Rac “Insert Region” Is a Novel Effector Region That Is Implicated in the Activation of NADPH Oxidase, but Not PAK65
The small GTPase Rac assembles with the cytosolic p47phox and p67phox and the membrane-associated flavocytochrome b558 to form the multicomponent respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26-45), homologous to an effector region in Ras, was previously shown to interfere with Rac binding to the oxidase. Herein we have elucidated an additional region in Rac involved in regulating oxidase activity. Rho family small GTPases contain a 12-amino acid “insert” region (residues 124-135) that is not present in Ras. Point mutations in and deletion of this region were constructed and used for in vitro studies of the activation of PAK65 and NADPH oxidase. Apparent binding constants (based on EC50 values) of the mutant Rac proteins for the oxidase are at least 13-25-fold higher than for wild-type Rac. Mutations in the insert region versus the 26-45 effector region resulted in distinct kinetic consequences, pointing to different roles for these two protein regions: mutations in the insert region but not the 26-45 effector region resulted in an increase in the EC50 for p67phox. Although mutations in the 26-45 amino acid effector region showed markedly diminished activation of both PAK and the NADPH oxidase, insert region mutations did not affect activation of PAK. We propose that the combinatorial use of the 26-45 effector region and the insert region provides the Rho family GTPases with versatility in their specificity for several downstream targets. The small GTPase Rac assembles with the cytosolic p47phox and p67phox and the membrane-associated flavocytochrome b558 to form the multicomponent respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26-45), homologous to an effector region in Ras, was previously shown to interfere with Rac binding to the oxidase. Herein we have elucidated an additional region in Rac involved in regulating oxidase activity. Rho family small GTPases contain a 12-amino acid “insert” region (residues 124-135) that is not present in Ras. Point mutations in and deletion of this region were constructed and used for in vitro studies of the activation of PAK65 and NADPH oxidase. Apparent binding constants (based on EC50 values) of the mutant Rac proteins for the oxidase are at least 13-25-fold higher than for wild-type Rac. Mutations in the insert region versus the 26-45 effector region resulted in distinct kinetic consequences, pointing to different roles for these two protein regions: mutations in the insert region but not the 26-45 effector region resulted in an increase in the EC50 for p67phox. Although mutations in the 26-45 amino acid effector region showed markedly diminished activation of both PAK and the NADPH oxidase, insert region mutations did not affect activation of PAK. We propose that the combinatorial use of the 26-45 effector region and the insert region provides the Rho family GTPases with versatility in their specificity for several downstream targets. INTRODUCTIONDuring the respiratory burst, neutrophils and other phagocytic cells reduce molecular oxygen to generate superoxide anion, with subsequent production of secondary products such as hydrogen peroxide and hydroxyl radical, all of which participate in microbial killing. The enzyme that initiates the respiratory burst, the NADPH oxidase, is a multicomponent enzyme that utilizes reducing equivalents from NADPH to reduce oxygen to superoxide (reviewed in Ref. 1Quinn M. J. Leukocyte Biol. 1995; 58: 263-276Crossref PubMed Scopus (56) Google Scholar). The oxidase is dormant in resting cells but becomes activated in response to microorganisms or to a variety of soluble agonists. Activation involves the assembly of three cytosolic proteins, p47phox, p67phox, and the GTP-binding protein Rac (1 or 2), with the membrane-bound flavocytochrome b558. The flavocytochrome contains flavin, heme, and the NADPH binding site but catalyzes oxygen reduction only when the cytosolic components are associated. In response to cell activation, p47phox, p67phox, and Rac translocate to the cytochrome (2Heyworth P.G. Curnutte J.T. Nauseef W.M. Volpp B.D. Pearson D.W. Rosen H. Clark R.A. J. Clin. Invest. 