Activation of the superoxide-generating NADPH oxidase by chimeric proteins consisting of segments of the cytosolic component p67(phox) and the small GTPase Rac1.

Activation of the superoxide (O2(-))-generating NADPH oxidase of phagocytes is the consequence of the assembly of a membrane-associated flavocytochrome b(559) with the cytosolic proteins p47(phox) and p67(phox) and the small GTPase Rac (1 or 2). We proposed that Rac1 serves as a membrane-targeting molecule for p67(phox). This hypothesis was tested by constructing recombinant chimeric proteins, joining various functional domains of p67(phox) and Rac1, and expressing these in Escherichia coli. Chimeras were assayed for the ability to support O2(-) production by phagocyte membranes in an amphiphile-activated cell-free system in the presence or absence of p47(phox). A chimera consisting of p67(phox) truncated at residue 212 and fused to a full-length Rac1 [p67(phox)(1-212)-Rac1(1-192)] was a potent NADPH oxidase activator. A p67(phox)(1-212)-Rac1(178-192) chimera, to which Rac1 contributed only the C-terminal polybasic domain, was a weaker but consistent activator. Chimeras comprising the full length of Rac1 bound GTP/GDP, like bona fide GTPases. The activity of p67(phox)-Rac1 chimeras was dependent on the presence of the tetratricopeptide repeat and activation domains, in the p67(phox) segment, and on an intact polybasic region, at the C terminus of the Rac1 segment, but not on the insert region of Rac1. Partial activation by chimeras, in the GTP-bound form, was also possible in the absence of p47(phox). Evidence is offered in support of the proposal that the GTP- and GDP-bound forms of chimera p67(phox)(1-212)-Rac1(1-192) have distinct conformations, corresponding to the presence and absence of intrachimeric bonds, respectively.

Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly.

The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox), p67(phox), and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O(2) production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O(2) upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559).

Mapping of functional domains in p47(phox) involved in the activation of NADPH oxidase by "peptide walking".

The superoxide generating NADPH oxidase of phagocytes consists, in resting cells, of a membrane-associated electron transporting flavocytochrome (cytochrome b559) and four cytosolic proteins as follows: p47(phox), p67(phox), p40(phox), and the small GTPase, Rac(1 or 2). Activation of the oxidase is consequent to the assembly of a membrane-localized multimolecular complex consisting of cytochrome b559 and the cytosolic components. We used "peptide walking" (Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082) for mapping domains in the amino acid sequence of p47(phox) participating in the molecular events leading to the activation of NADPH oxidase. Ninety-five overlapping pentadecapeptides, with a four-residue offset between neighboring peptides, spanning the complete p47(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in a cell-free system. This consisted of solubilized macrophage membranes, recombinant p47(phox), p67(phox), and Rac1, and lithium dodecyl sulfate, as the activator. Eight functional domains were identified and labeled a-h. These were (N- and C-terminal residue numbers are given for each domain) as follows: a (21-35); b (105-119); c (149-159); d (193-207); e (253-267); f (305-319); g (325-339), and h (373-387). Four of these domains (c, d, e, and g) correspond to or form parts of regions shown before to participate in NADPH oxidase assembly. Thus, domain c corresponds to a region on the N-terminal boundary of the first src homology 3 (SH3) domain, whereas domains d and e represent more precisely defined sites within the full-length first and second SH3 domains, respectively. Domain g overlaps an extensively investigated arginine-rich region. Domains a and b, in the N-terminal half of p47(phox), and domains f and h, in the C-terminal half, represent newly identified entities, for which there is no earlier experimental evidence of involvement in NADPH oxidase activation. "Peptide walking" was also applied to the identification of domains in p47(phox) mediating binding to p67(phox). This was done by quantifying, by enzyme-linked immunosorbent assay, the binding of p67(phox), in solution, to a series of 95 overlapping biotinylated p47(phox) peptides, attached to streptavidin-coated 96-well plates. A single proline-rich domain (residues 357-371) was found to bind p67(phox) in the absence and presence of lithium dodecyl sulfate.

Mutational analysis of novel effector domains in Rac1 involved in the activation of nicotinamide adenine dinucleotide phosphate (reduced) oxidase.

The small molecular weight GTP-binding protein Rac (1 or 2) is an obligatory participant in the activation of the superoxide-generating NADPH oxidase. Active NADPH oxidase can be reconstituted in a cell-free system, consisting of phagocyte-derived membranes, containing cytochrome b559, and the recombinant cytosolic proteins p47-phox, p67-phox, and Rac, supplemented with an anionic amphiphile as an activator. The cell-free system was used before for the analysis of structural requirements of individual components participating in the assembly of NADPH oxidase. In earlier work, we mapped four previously unidentified domains in Rac1, encompassing residues 73-81 (a), 103-107 (b), 123-133 (c), and 163-169 (d), as important for cell-free NADPH oxidase activation. The domains were defined by assessing the activation inhibitory effect of a series of overlapping peptides, spanning the entire length of Rac1 [Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082]. We now used the construction of Rac1/H-Ras chimeras, domain deletion, and point mutations, to ascertain the functional relevance of three domains (b, c, and d) predicted by "peptide walking" and to determine the importance of specific residues within these domains. This methodology firmly establishes the involvement of domains b and d in the activation of NADPH oxidase by Rac1 and identifies H103 and K166, respectively, as residues critical for the effector function of these two domains. The functional significance of domain c (insert region) could not be confirmed, as shown by the minor effect of deleting this domain on NADPH oxidase activation. Analysis of the three-dimensional structure of Rac1 reveals that residues H103 and K166 are exposed on the surface of the molecule. Modeling of the activity-impairing point mutations suggests that the effect on the ability to activate NADPH oxidase depends on the side chains of the mutated amino acids and not on changes in the global structure of the protein. In conclusion, we demonstrate the existence of two novel effector sites in Rac1, necessary for supporting NADPH oxidase activation, supplementing the canonical N-terminal effector region.

Inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of small GTP-binding proteins. Lack of amino acid sequence specificity and importance of polybasic motif.

The small GTP-binding protein (G protein) Rac1 is an obligatory participant in the assembly of the superoxide (O2-.)-generating NADPH oxidase complex of macrophages. We investigated the effect of synthetic peptides, mapping within the near carboxyl-terminal domains of Rac1 and of related G proteins, on the activity of NADPH oxidase in a cell-free system consisting of solubilized guinea pig macrophage membrane, a cytosolic fraction enriched in p47phox and p67phox (or total cytosol), highly purified Rac1-GDP dissociation inhibitor for Rho (Rho GDI) complex, and the activating amphiphile, lithium dodecyl sulfate. Peptides Rac1-(178-188) and Rac1-(178-191), but not Rac2-(178-188), inhibited NADPH oxidase activity in a Rac1-dependent system when added prior to or simultaneously with the initiation of activation. However, undecapeptides corresponding to the near carboxyl-terminal domains of RhoA and RhoC and, most notably, a peptide containing the same amino acids as Rac1-(178-188), but in reversed orientation, were also inhibitory. Surprisingly, O2-. production in a Rac2-dependent cell-free system was inhibited by Rac1-(178-188) but not by Rac2-(178-188). Finally, basic polyamino acids containing lysine, histidine, or arginine, also inhibited NADPH oxidase activation. We conclude that inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of certain small G proteins is not amino acid sequence-specific but related to the presence of a polybasic motif. It has been proposed that such a motif serves as a plasma membrane targeting signal for a number of small G proteins (Hancock, J.F., Paterson, H., and Marshall, C.J. (1990) Cell 63, 133-139).

The GDP-bound form of the small G protein Rac1 p21 is a potent activator of the superoxide-forming NADPH oxidase of macrophages.

Phagocytes produce superoxide by the assembly of a multicomponent complex that utilizes NADPH for the reduction of molecular oxygen (NADPH oxidase). The components participating in the assembly are a membrane-bound flavocytochrome and three cytosolic proteins, one of which was shown to be a dimer of the small GTP-binding protein (G protein) Rac1 p21 or Rac2 p21 with GDP dissociation inhibitor for Rho (Rho GDI). We determined the identity and quantity of the nucleotide bound to Rac1 p21 by high performance anion exchange chromatography of extracts prepared from highly purified Rac1 p21-Rho GDI, isolated from guinea pig macrophage cytosol. Rac1 p21 contained only GDP at a ratio of close to 1 mol of GDP per mol of G protein. The GDP-bound form of Rac1 p21 complexed to Rho GDI functioned as a potent activator of NADPH oxidase in a cell-free system that contained no free GTP or ATP. We propose that the GDP-bound form of Rac1 p21 might be the physiological activator of NADPH oxidase in macrophages, following its dissociation from Rho GDI, and that nucleotide exchange or conversion to GTP is not necessarily involved.

Role of the rac1 p21-GDP-dissociation inhibitor for rho heterodimer in the activation of the superoxide-forming NADPH oxidase of macrophages.

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes requires the interaction of membrane-associated cytochrome b559 with three cytosolic components; p47-phox, p67-phox and sigma 1. We proposed that sigma 1 was a heterodimer composed of proteins of 22 kDa and 24 kDa that were tentatively identified as the small GTP-binding protein (G protein) rac1 p21 and GDP-dissociation inhibitor for rho (rho GDI). We now describe a modified procedure for the rapid purification of sigma 1 and demonstrate that the NADPH-oxidase-activating capacity is associated, throughout the purification sequence, with a protein binding 35S-labelled guanosine 5'-[3-O-thio]triphosphate. SDS/PAGE analysis confirmed the absolute association of sigma 1 activity with the presence of both the 22 kDa and 24 kDa proteins. Immunoblotting with a battery of antibodies against the small G proteins demonstrated that the 22-kDa protein was only recognized by antibodies reacting with rac1 p21; no reaction was found with anti-(rac2 p21), anti-[v-ras(H) p21] and anti anti-(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was not detected at any stage of cytosol fractionation. The proteins comprising the sigma 1 heterodimer could be separated by reverse-phase chromatography and amino acid sequencing was performed on peptides derived by trypsin digestion of each of the isolated proteins. This demonstrated the identity of the 22-kDa protein with rac1 p21 and that of the 24-kDa protein with rho GDI. Purified heterodimeric sigma 1 did not require exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the sigma 1 heterodimer with 1% sodium cholate, followed by gel filtration or anion-exchange chromatography in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was able to stimulate cell-free O2- generation. Artificial heterodimeric sigma 1, capable of NADPH oxidase activation, could be reconstituted in vitro by recombining purified monomeric rac1 p21 and rho GDI and removing the sodium cholate used to dissociate the native sigma 1 dimer. Monomeric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nucleotide in low Mg2+ solution. Under similar conditions, heterodimeric sigma 1 was resistant to nucleotide exchange.(ABSTRACT TRUNCATED AT 400 WORDS)