©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Effector-specifying Domain of Rac Involved in NADPH Oxidase Activation (*)

(Received for publication, April 24, 1995; and in revised form, June 23, 1995)

Cheung H. Kwong Anthony G. Adams Thomas L. Leto

From the Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Production of microbicidal oxidants by phagocytic leukocytes requires activation of a latent NADPH oxidase by the coordinated assembly of a membrane-associated flavocytochrome b, with three cytosolic components, p47, p67, and the low molecular weight GTP-binding protein Rac. Rac1 and Rac2 have 92% sequence identity and are both active in supporting the oxidase, while CDC42Hs, the closest relative to Rac with 70% sequence identity, only weakly supports oxidase activation in vitro. We have used CDC42Hs as a foil to identify residues in Rac that are critical for oxidase activation. Most of the divergent sequences of CDC42Hs could be incorporated into Rac-CDC42Hs chimeric proteins without affecting cell-free NADPH oxidase activity. However, incorporation of the amino-terminal segment of CDC42Hs (residues 1-40), which differs from Rac1 by only four residues (positions 3, 27, 30, and 33), resulted in a marked loss of oxidase activation capacity. Point mutagenesis studies showed that this was due to changes at residues 27 and 30, but not residues 3 and 33. Conversely, incorporation of the amino terminus of Rac1 (residues 1-40) into CDC42Hs increased its activity to that of Rac1, indicating that this terminus contains the effector-specifying domain of Rac. Taken together, these studies show that the difference in the activity between CDC42Hs and Rac1 is due entirely to differences in amino acids at position 27 and 30.


INTRODUCTION

In response to a variety of stimuli, phagocytic white blood cells produce superoxide and a variety of microbicidal oxidants(1) . The superoxide-generating NADPH oxidase of phagocytes consists of both membrane and cytosolic proteins. Cytochrome b is the sole membrane component required for superoxide generation (2, 3, 4) . It is an integral membrane protein complex composed of a 22-kDa protein (alpha-subunit) and a 91-kDa glycoprotein (beta-subunit) that catalyzes the one-electron reduction of oxygen to produce superoxide anion. Studies by Rotrosen et al.(3) and Segal et al.(4) showed that cytochrome b bound FAD and that structural similarities exist between the beta-subunit and the putative NADPH- and FAD-binding sites of the ferredoxin-NADP reductase family. Two of the cytosolic proteins, p47 and p67, were identified based on their deficiencies in most patients with autosomal, cytochrome b-positive forms of chronic granulomatous disease, an inherited disease characterized by the inability of phagocytes to produce superoxide(5, 6, 7, 8, 9) . Both cytosolic proteins become tightly associated with membranes upon cell activation(10, 11) . Several studies have indicated that aside from p47 and p67, at least one other cytosolic protein is required to complement membranes for cell-free production of superoxide(6, 12, 13) . This component was identified as Rac1 in guinea pig macrophages (14) and Rac2 in human neutrophils(15, 16) . These two proteins belong to the Rho subfamily of Ras-related GTP binding proteins and are 95% identical to one another in their amino acid sequences. Rac1 and Rac2 were purified as complexes with Rho-GDI, (^1)although Rho-GDI is not required for in vitro activation of the oxidase(14, 16) . Upon activation of phagocytes, p47, p67, and Rac, but not Rho-GDI migrate to the membrane(10, 11, 17, 18) . Although interactions between Rac and p67 have been observed(19) , studies with chronic granulomatous disease neutrophils suggest that Rac membrane translocation is independent of p47 and p67, and that cytochrome b may stabilize this membrane interaction (20) .

Cytochrome b alone, reconstituted with certain lipids, is capable of superoxide generation in vitro(21) , indicating that it is the sole electron carrier of the oxidase. However, optimal superoxide production also requires the presence of p47 and p67 and Rac(3, 22) . p47and p67 appear to play distinct roles in the regulation of electron flow in NADPH oxidase(23) . A role for Rac in whole cell oxidase activation is supported by studies in intact cells where inhibition of Rac synthesis by antisense DNA in lymphocytes (24) and the expression of a dominant-negative mutant of Rac(N17) in HL-60 cells diminished oxidase activity(25) . Like Ras, Rac1 and Rac2 are active in their GTP-bound states, and their effects on oxidase activation can be regulated by proteins that modulate guanine nucleotide exchange and hydrolysis(26, 27, 28) .

