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.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

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, N88p8, N200p8, N304p8, and N387p8. 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/N88p8, pGEX5G/LIC/N200p8, pGEX5G/LIC/N304p8, and pGEX5G/LIC/N387p8. 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 Amp^r 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 GTPS 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.

	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.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effect of affinity-purified antibodies on p5-stimulated GTPase activity of p3. ARD1 contains an amino-terminal GAP domain (p5) and a carboxyl-terminal, GTP-binding ARF domain (p3). Molecular masses of p3, p5, and p8 expressed as recombinant proteins are indicated. 55 pmol of p3 (~1 µg) was incubated with [-32P]GTP for 40 min at 30 °C in 60 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 and then for 30 min at 4 °C with the indicated amount of affinity-purified antibodies raised against recombinant p3 or the undecapeptide corresponding to the carboxyl terminus of p3 (CtARD1) (200 µl, final volume). 110 pmol of p5 (~5 µg) was incubated (30 min at 4 °C) with the indicated amount of affinity-purified antibody raised against recombinant p5 or the undecapeptide corresponding to the amino terminus of p5 (NtARD1) before addition to p3 with [-32P]GTP bound. GTP hydrolysis during the next 60 min at room temperature is expressed as the increase in GDP bound to p3 relative to that during incubation with p5 without antibody (=100%) based on PhosphorImager quantification. Data are means of duplicate values ± one-half the range. Error bars smaller than symbols are not shown. Each experiment was repeated at least once.

View larger version (61K): [in this window] [in a new window]   Fig. 2.   Effect of amino-terminal deletions on the intrinsic GTPase activity of ARD1. Panel A, deletion of 88, 200, 304, or 387 amino acids from the amino terminus of ARD1 yielded N88p8, N200p8, N304p8, and N387p8. Panel B, 55 pmol of p3, ARD1 (p8) or mutated ARD1 with [-32P]GTP bound was incubated for 60 min at room temperature before bound nucleotides were separated by TLC. Positions of standard GTP and GDP are indicated on the left. Data are duplicate assays representative of at least three different protein preparations. Panel C, after the protein (70 pmol) was incubated with 100 µM GTP or GTPS, stimulation of cholera toxin-catalyzed ADP-ribosylagmatine formation was assayed for 60 min at 30 °C. ARD activity is the difference between CTA-catalyzed formation of [14C]ADP-ribosylagmatine without and with ARD1 protein (nmol/h). Data are means of quadruplicates ± one-half the range. These findings were replicated twice with two independent preparations of proteins.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Intrinsic GTPase activity of amino-terminal deletion mutants of ARD1. Deletion of 101, 124, 146, or 161 amino acids from the amino terminus of ARD1 yielded N101p8, N124p8, N146p8, and N161p8, respectively. 55 pmol of ARD1 or mutated protein with [-32P]GTP bound was incubated for 60 min at room temperature before separation of bound nucleotides by TLC. GTPase activity is expressed as the increase in bound GDP relative to the increase of GDP bound to p8 (=100%) based on PhosphorImager quantification. Data are means of duplicates ± one-half the range in one experiment representative of two with two independent protein preparations.

View larger version (68K): [in this window] [in a new window]   Fig. 4.   Effect of amino- and carboxyl-terminal deletion mutants of p5 on GTPase activity of p3. Panel A, deletion of 88, 200, or 304 amino acids from the amino terminus of p5 yielded N88p5, N200p5, and N304p5; deletion of 69, 191, or 293 amino acids from the carboxyl terminus of p5 yielded C69p5, C191p5, and C293p5, respectively. Panel B, 55 pmol of p3 (~1 µg) with [-32P]GTP bound was incubated with 110 pmol (30 µl) of p5, mutated proteins, or water (control) for 60 min at room temperature before separation of bound nucleotides by TLC. Positions of standard GTP and GDP are indicated on the left. Data are means of values from duplicate assays representative of those obtained with three independent protein preparations. Panel C, after 70 pmol of p3 was incubated with 100 µM GTP or GTPS for 30 min at 30 °C and then with 30 µl (140 pmol) of p5, mutated protein, or water (control) for 20 min at room temperature, ARD stimulation of cholera toxin-catalyzed ADP-ribosylagmatine formation was assayed for 60 min at 30 °C as described in Fig. 2. Toxin activity without added p3 in each condition was subtracted. Data are means of values from quadruplicate assays ± one-half the range in one experiment representative of two with two different protein preparations.

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 N200GST-p5 both clearly interacted physically with p3, whereas N304GST-p5 did not (Fig. 5). C69GST-p5, but not C191GST-p5 and C293GST-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 N200GST-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.

