (16) and that both Ras GAP and the
helical domain introduce into the catalytic cleft an arginine residue that helps to stabilize the transition state (35).
ARD1 exhibits significantly greater GTPase activity than other members of the Ras family (14). Although its GTP-binding domain (p3) has no GTPase activity, addition of the amino-terminal extension (p5) promoted hydrolysis of GTP bound to p3. Deletion of 101 and 69 amino acids from, respectively, the amino and carboxyl termini of p5 did not prevent physical and functional interactions with p3, thus demarcating a minimal domain required for GAP activity. The smallest GAP domain of ARD1 (232 residues) is comparable in size to the minimal catalytic domains of the Ras GAPs, p120GAP, and neurofibromin, respectively, 272 and 229 amino acids (44).
The unusual GTPase activity of ARD1 made possible the identification of
a region specifically involved in both functional and physical
interaction between the GTP binding and the GAP domains of ARD1.
Specific mutations of amino acids in the effector region of the ARF
domain of ARD1 provided evidence for a function of two negatively
charged residues (Asp427 and Glu428), as well
as of Pro432, which presumably creates a curve in the
-sheet structure which could place charged residues in correct
position for interaction with the GAP domain (22). Our data show that
these residues might form salt bridges with Arg249 and
Lys250 in p5. Accordingly, it has been demonstrated that
the Ras/GAP association is based on interaction between positively
charged Arg and Lys, conserved in GAPs, and negatively charged residues in the effector region of Ras (45). It has also been suggested that
hydrophobic residues in the effector domain of ARD1 (Phe429
and Met430) could be involved in the interaction with p5
(22). We postulate that they might interact with Leu251 and
Val252, which directly follow the two positively charged
amino acids in p5, as expected. GTPase activity always required
physical interaction between the two domains of ARD1, and binding of p5
to the effector domain appeared necessary for GTP hydrolysis. The
peptide p5, corresponding to the region of interaction of p5 with p3,
effectively prevented association of the two proteins and GTP
hydrolysis. The peptide also significantly reduced (~40%) the
intrinsic GTP hydrolysis by ARD1 and could be useful to assess the
importance of the intrinsic GTPase activity of ARD1 in its biological
activity.
Despite very little amino acid identity, the minimal GAP domain of ARD1 does exhibit similarities to GAPs characterized previously. Indeed, a zinc finger motif has been identified in the recently cloned mammalian ARF GAP (11) as well as in the yeast ARF GAP Gcs1 (46). Replacement of cysteines that are expected to form a zinc finger structure (33) resulted in a complete loss of GAP activity in ARD1 and in ARF GAP (11). The 139CX2CX4CX2C150 motif in the GAP domain of ARD1 also resembles a ferrodoxin signature (iron-sulfur) domain. However, up to 10 µM, zinc or iron sulfate had little effect on GTP binding or GTPase activity of ARD1.2 The exact function of the domain is not known, and the importance of metal binding to zinc finger motifs in ARF GAPs remains to be determined. A clue may be provided by the recent demonstration that Rab GEPs from mammals and yeast, respectively, Mss4 and Dss4, also have a critical zinc binding motif, which may bind to the GTPase at the region that surrounds its effector domain (47). ARD1 also contains a second potential zinc-binding domain (31CX2CX16CXHX2CX2CX12CX375) near the amino terminus, the function of which remains unknown but seems unlikely to involve GAP activity, as its deletion did not affect GTP hydrolysis.
The region sharing partial identity with the second consensus sequence
of Rho/Rac GAPs also appeared to play an important role in p5 GAP
activity. The crystal structure of p50rhoGAP shows that residues
conserved among members of the Rho GAP family, which are confined to
one face of the protein, are likely involved in binding to G proteins
and enhancing GTPase activity (48). We speculate that
Lys158, Pro167, and Leu168 may play
an equivalent role in the GAP domain of ARD1. Replacement of either of
the two arginines, located precisely in this domain, prevented GTP
hydrolysis. It is conceivable that they both contribute to catalysis as
has been suggested for Arg789 and Arg903 in Ras
GAP (49) and for Arg201 in Gs (16). The
crystal structure of Ras associated with the GAP domain of Ras GAP
confirmed that Arg789 of GAP-334 is positioned in the
active site of Ras to neutralize developing charges in the transition
state, whereas Arg903 stabilized the arginine finger motif
(50).