Rac and CDC42Hs have been implicated in various aspects of reorganization of the actin-based cytoskeleton. Rac participates in growth factor-induced membrane ruffling in fibroblasts(29) , and CDC42Hs functions in the polarization of actin filaments preceding bud site assembly in yeast(30) . CDC42Hs, with 72% homology to Rac1, has a low capacity for oxidase activation(16) . Confirmation that CDC42Hs and Rac have distinct effectors is shown by the fact that human CDC42Hs, but neither Rac1 nor Rac2, complements the lethal mutation of CDC42Sc in Saccharomyces cerevisiae(31) . Alignment of the amino acid sequences of Rac1 and Rac2 with that of CDC42Hs revealed three regions containing most of the structural divergence between these proteins, suggesting that one or more of these sequences may determine the functional specificity of these Ras relatives(16) .

To date, most studies addressing structure-function relationships of Rac in oxidase activation have been based on analogy to Ras, which exhibits only 30% homology to Rac and other Rho subfamily members. Consistent with the Ras paradigm, point mutations within the amino terminus of Rac reduced its oxidase-activating capacity, although these experiments have provided only indirect evidence that the effector-specifying domain of Rac is located within the amino terminus (19, 32) . In the present study, we systematically compared a series of chimeras produced from Rac1 and CDC42Hs in order to determine the domain of Rac that functionally distinguishes it from CDC42Hs. This domain was anticipated to confer a Rac-like oxidase activating capacity to CDC42Hs. Analysis of these chimeras has allowed us to assess the impact of mutational changes throughout various regions of Rac.


EXPERIMENTAL PROCEDURES

Materials

Chemicals, enzymes, and molecular biology reagents were obtained from the following sources: pGEX2T vector (Pharmacia Biotech Inc.); SacII and BssHII (New England Biolabs); ferricytochrome c (horse heart; type VI), FAD, superoxide dismutase, phenylmethylsulfonyl fluoride, and bovine thrombin (1819 NIH units/mg; Calbiochem); NADPH, GTPS, BamHI, EcoRI (Boehringer Mannheim); dithiothreitol (Schwarz/Mann Biotech, Cleveland, OH); Bio-Rad protein assay kit (Bio-Rad); Ecoscint A (National Diagnostics, Atlanta, GA); Novex precast polyacrylamide gels (Novel Experimental Technology, San Diego, CA); PCR kit with Taq polymerase (Perkin Elmer Cetus); subcloning efficiency DH5alpha Escherichia coli (Life Technologies Inc.). Recombinant proteins rp47 and rp67 were produced and purified from a baculovirus expression system as described (13) .

Methods

Expression and Purification of Recombinant Rac1, CDC42Hs, and Their Chimeras

Expression of glutathione S-transferase fusion proteins and cleavage of GTP-binding proteins from glutathione S-transferase was performed essentially as described previously(16) . cDNA of Rac1, CDC42Hs, or chimeric cDNA were subcloned using oligonucleotide linkers containing BamHI (5`) and EcoRI (3`) sites to enable forced orientation in the bacterial expression vector pGEX2T. Rac1/CDC42Hs chimeric cDNAs were produced by a PCR-based ``gene splicing by overlap extension'' technique described by Horton et al.(33) . Site-directed mutagenesis was performed with primers that contained the desired mutations, and the final cDNA insert was produced by a second stage overlap extension amplification technique. The entire sequences of all cDNA inserts were confirmed prior to expression experiments. Expression and purification of bacterial fusion proteins from isopropyl-1-thio-beta-D-galactopyranoside-induced E. coli, as well as thrombin-mediated release of GTP-binding proteins from glutathione S-transferase were performed as described previously(16) .

Protein Quantitation and Electrophoresis

The concentration of GTP-binding proteins was determined with the Bio-Rad protein assay kit using bovine serum albumin as a standard. Electrophoresis (SDS-polyacrylamide gel electrophoresis) was performed with precast SDS-Tris-glycine 8-16% polyacrylamide gels.