1991; 87: 352-356Crossref PubMed Scopus (306) Google Scholar, 3Clark R.A. Volpp B.D. Leidal K.G. Nauseef W.M. J. Clin. Invest. 1990; 85: 714-721Crossref PubMed Scopus (324) Google Scholar, 4Bokoch G. Bohl B. Chuang T. J. Biol. Chem. 1994; 269: 31674-31679Abstract Full Text PDF PubMed Google Scholar, 5Heyworth P. Bohl B. Bokoch G. Curnutte J. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google Scholar). SH3 domains in p47phox bind directly to proline-rich sequences in the p22phox subunit of the cytochrome (6Leto T. Adams A. Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (243) Google Scholar), and p67phox binds directly to p47phox (7Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar), also utilizing SH3 domains in the former to bind to proline-rich regions in the latter. Kinetic evidence also supports a direct interaction between p67phox and cytochrome b558 (8Uhlinger D. Taylor K. Lambeth J.D. J. Biol. Chem. 1994; 269: 22095-22098Abstract Full Text PDF PubMed Google Scholar). Based on experiments using cells from chronic granulomatous disease patients lacking either p47phox or p67phox, Rac translocation occurs independently of these components (5Heyworth P. Bohl B. Bokoch G. Curnutte J. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google Scholar). NADPH oxidase activation (9Abo A. Pick E. Hall A. Totty N. Teahan C.G. Segal A.W. Nature. 1991; 353: 668-670Crossref PubMed Scopus (758) Google Scholar, 10Abo A. Boyhan A. West I. Thrasher A.J. Segal A.W. J. Biol. Chem. 1992; 267: 16767-16770Abstract Full Text PDF PubMed Google Scholar, 11Kreck M.L. Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 4161-4168Abstract Full Text PDF PubMed Google Scholar) and partial assembly (12Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1993; 268: 8624-8631Abstract Full Text PDF PubMed Google Scholar, 13Quinn M.T. Evans T. Loetterle L.R. Jesaitis A.J. Bokoch G.M. J. Biol. Chem. 1993; 268: 20983-20987Abstract Full Text PDF PubMed Google Scholar) can be reconstituted in vitro using recombinant p47phox, p67phox, Rac (1 or 2), and either neutrophil plasma membrane or purified, lipid-reconstituted cytochrome b558 plus the activators GTPγS 1The abbreviations used are: GTPγSguanosine 5′-O-(3-thiotriphosphate)PAKp21-activated kinaseMantN-methylanthraniloylGppNHp5′-guanylylimidodiphosphatePCRpolymerase chain reactionPIPES1,4-piperazinediethanesulfonic acid. and an anionic amphiphile such as arachidonate.Rac is a small GTPase belonging to the Rho family of proteins. Rho family GTPases regulate cytoskeletal rearrangements (14Ridley A. Curr. Opin. Genet. & Dev. 1995; 5: 24-30Crossref PubMed Scopus (153) Google Scholar, 15Hall A. Annu. Rev. Cell Biol. 1994; 10: 31-54Crossref PubMed Scopus (764) Google Scholar, 16Takai Y. Sasaki T. Tanaka K. Nakanishi H. Trends Biochem. Sci. 1995; 20: 227-230Abstract Full Text PDF PubMed Scopus (366) Google Scholar) and participate in cell growth and transformation (17Qiu R. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (812) Google Scholar, 18Vojtek A. Cooper J. Cell. 1995; 82: 527-529Abstract Full Text PDF PubMed Scopus (253) Google Scholar). Direct targets of this family of GTPases include phospholipase D (Rho) (19Bowman E.P. Uhlinger D.J. Lambeth J.D. J. Biol. Chem. 1993; 268: 21509-21512Abstract Full Text PDF PubMed Google Scholar, 20Malcolm K. Ross A. Qiu R. Symons M. Exton J. J. Biol. Chem. 1994; 269: 25951-25954Abstract Full Text PDF PubMed Google Scholar, 21Kwak J.-Y. Lopez I. Uhlinger D.J. Ryu S.H. Lambeth J.D. J. Biol. Chem. 1995; 270: 27093-27098Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and the protein kinase PAK (22Martin G. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (300) Google Scholar, 23Manser E. Leung T. Salihuddln H. Zhao Z. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1292) Google Scholar) as well as the respiratory burst oxidase (Rac). The Rho type GTPases are approximately 30% homologous to Ras and 50% homologous to each other. As shown in Fig. 1, Ras contains a well studied region within residues 26-45, which participates in the binding of downstream effector enzymes such as Raf (24Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Abstract Full Text PDF PubMed Scopus (193) Google Scholar). Contained within this span is the Switch I region, which undergoes a conformational change depending on whether GTP or GDP is bound. The amino acid sequence of Rac in this region is highly homologous to that in Ras, suggesting conservation of the effector binding function of this region. Site-specific mutations (25Xu X. Barry D. Settleman J. Schwartz M. Bokoch G. J. Biol. Chem. 1994; 269: 23569-23576Abstract Full Text PDF PubMed Google Scholar, 26Freeman J.L.R. Uhlinger D.J. Lambeth J.D. Biochemistry. 1994; 33: 13431-13435Crossref PubMed Scopus (39) Google Scholar, 27Diekmann D. Abo A. Johnston C. Segal A.W. Hall A. Science. 1994; 265: 531-532Crossref PubMed Scopus (346) Google Scholar) and chimeric proteins of Rac/Cdc42 (28Kwong C. Adams A. Leto T. J. Biol. Chem. 1995; 270: 19868-19872Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) assayed for the ability to support superoxide generation demonstrate that this region is important for the interactions within the NADPH oxidase complex. Rac can bind directly to p67phox, and mutations in amino acids within this region eliminate this interaction (27Diekmann D. Abo A. Johnston C. Segal A.W. Hall A. Science. 1994; 265: 531-532Crossref PubMed Scopus (346) Google Scholar).One of the major differences between Rac and Rho family proteins is the presence of an additional 12-amino acid “insert region” in the Rho family proteins (Fig. 1). This region is predicted by molecular modeling to form a surface loop near the GTP binding site, 2J. D. Lambeth and J. L. Freeman, unpublished results. but the function of this region has not been investigated. In the present studies, we have used site-directed mutagenesis and deletion analysis to examine the effector function of the insert region, using both the NADPH oxidase and PAK as target enzymes. We find that for the NADPH oxidase but not for PAK, the insert region functions as a novel effector region.DISCUSSIONLike other members of the Rho family, Rac contains 12 amino acids (residues 124-135) C-terminal to the GTP binding domain that are not found in other small GTPases. This insert region represents one of several areas of divergence among Rho family members (41Kwong C.H. Malech H.L. Rotrosen D. Leto T.L. Biochemistry. 1993; 32: 5711-5717Crossref PubMed Scopus (93) Google Scholar). Because Rac is the only Rho family member able to support significant NADPH oxidase activity and because other regions of sequence divergence between Rac and the other Rho family members are important for its specificity, we examined these 12 amino acids (124-135) by mutational and deletional analysis. Single site mutants of amino acids in this region, as well as deletion of the entire insert region, result in a significantly diminished ability of Rac to support superoxide generation by the NADPH oxidase. This effect is largely due to a decreased affinity of Rac for the NADPH oxidase complex (as reflected in an increased EC50 for the small GTPase). In contrast, there was little or no effect of these mutations on the ability of Rac to activate PAK. The latter can also be activated by Cdc42 (22Martin G. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (300) Google Scholar, 23Manser E. Leung T. Salihuddln H. Zhao Z. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1292) Google Scholar). Additional evidence for a role for this region in NADPH oxidase function comes from recently published “peptide scanning” studies (42Joseph G. Gorzalczany Y. Koshkin V. Pick E. J. Biol. Chem. 1994; 269: 29024-29031Abstract Full Text PDF PubMed Google Scholar), in which peptides spanning this region of Rac inhibited cell-free NADPH oxidase activity. In contrast, studies using chimeric expressed proteins composed of Rac and Rho failed to identify this region as being functionally relevant but did identify an additional region near the C terminus (contained within residues 143-175), which, like the Ras-like effector region (residues 26 to 45), was found to mediate the interaction of Rac with both p67phox and PAK (43Diekmann D. Nobes C. Burbelo P. Abo A. Hall A. EMBO J. 1995; 14: 5297-5305Crossref PubMed Scopus (115) Google Scholar).It has become increasingly evident that each of the small GTPases can bind to and regulate multiple effector targets. Ras, for example, interacts not only with Raf but also with PI 3-kinase (44Rodriguez-Viciana P. Warne P. Dhand R. Vanhaesebroeck B. Gout I. Fry M. Waterfield M. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1717) Google Scholar), Ral GDS (45Spaargaren M. Bischoff J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12609-12613Crossref PubMed Scopus (248) Google Scholar, 46Hofer F. Fields S. Schneider C. Martin G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11089-11093Crossref PubMed Scopus (245) Google Scholar, 47Kikuchi A. Demo S. Ye Z. Chen Y. Williams L. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar), and L. J. Mol. Cell. Biol. 1995; PubMed Google Scholar). This important and the of specificity and in a studies that insert region amino acids a novel effector region for Rac and that this region also be important in of the functions of the other Rho family proteins. The use of the Ras-like effector region in with the insert effector region the Rho family proteins with in of the of proteins with which example, Rac and which are homologous in the Ras-like effector region, both activate PAK. The Ras-like effector region participates in this but the insert region not binding In contrast, Rac but not Cdc42 binds to and the NADPH oxidase. Rac and Cdc42 are 50% homologous within the insert region. The use of both the Ras-like effector region and the insert effector region the specificity and affinity to Rac but not Cdc42 to function as an NADPH oxidase the use of two or effector regions on small GTPases Rac and Cdc42 to a combinatorial to with cytosolic components p47phox and p67phox in the cell-free demonstrate that the Rac insert region mutations have a but effect to increase the EC50 of p67phox for the NADPH oxidase complex. effect was on the EC50 for region mutations little effect on the Rac In contrast, mutations in the Ras-like effector region have no effect on the EC50 for p67phox or p47phox J.L.R. Uhlinger D.J. Lambeth J.D. Biochemistry. 1994; 33: 13431-13435Crossref PubMed Scopus (39) Google Scholar). the kinetic of mutations in these two effector regions are significantly Although there are several molecular for these kinetic are to different functions for these two molecular on the Ras the insert region is predicted to form a surface loop that is on the of Rac from the Ras-like effector is that Rac binds these two regions to different within the NADPH oxidase and to different components of this multicomponent The use of two regions Rac to participate in the assembly or of oxidase components to form the activated NADPH oxidase this there is evidence for the binding of Rac to at least two components of the oxidase. direct interaction between p67phox and Rac has been by several using p67phox affinity (27Diekmann D. Abo A. Johnston C. Segal A.W. Hall A. Science. 1994; 265: 531-532Crossref PubMed Scopus (346) Google Scholar), binding to protein (43Diekmann D. Nobes C. Burbelo P. Abo A. Hall A. EMBO J. 1995; 14: 5297-5305Crossref PubMed Scopus (115) Google Scholar, E. S. A. R. E. Segal A. Lim L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), and the O. L. Bokoch G. J. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In these studies, in residues 26-45 and inhibited binding to p67phox, that the interaction occurs directly with the Ras homologous effector region and an effector region C-terminal to the GTP binding In studies of Rac translocation to the plasma membrane using and chronic granulomatous disease Rac translocation was diminished in cells lacking cytochrome b558 (5Heyworth P. Bohl B. Bokoch G. Curnutte J. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google Scholar). This result was not in cells lacking either p47phox or p67phox, and were as a direct interaction between cytochrome b558 and Rac. Although these binding are as the of an additional binding surface that is important for oxidase activity that both binding are and that Rac to between the cytochrome b558 and of the is important to the effect of insert region mutations on the EC50 for p67phox. This result from one of at least three this region of Rac be directly with p67phox, the insert region bind to and the of of the NADPH oxidase to which p67phox binds or Rac bind in but not directly to p67phox, and the binding of p67phox be due to when of the in the insert region are to The latter can be because the deletion that not also in the increased EC50 for p67phox. As there is no evidence that the insert region binds directly to p67phox. the present not between a direct binding to p67phox and an effect due to conformational or other in the are with a in which Rac binds to both the cytochrome and to p67phox. are to the direct interactions of Rac with other components of the respiratory burst the present studies have a novel effector region present on Rac that is important for activation of the respiratory burst oxidase but not PAK. This region, with the effector region, small GTPases to a combinatorial to target The use of molecular on Rac to activate the multicomponent respiratory burst oxidase a in which Rac functions to two or other components of the oxidase in a INTRODUCTIONDuring the respiratory burst, neutrophils and other phagocytic cells reduce molecular oxygen to generate superoxide anion, with subsequent production of secondary products such as hydrogen peroxide and hydroxyl radical, all of which participate in microbial killing. The enzyme that initiates the respiratory burst, the NADPH oxidase, is a multicomponent enzyme that utilizes reducing equivalents from NADPH to reduce oxygen to superoxide (reviewed in Ref. 1Quinn M. J. Leukocyte Biol. 1995; 58: 263-276Crossref PubMed Scopus (56) Google Scholar). The oxidase is dormant in resting cells but becomes activated in response to microorganisms or to a variety of soluble agonists. Activation involves the assembly of three cytosolic proteins, p47phox, p67phox, and the GTP-binding protein Rac (1 or 2), with the membrane-bound flavocytochrome b558. The flavocytochrome contains flavin, heme, and the NADPH binding site but catalyzes oxygen reduction only when the cytosolic components are associated. In response to cell activation, p47phox, p67phox, and Rac translocate to the cytochrome (2Heyworth P.G. Curnutte J.T. Nauseef W.M. Volpp B.D. Pearson D.W. Rosen H. Clark R.A. J. Clin. Invest. 1991; 87: 352-356Crossref PubMed Scopus (306) Google Scholar, 3Clark R.A. Volpp B.D. Leidal K.G. Nauseef W.M. J. Clin. Invest. 1990; 85: 714-721Crossref PubMed Scopus (324) Google Scholar, 4Bokoch G. Bohl B. Chuang T. J. Biol. Chem. 1994; 269: 31674-31679Abstract Full Text PDF PubMed Google Scholar, 5Heyworth P. Bohl B. Bokoch G. Curnutte J. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google Scholar). SH3 domains in p47phox bind directly to proline-rich sequences in the p22phox subunit of the cytochrome (6Leto T. Adams A. Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (243) Google Scholar), and p67phox binds directly to p47phox (7Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar), also utilizing SH3 domains in the former to bind to proline-rich regions in the latter. Kinetic evidence also supports a direct interaction between p67phox and cytochrome b558 (8Uhlinger D. Taylor K. Lambeth J.D. J. Biol. Chem. 1994; 269: 22095-22098Abstract Full Text PDF PubMed Google Scholar). Based on experiments using cells from chronic granulomatous disease patients lacking either p47phox or p67phox, Rac translocation occurs independently of these components (5Heyworth P. Bohl B. Bokoch G. Curnutte J. J. Biol. Chem. 1994; 269: 30749-30752Abstract Full Text PDF PubMed Google Scholar). NADPH oxidase activation (9Abo A. Pick E. Hall A. Totty N. Teahan C.G. Segal A.W. Nature. 1991; 353: 668-670Crossref PubMed Scopus (758) Google Scholar, 10Abo A. Boyhan A. West I. Thrasher A.J. Segal A.W. J. Biol. Chem. 1992; 267: 16767-16770Abstract Full Text PDF PubMed Google Scholar, 11Kreck M.L. Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1994; 269: 4161-4168Abstract Full Text PDF PubMed Google Scholar) and partial assembly (12Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. J. Biol. Chem. 1993; 268: 8624-8631Abstract Full Text PDF PubMed Google Scholar, 13Quinn M.T. Evans T. Loetterle L.R. Jesaitis A.J. Bokoch G.M. J. Biol. Chem. 1993; 268: 20983-20987Abstract Full Text PDF PubMed Google Scholar) can be reconstituted in vitro using recombinant p47phox, p67phox, Rac (1 or 2), and either neutrophil plasma membrane or purified, lipid-reconstituted cytochrome b558 plus the activators GTPγS 1The abbreviations used are: GTPγSguanosine 5′-O-(3-thiotriphosphate)PAKp21-activated kinaseMantN-methylanthraniloylGppNHp5′-guanylylimidodiphosphatePCRpolymerase chain reactionPIPES1,4-piperazinediethanesulfonic acid. and an anionic amphiphile such as arachidonate.Rac is a small GTPase belonging to the Rho family of proteins. Rho family GTPases regulate cytoskeletal rearrangements (14Ridley A. Curr. Opin. Genet. & Dev. 1995; 5: 24-30Crossref PubMed Scopus (153) Google Scholar, 15Hall A. Annu. Rev. Cell Biol. 1994; 10: 31-54Crossref PubMed Scopus (764) Google Scholar, 16Takai Y. Sasaki T. Tanaka K. Nakanishi H. Trends Biochem. Sci. 1995; 20: 227-230Abstract Full Text PDF PubMed Scopus (366) Google Scholar) and participate in cell growth and transformation (17Qiu R. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (812) Google Scholar, 18Vojtek A. Cooper J. Cell. 1995; 82: 527-529Abstract Full Text PDF PubMed Scopus (253) Google Scholar). Direct targets of this family of GTPases include phospholipase D (Rho) (19Bowman E.P. Uhlinger D.J. Lambeth J.D. J. Biol. Chem. 1993; 268: 21509-21512Abstract Full Text PDF PubMed Google Scholar, 20Malcolm K. Ross A. Qiu R. Symons M. Exton J. J. Biol. Chem. 1994; 269: 25951-25954Abstract Full Text PDF PubMed Google Scholar, 21Kwak J.-Y. Lopez I. Uhlinger D.J. Ryu S.H. Lambeth J.D. J. Biol. Chem. 1995; 270: 27093-27098Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and the protein kinase PAK (22Martin G. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (300) Google Scholar, 23Manser E. Leung T. Salihuddln H. Zhao Z. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1292) Google Scholar) as well as the respiratory burst oxidase (Rac). The Rho type GTPases are approximately 30% homologous to Ras and 50% homologous to each other. As shown in Fig. 1, Ras contains a well studied region within residues 26-45, which participates in the binding of downstream effector enzymes such as Raf (24Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Abstract Full Text PDF PubMed Scopus (193) Google Scholar). Contained within this span is the Switch I region, which undergoes a conformational change depending on whether GTP or GDP is bound. The amino acid sequence of Rac in this region is highly homologous to that in Ras, suggesting conservation of the effector binding function of this region. Site-specific mutations (25Xu X. Barry D. Settleman J. Schwartz M. Bokoch G. J. Biol. Chem. 1994; 269: 23569-23576Abstract Full Text PDF PubMed Google Scholar, 26Freeman J.L.R. Uhlinger D.J. Lambeth J.D. Biochemistry. 1994; 33: 13431-13435Crossref PubMed Scopus (39) Google Scholar, 27Diekmann D. Abo A. Johnston C. Segal A.W. Hall A. Science. 1994; 265: 531-532Crossref PubMed Scopus (346) Google Scholar) and chimeric proteins of Rac/Cdc42 (28Kwong C. Adams A. Leto T. J. Biol. Chem. 1995; 270: 19868-19872Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) assayed for the ability to support superoxide generation demonstrate that this region is important for the interactions within the NADPH oxidase complex. Rac can bind directly to p67phox, and mutations in amino acids within this region eliminate this interaction (27Diekmann D. Abo A. Johnston C. Segal A.W. Hall A. Science. 1994; 265: 531-532Crossref PubMed Scopus (346) Google Scholar).One of the major differences between Rac and Rho family proteins is the presence of an additional 12-amino acid “insert region” in the Rho family proteins (Fig. 1). This region is predicted by molecular modeling to form a surface loop near the GTP binding site, 2J. D. Lambeth and J. L. Freeman, unpublished results. but the function of this region has not been investigated. In the present studies, we have used site-directed mutagenesis and deletion analysis to examine the effector function of the insert region, using both the NADPH oxidase and PAK as target enzymes. We find that for the NADPH oxidase but not for PAK, the insert region functions as a novel effector region.