Ras GAP contacts the GTP-binding pocket and the effector domain of Ras,
a loop that undergoes significant conformational change upon GTP
hydrolysis (49, 50). In G, the helical domain interacts with the
GTP-binding pocket, but not with "switch" regions that undergo
conformational change during GTP hydrolysis. Hence, an RGS protein
could accelerate GTP hydrolysis of G
by binding to one or more of
the switch elements and/or by introducing additional arginine(s) to the
catalytic center. As the GTPase activity of ARD1 is much lower than
that of the Ras·Ras GAP complex, it is possible that, like
heterotrimeric G proteins, ARD1 has an RGS-like protein that stimulates
GTP hydrolysis. A recently purified ARF GAP (12), as well as p5
expressed separately in Escherichia coli, however, failed to
increase intrinsic GTPase activity of ARD1.2 Further
studies will be required to identify partners of ARD1 involved in its
alternation between GDP- and GTP-bound forms and to demonstrate the
role of the intrinsic GTPase activity in the intracellular function of
ARD1. In Ras and G
, GTP is hydrolyzed by in-line attack of its
phosphate by a nucleophilic water molecule (35). A glutamine residue
(Gln61 in Ras, Gln204 in G
i1,
and Gln71 in ARF1) located in the amino terminus of switch
II seems to abstract a proton from this attacking water molecule in all
G proteins (35). In ARD1, the equivalent residue is
Lys458, which might also explain the extremely low rate of
GTP hydrolysis by the ARF domain (p3) and the relatively modest rate by
ARD1 itself relative to that of the Ras·GAP complex.
ARFs are possible sites at which phospholipids may function in membrane traffic. The interaction of ARF1 with three different GAPs (9, 10, 12), and phospholipase D (5, 6) has been shown to be PIP2-dependent. Two lipid-binding sites on ARF1 have been identified, and GAP activity depended on occupancy of both sites (9, 51). The effect of PIP2 on nucleotide dissociation from ARF1 has been taken as an evidence of PIP2 binding to ARF1 (51). Furthermore, the crystal structure of ARF1 has revealed that basic amino acids in positions 10, 15, 16, 59, 178, and 181 form a solvent-exposed patch of positive charges (52, 53), which is reminiscent of a pleckstrin-homology domain. Four of these residues were critical for PIP2 binding (54). PIP2 also accelerated dissociation of GTP and GDP from p3 or p8(1), but was not required for GTP hydrolysis induced by p5 (14). Interestingly, three of the four positively charged residues that were implicated in PIP2-dependent GAP binding (54) are not present in ARD1. Phospholipids, however, are known to affect GTP binding to ARFs (10) as well as to ARD1 (22).
The unusual intrinsic GTPase activity of ARD1 may result from the
covalent attachment of a GAP-like domain to the GTPase core of an ARF
protein, by exon shuffling during evolution (55). The mechanism by
which GAPs accelerate the GTPase reaction of monomeric G proteins has
been a matter of considerable debate. Our data seem to favor the
arginine finger hypothesis (35, 50) in which arginines are expected to
stabilize the transition state in GTP hydrolysis. The GAP site of ARD1,
between amino acids 101 and 333, can be divided into a region important
for physical association with the ARF domain (residues 200-333) and a
domain directly involved in stimulation of GTP hydrolysis (residues
101-200). The latter contains a zinc finger motif reminiscent of one
found in ARF GAP and a region that resembles the second consensus
sequence in Rho/Rac·GAPs. The function of the amino-terminal 101 residues, as well as that of amino acids 333-387, remains to be
determined, whereas the hydrophobic -helical structure (residues
387-402) preceding the ARF domain has been demonstrated to have a GDP
dissociation inhibitor-like effect (15). Crystal structures of
Ras·GAP (36, 37, 49) and RGS4 (42) as well as those of G
subunits
(38-41), have revealed that GAPs, RGS, and GAP-like structures contain
exclusively helical secondary structure elements. It will be
interesting to learn whether the GAP domain of ARD1 also has that
structure. Structural information about ARD1 in GDP- and GTP-bound
forms will surely be helpful in understanding the interaction between
the GTP- binding and GAP domains, as well as alterations associated
with the GDP-GTP transition.
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ACKNOWLEDGEMENT |
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We thank Dr. V. C. Manganiello for critical review of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Room 5N-307, Bldg. 10, 10 Center Dr., MSC 1434, National Institutes of Health, Bethesda, MD
20892-1434. Tel.: 301-402-1157; Fax: 301-402-1610; E-mail:
vitalen{at}fido.nhlbi.nih.gov.
1
The abbreviations used are: ARF,
ADP-ribosylation factor; CTA, cholera toxin A subunit; GAP,
GTPase-activating protein; GEP, guanine nucleotide-exchange protein;
GST, GSH S-transferase; GTPS, guanosine
5
-3-O-(thio)triphosphate; G protein, guanine
nucleotide-binding protein; p3, a recombinant protein corresponding to
the carboxyl-terminal 18-kDa ARF domain of ARD1; p5, a recombinant
protein corresponding to the amino-terminal 46-kDa GAP domain of ARD1;
p8, a recombinant ARD1; PAGE, polyacrylamide gel electrophoresis;
PIP2, phosphatidylinositol 4,5-bisphosphate; RGS, regulator
of G protein signaling.
2 N. Vitale, J. Moss, and M. Vaughan, unpublished observations.
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