NADPH Oxidase Reconstitution Studies

Cell-free superoxide production was measured in 96-well plates with a Molecular Devices Thermomax microplate reader (Menlo Park, CA) essentially as described earlier(16) . Typical reactions (100 µl) contained 5 10^5 cell equivalents of deoxycholate-solubilized neutrophil membranes(6) , 5 µg/ml pure recombinant p47 and p67(13) , and variable amounts of E. coli-expressed Rac1, CDC42Hs, or Rac1/CDC42Hs chimeric proteins. Reaction mixtures contained 75 mM potassium phosphate, pH 7, 0.2 mM cytochrome c, 10 mM MgCl(2), 10 µM FAD, 5 µM GTPS, and 200 µM NADPH. The reactions were initiated by addition of arachidonic acid to yield a final concentration of 40 µM. Control reactions contained 2.5 µg (12.5 units) of superoxide dismutase. Superoxide generation was calculated from the superoxide dismutase-inhibitable changes in cytochrome c absorbance observed at 550 nm, with a 1-nm bandwidth filter, using an extinction coefficient of 21.1 mM. Absorbance readings were taken for 10 min at 30-s intervals following addition of arachidonic acid. Maximum rates of superoxide generation were calculated from the linear region of the absorbance curves by least-squares fit and converted to micromoles of superoxide generated/minute/milligram of membrane protein.

[S]GTPS Binding Assay

1 µg of thrombin-released GTP-binding protein (5 µl) was incubated with 32 µl of nucleotide exchange buffer (50 mM Tris, pH 7.5, 1 mM dithiothreitol, 0.3 M NaCl, 1 mM MgCl(2), and 2 mM EDTA) and 3.3 µl of [S] GTPS (13.9 Ci/mmol, 1.25 µCi/µl) for 10 min in room temperature. Then 4 µl of 100 mM MgCl(2) was added to promote nucleotide binding. 20 min later, 22 µl of this reaction mixture was drawn through a Millipore HA filter (2.5-cm disc; over Millipore Sampling Manifold; Marlborough, MA) presoaked with Washing Buffer (50 mM Tris, pH 7.4, 1 mM dithiothreitol, 0.3 M NaCl, and 10 mM MgCl(2)). After washing each filter twice with 10 ml of Washing Buffer, the radioactivity of protein-bound [S]GTPS was determined by scintillation counting in 10 ml of Ecoscint A. Nonspecific binding of [S]GTPS to the protein or filter (representing <3% of specific binding) was determined from binding assays performed in the presence of a 1000-fold excess of unlabeled GTPS.


RESULTS AND DISCUSSION

In our previous report(16) , we identified three regions (residues 27-52, 130-150, and 174-190) that are relatively conserved between Rac1 and Rac2, but divergent in CDC42Hs (Fig. 1). Rac and CDC42 clearly have distinct effectors, since human CDC42 but not Rac is capable of complementing a lethal mutation of CDC42 in S. cerevisiae, and Rac but not CDC42 supports oxidase activation. Our working hypothesis is that the domain of Rac that confers functional specificity is conserved between Rac1 and Rac2 but is structurally divergent in CDC42Hs. In order to assess the contributions of various regions of Rac1 in their interaction with the NADPH oxidase, selected regions of Rac1 were substituted sequentially with the corresponding regions of CDC42Hs (Fig. 2A). Substitution of residues 41-52 of Rac1 (Chimera B), residues 53-173 (Chimera C), and residues 41-192 (Chimera E) with the corresponding residues of CDC42Hs had no discernible effect on its ability to activate the oxidase in a cell-free assay (Fig. 2B). Together, these three active chimeras incorporated a majority of the sequence differences found in CDC42Hs. In contrast, substitution of the NH(2)-terminal 40 amino acid segment of Rac1 with that of CDC42Hs resulted in a protein (Chimera A) that had activity reduced to that of CDC42Hs. The lower activity of Chimera D raised the possibility that the terminal 17 amino acids of Rac represent another functional domain. However, this possibility was not supported by the observation that Chimera E, which lacks an even more extensive COOH-terminal Rac sequence, had activity comparable to that of wild type Rac1. The reduced activity of Chimera D may reflect how well sequence difference at the COOH terminus are complemented by the rest of protein molecule for proper folding and function.


Figure 1: Comparison of amino acid sequence of Rac1 with that of CDC42Hs. Identical amino acids are represented as soliddots. A gap that optimized the alignment of CDC42Hs is denoted by a dash.