View larger version (61K): [in this window] [in a new window]   Fig. 5.   Binding of the ARF domain (p3) of ARD1 to immobilized GST-p5 mutant proteins. GSH-Sepharose beads (200 µl) with bound N88GST-p5, N200GST-p5, N304GST-p5, C69GST-p5, C191GST-p5, or C293GST-p5 (100 µg) were incubated for 1 h at room temperature with 50 µg of p3 in 1 ml of 20 mM Tris, pH 8.0, with 2.5 mM EDTA. Beads were then washed four times with 20 volumes (~4 ml) of phosphate-buffered saline before elution of bound proteins in 0.5 ml of 10 mM GSH with 50 mM Tris, pH 8.0. Samples (~3 µg) of the eluted proteins were separated by SDS-PAGE in 4-20% gels and stained with Coomassie Blue. Positions of protein standards are on the right. The molecular mass of p3 is 18 kDa. These findings have been replicated twice with two independent preparations of proteins.

View larger version (53K): [in this window] [in a new window]   Fig. 6.   Effect of mutagenesis on physical and functional interaction between the two domains of ARD1. Panel A, three mutant proteins of p5 [(K210G/H211A)p5, (H214A/K215G/H216G)p5, and (R249A/K250G)p5] were synthesized, respectively, using the primers 5-TGCAAAGAATATGGAGCCCAGGGTCACAAGCAT-3, 5-GGAAAACACCAGGG TGCCGGGGCTTCAGTATTGGAACCA-3, and 5-ATCTCAGATTATTCCGCAGGATTAGTTGGAATTGTG-3 (differences from original sequence are underlined). The proteins, expressed as GST fusion proteins, were used to estimate physical interaction with p3 as described in Fig. 5. GSTp-5 (lane 1), (K210G/H211A)GST-p5 (lane 2), (H214A/K215G/H216G)GST-p5 (lane 3) interacted with p3, whereas (R249A/-K250G)GST-p5 did not (lane 4). Positions of protein standards are on the right (lane 5). The molecular mass of p3 is 18 kDa. Data are representative of those from three independent experiments. Panel B, 55 pmol of p3 with [-32P]GTP bound were incubated with 30 µl of water (lane 1) or 110 pmol of p5 (lane 2), (K210G/H211A)p5 (lane 3), (H214A/K215G/H216G)p5 (lane 4), or (R249A/K250G)p5 (lane 5) for 60 min at room temperature, before separation of bound nucleotides by TLC with duplicate samples in each lane. Data are representative of those obtained with at least two independent protein preparations.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effect of synthetic peptides that correspond to domains involved in physical interaction on GTPase activity. 55 pmol of p3 (panel A), ARF1(39-45p3) (panel B), and ARD1 (panel C) with [-32P]GTP bound were incubated with 110 pmol of p5 (5 µg) or water (30 µl) with the indicated concentration of peptide. rARF1p and p3p are tridecapeptides corresponding to the effector regions of ARF1 and p3, respectively. p5p is a dodecapeptide corresponding to amino acids 245-256 of ARD1, and Rp5p is a dodecapeptide with the same residues in random sequence. GTPase activity is expressed as the increase in bound GDP relative to that without peptide (=100%), based on PhosphorImager quantification. Data are means of values from three experiments performed in duplicate ± one-half the range. Error bars smaller than symbols are not shown.

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, N124p8, and N146p8 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.

View this table: [in this window] [in a new window]   Table I Effect of mutation of cysteines in a zinc finger motif in the p5 domain on physical and functional interactions with p3 p5 mutant proteins were created using the indicated oligonucleotides, with differences from the original sequence underlined. Cysteines were replaced with alanine, resulting in (C139A)p5, (C142A)p5, (C147A)p5, (C150A)p5, (C178A)p5, and (C190A)p5. 55 pmol of p3 (1 µg) with [-32P]GTP bound was incubated for 60 min, at room temperature, with 110 pmol (5 µg) of p5 or mutant before separation of bound nucleotide by TLC. Amounts of bound GTP and GDP were quantified by PhosphorImaging. GAP activity of mutant proteins is the increase in bound GDP caused by incubation with a p5 mutant relative to that with p5 (=100%). In the presence of p5, ~60% of GTP bound to p3 was hydrolyzed to GDP. Data are means of values from three experiments carried out in duplicate ± one-half the range. GSH-Sepharose beads (200 µl) with bound GST-p5 or GST-mutant p5 (100 µg) were incubated for 1 h, at room temperature, with 50 µg of p3, in 1 ml of 20 mM Tris, pH 8.0, 2.5 mM EDTA. Beads were washed, and bound proteins were eluted as described in Fig. 5. Samples (2.5 µg) of eluted proteins were separated by SDS-PAGE in 4-20% gels and stained with Coomassie Blue. Interacting p3 was quantified by densitometry (Color Onescanner, Macintosh). The amount of p3 bound to GST-mutant p5 proteins is expressed relative to that bound to GST-p5 (=100%). Data are means of values from two experiments carried out in duplicate ± one-half the range.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

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, p120^GAP, 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 G_s (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.