Figure 2: A, schematic depiction of chimeric Ras-related GTP-binding proteins constructed from Rac1 and CDC42Hs. Shadedsegment, CDC42Hs; solidsegments, Rac1. B, comparison of oxidase-activating capacities of chimeric Rac1/CDC42Hs proteins. Proteins studied were: Rac1 (), CDC42Hs (bullet), Chimera A (), Chimera B (), Chimera C (), Chimera D (black square), and Chimera E (up triangle, filled). The data are representative of a minimum of three experiments and are averages of duplicates.



To further explore the role of the amino terminus, we replaced the NH(2)-terminal 40 amino acids of CDC42Hs with those of Rac1. The resulting protein (Chimera E) had activity equivalent to that of Rac1 (Fig. 2B), indicating that this segment of Rac1 contains the effector domain of Rac specifying oxidase activation. This unique Rac segment may interact directly with its effector, or alternatively, may act intramolecularly to affect the interaction of other region(s) of Rac with its effector. Our study did not distinguish between these two possibilities. In this connection, it is interesting to note that a Rac peptide spanning residues 18-40, at a concentration of 100 µM, did not inhibit cytochrome b dependent-NADPH oxidase activity in a cell-free oxidase assay containing neutrophil membranes and cytosol (data not shown). The amino terminus of Rac1 (residues 1-40) is identical to that of Rac2 and differs from CDC42Hs by only four amino acids (residues 3, 27, 30, and 33). Point mutants of Rac1 were made by substituting each of these residues with the corresponding residues of CDC42Hs, resulting in Rac point mutants A3T (alanine to threonine), A27K (alanine to lysine), G30S (glycine to serine), and I33V (isoleucine to valine). The activities of A3T and I33V were comparable to that of wild type Rac1 (Fig. 3), indicating that these amino acid substitutions were conservative with respect to their effect on oxidase activation. In contrast, mutations at either residue 27 (A27K) or residue 30 (G30S) reduced the activity of the point mutants to that of CDC42Hs (Fig. 3).


Figure 3: Activity of point mutants of Rac1. Proteins studied were: Rac1 (), CDC42Hs (bullet), A3T (black square, with substitution of alanine at residue 3 of Rac1 by threonine), I33V (up triangle, filled, with substitution of isoleucine at residue 33 by valine), G30S (, with substitution of glycine at residue 30 by serine), and A27K (, with substitution of alanine at residue 27 by lysine). Data are representative of three experiments and are averages of duplicates.



Until now, only a limited number of point mutants of Rac have been studied with regard to its oxidase effector function. Based on the characterization of the Ras effector region, Diekmann et al.(19) showed that point mutations at residue 35, 38, or 40 of Rac inhibited cell-free oxidase activation and block the interaction of Rac with p67, its putative target. Similarly, Xu et al.(32) showed that point mutations at residue 28, 35, 36, or 38 diminished its activity in oxidase activation. In the present study, Chimeras A-E contain extensive mutations throughout Rac that had not been studied previously. Chimera E contains most of the sequence divergence between Rac and CDC42Hs, yet this protein retained the oxidase-activating capacity of Rac. This study showed that the Rac-like oxidase regulating activity could be conferred to CDC42Hs when only four amino acids within the amino terminus of CDC42Hs were substituted by the corresponding residues in Rac (Chimera E). Of these, only two residues (27 and 30) account for the difference in oxidase activation between Rac and CDC42Hs; these were located within a region analogous to the Ras effector domain.

As necessary controls for loss of function observed in some of these proteins, the integrity of the thrombin-released proteins was assessed by SDS-polyacrylamide gel electrophoresis, which confirmed that the recombinant proteins were generated with the predicted size (approximately 21-22 kDa) and of high purity (>90%; Fig. 4). Furthermore, to rule out the possibility that the reduced activities of Chimera A and point mutants G30S and A27K were due to impairment of guanine nucleotide binding, we compared their abilities to bind [S]GTPS. The results given in Fig. 5showed that these mutations of Rac1 did not affect the amount of guanine nucleotide binding. These studies also showed that the specific activity of Rac1 in oxidase activation is actually much higher than what is shown in Fig. 2B, since only 11% of the recombinant Rac1 bound [S]GTPS, whereas 93% of recombinant CDC42Hs had binding activity (data not shown).


Figure 4: Coomassie Blue-stained electrophoretogram (8-16% Tris-glycine-SDS-polyacrylamide gel electrophoresis) of recombinant Rac1/CDC42Hs proteins. Lane1, Rac1; lane2, CDC42Hs; lanes 3-7, Chimeras A-E; lane8, A3T; lane9, A27K; lane10, G30S; lane11, I33V.




Figure 5: Comparison of [S]GTPS binding activity of 0.5 µg of Rac1, Chimera A, and Rac point mutants. dpm, protein-bound radioactivity of [S]GTPS, expressed as disintegrations/minute. Each bar represents the mean of data from two experiments. The interval represents the range. Nonspecific binding, as determined in the presence of 100-fold excess of cold GTPS, accounted for <3% of the total binding.



The identification of the amino terminus of Rac as being essential for optimal oxidase activation in the present studies is analogous to studies that mapped effector sites within Ras, where the amino terminus (residues 21-31 and 45-54) was shown to be essential for Ras transforming activity(34) . Consistent with this, transforming activity could be conferred to Rho (a protein with only 30% homology) when residues 23-46 were substituted with the corresponding sequence of Ras, indicating that this region contains the effector-specifying domain of Ras(35) . Furthermore, a peptide containing residues 17-44 of Ras inhibits its interaction with its effector, Raf(36) .

At this time it is unclear to what extent the large body of structural and functional information available on Ras can be applied to members of the Rho subfamily, including Rac. The identification of critical Rac effector domain residues in this report and others (residues 28, 35, 36, 38, 40) (19, 32) is consistent with some aspects of the Ras model. Two regions in Ras undergo significant changes in conformation upon binding of a non-hydrolyzable GTP analog to Ras. These are residues 30-38 (switch I region) and residues 60-76 (switch II region)(37) . Both regions form a continuous strip on the surface most likely to be recognized by effectors. The recent report by Xu et al.(32) showed that a mutation at residue 61 of Rac (substitution of glutamine by leucine) can overcome an inactivating mutation at residue 38 (aspartic acid to alanine), raising the possibility that the effector-specifying domain of Rac includes both switch regions. Due to the extensive structural homology between Rac and CDC42Hs around residue 61(switch II region), our studies do not exclude this possibility. One important distinction between Ras and members of the Rho subfamily concerns their sites for interaction with their respective GTPase-activating proteins (GAPs). The binding site of RasGAP to Ras appears to overlap with its amino-terminal ``effector domain,'' while mutations in the effector-specifying region of Rac do not affect its responsiveness to its GAPs(32) .

The roles of the other regions of Rac that are divergent from CDC42Hs remain to be determined. Substitution of the region spanning residues 41-52 of Rac with the corresponding divergent sequence of CDC42Hs did not diminish cell-free oxidase activity (Chimera B, Fig. 2B). The divergent region at the carboxyl terminus (residues 174-192), which also exhibits significant sequence differences between Rac1 and Rac2, is modified by geranylgeranylation, a post-translational process required for Rac's association with both membranes and GDP/GTP exchange proteins(38) . However, this processing is not required for cell-free oxidase reconstitution, since the unmodified forms of both recombinant Rac1 and Rac2 support oxidase activity(14, 15, 16, 39, 40) . This region nonetheless does serve an important oxidase-related function even in the absence of geranylgeranylation, since the truncated form of Rac lacking residues 175-195 does not support cell-free oxidase reconstitution (data not shown). One or more of these divergent regions between Rac and CDC42Hs may specify interactions with several regulator proteins, such as p120, RhoGAP, and Bcr. p120 is a protein kinase that binds to CDC42Hs, but not to Rac(41) . RhoGAP is a GTPase-activating protein exhibiting a striking preference for CDC42(42) , while Bcr is another GAP implicated as a down-regulator of the neutrophil respiratory burst(43) .

In conclusion, we present direct evidence in this study that the effector-specifying domain of Rac in NADPH oxidase activation is located in the amino terminus. Furthermore, we demonstrate that residues 27 and 30 alone account for the difference in activity between Rac1 and CDC42Hs in oxidase activation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: GDI, GDP dissociation inhibitor; GTPS, guanosine 5`-3-O-(thio)triphosphate.


REFERENCES

  1. Clark, R. A. (1990) J. Infect. Dis. 161,1140-1147 [Medline] [Order article via Infotrieve]
  2. Knoller, S., Shpungin, S., and Pick, E. (1991) J. Biol. Chem. 266,2795-2804 [Abstract/Free Full Text]
  3. Rotrosen, D., Yeung, C. L., Leto, T. L., Malech, H. L., and Kwong, C. H. (1992) Science 256,1459-1462 [Medline] [Order article via Infotrieve]
  4. Segal, A. W., West, I., Wientjes, F., Nugent, J. H. A., Chavan, A. J., Haley, B., Garcia, R. C., Rosen, H., and Scrace, G. (1992) Biochem. J. 284,781-788 [Medline] [Order article via Infotrieve]
  5. Leto, T. L., Lomax, K. J., Volpp, B. D., Nunoi, H., Sechler, J. M. G., Nauseef, W. M., Clark, R. A., Gallin, J. I., and Malech, H. L. (1990) Science 248,727-730 [Medline] [Order article via Infotrieve]
  6. Nunoi, H., Rotrosen, D., Gallin, J. I., and Malech, H. L. (1988) Science 242,1298-1301 [Medline] [Order article via Infotrieve]
  7. Lomax, K. J., Leto, T. L., Nunoi, H., Gallin, J. I., and Malech, H. L. (1989) Science 245,409-412 [Medline] [Order article via Infotrieve]
  8. Volpp, B. D., Nauseef, W. M., and Clark, R. A. (1988) Science 242,1295-1297 [Medline] [Order article via Infotrieve]
  9. Volpp, B. D., Nauseef, W. M., Donelson, J. E., Moser, D. R., and Clark, R. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,7195-7199 [Abstract]
  10. Heyworth, P. G., Curnutte, J. T., Nauseef, W. M., Volpp, B. D., Pearson, D. W., Rosen, H., and Clark, R. A. (1991) J. Clin. Invest. 87,352-356 [Medline] [Order article via Infotrieve]
  11. Clark, R. A., Volpp, B. D., Leidal, K. G., and Nauseef, W. M. (1990) J. Clin. Invest. 85,714-721 [Medline] [Order article via Infotrieve]
  12. Bolscher, B. G. J. M., Zwieten, V., Kramer, I. M., Weening, R. S., Verhoeven, A. J., and Roos, D. (1989) J. Clin. Invest. 83,757-763 [Medline] [Order article via Infotrieve]
  13. Leto, T. L., Garrett, M. C., Fujii, H., and Nunoi, H. (1991) J. Biol. Chem. 266,19812-19818 [Abstract/Free Full Text]
  14. Abo, A., Pick, E., Hall, A., Totty, N., Teahan, C. G., and Segal, A. W. (1991) Nature 353,668-670 [CrossRef][Medline] [Order article via Infotrieve]
  15. Knaus, U. G., Heyworth, P. G., Evans, T., Curnutte, J. T., and Bokoch, G. M. (1991) Science 254,1512-1515 [Medline] [Order article via Infotrieve]
  16. Kwong, C. H., Malech, H. L., Rotrosen, D., and Leto, T. L. (1993) Biochemistry 32,5711-5717 [Medline] [Order article via Infotrieve]
  17. Abo, A., Webb, M. R., Grogan, A., and Segal, A. W. (1994) Biochem. J. 298,585-591 [Medline] [Order article via Infotrieve]
  18. Philips, M. R., Pillinger, M. H., Staud, R., Volker, C., Rosenfeld, M. G., Weissmann, G., and Stock, J. B. (1993) Science 259,977-980 [Medline] [Order article via Infotrieve]
  19. Diekmann, D., Abo, A., Johnston, C., Segal, A. W., and Hall, A. (1994) Science 265,531-533 [Medline] [Order article via Infotrieve]
  20. Heyworth, P. G., Bohl, B. P., Bokoch, G. M., and Curnutte, J. T. (1994) J. Biol. Chem. 269,30749-30752 [Abstract/Free Full Text]
  21. Koshkin, V., and Pick, E. (1993) FEBS Lett. 327,57-62 [CrossRef][Medline] [Order article via Infotrieve]
  22. Abo, A., Boyhan, A., West, I., Thrasher, A. J., and Segal, A. W. (1992) J. Biol. Chem. 267,16767-16770 [Abstract/Free Full Text]
  23. Cross, A. and Curnutte, J. T. (1995) J. Biol. Chem. 270,6543-6548 [Abstract/Free Full Text]
  24. Dorseuil, O., Vazquez, A., Lang, P., Bertoglio, J., Gacon, G., and Leca, G. (1992) J. Biol. Chem. 267,20540-20542 [Abstract/Free Full Text]
  25. Gabig, T. G., Crean, C. D., Mantel, P. L., and Rosli, R. (1995) Blood 85,804-811 [Abstract/Free Full Text]
  26. Mizuno, T., Kaibuchi, K., Ando, S., Musha, T., Hiraoka, K., Takaishi, K., Asada, M., Nunoi, H., Matsuda, I., and Takai, Y. (1992) J. Biol. Chem. 267,10215-10218 [Abstract/Free Full Text]
  27. Heyworth, P. G., Knaus, U. G., Settleman, J., Curnutte, J. T., and Bokoch, G. M. (1993) Mol. Biol. Cell 4,1217-1223 [Abstract]
  28. Voncken, J. W., Schaick, H. V., Kaartinen, V., Deemer, K., Coates, T., Landing, B., Pattengale, P., Dorseuil, O., Bokoch, G. M., Groffen, J., and Heisterkamp, N. (1995) Cell 80,719-728 [Medline] [Order article via Infotrieve]
  29. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992) Cell 70,401-410 [Medline] [Order article via Infotrieve]
  30. Johnson, D. I., and Pringle, J. R. (1990) J. Cell Biol. 111,143-152 [Abstract]
  31. Shinjo, K., Koland, J. G., Hart, M. J., Narasimhan, V., Johnson, D. I., Evans, T., and Cerione, R. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,9853-9857 [Abstract]
  32. Xu, X., Barry, D. C., Settleman, J., Schwartz, M. A., and Bokoch, G. M. (1994) J. Biol. Chem. 269,23569-23574 [Abstract/Free Full Text]
  33. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77,61-68 [CrossRef][Medline] [Order article via Infotrieve]
  34. Zhang, K., Noda, M., Vass, W. C., Papageorge, A. G., and Lowy, D. R. (1990) Science 249,162-165 [Medline] [Order article via Infotrieve]
  35. Self, A. J., Paterson, H. F., and Hall, A. (1993) Oncogene 8,655-661 [Medline] [Order article via Infotrieve]
  36. Warne, P. H., Viciana, P. R., and Downward, J. (1993) Nature 364,352-355 [CrossRef][Medline] [Order article via Infotrieve]
  37. Milburn, M. V., Tong, L., DeVos, A. M., Brunger, A., Yamaizumi, Z., Nishimura, S., and Kim, S.-H. (1990) Science 247,939-945 [Medline] [Order article via Infotrieve]
  38. Ando, S., Kaibuchi, K., Sasaki, T., Hiraoka, K., Nishiyama, T., Mizuno, T., Asada, M., Nunoi, H., Matsuda, I., Matsuura, Y., Polakis, P., McCormick, F., and Takai, Y. (1992) J. Biol. Chem. 267,25709-25713 [Abstract/Free Full Text]
  39. Heyworth, P. G., Knaus, U. G., Xu, X., Uhlinger, D. J., Conroy, L., Bokoch, G. M., and Curnutte, J. T. (1993) Mol. Biol. Cell 4,261-269 [Abstract]
  40. Kreck, M. L., Uhlinger, D. J., Tyagi, S. R., Inge, K. L., and Lambeth, J. D. (1994) J. Biol. Chem. 269,4161-4168 [Abstract/Free Full Text]
  41. Manser, E., Leung, T., Salihuddin, H., Zhao, Z. S., and Lim, L. (1994) Nature 367,40-46 [CrossRef][Medline] [Order article via Infotrieve]
  42. Ridley, A. J., Self, A. J., Kasmi, F., Paterson, H. F., Hall, A., Marshall, C. J., and Ellis, C. (1993) EMBO J. 12,5151-5160 [Abstract]
  43. Voncken, J. W., van Schaick, H., Kaartinen, V., Deemer, K., Coates, T., Landing, B., Pattengale, P., Dorseuil, O., Bokoch, G. M., Groffen, J., and Heisterkamp, N. (1995) Cell 80,719-728 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.