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The phagocyte NADPH oxidase consists of multiple protein subunits that interact with each other to form a functional superoxide-generating complex. Although the essential components for superoxide production have been well characterized, other proteins potentially involved in the regulation of NADPH oxidase activation remain to be identified. We report here that the Gαi subunit of heterotrimeric G proteins is a novel binding partner for p67phox in transfected HEK293T cells and peripheral blood polymorphonuclear leukocytes. p67phox preferably interacted with inactive Gαi. Expression of p67phox caused a dose-dependent decrease in intracellular cyclic AMP concentration, suggesting altered function of Gαi. We identified a fragment of p67phox, consisting of the PB1 domain and the C-terminal SH3 domain, to be critical for the interaction with Gαi. Because these domains are involved in the interaction with p47phox and p40phox, the relationship between the respective binding events was investigated. Wild-type Gαi, but not its QL mutant, could promote the interaction between p67phox and p47phox. However, the interaction between p67phox and p40phox was not affected by either Gαi form. These results provide the first evidence for an interaction between p67phox and an alpha subunit of heterotrimeric G proteins, suggesting a potential role for Gαi in the regulation or activation of NADPH oxidase.
Professional phagocytes play a critical role in the innate immune response to pathogens. The detection of microbial products such as fMet-Leu-Phe (fMLF) by resting neutrophils is an essential activating event that results in a spectrum of activities aimed to eliminate the causes of infection. In particular, neutrophils have the ability to generate toxic oxygen intermediates via activation of the NADPH oxidase (2), a tightly regulated multiprotein enzyme complex (32). Numerous studies have established that the active oxidase is composed of at least five essential subunits: membrane-associated gp91phox and p22phox, which form the redox core flavocytochrome b558 of the enzyme, and the cytosolic factors p67phox, p47phox, and p40phox (23, 29, 42, 43). Membrane translocation of the cytosolic subunits together with the active monomeric G protein Rac1/2 is a crucial step for assembly of a fully functional enzyme (1, 17).
p67phox is a multidomain protein implicated in essential NADPH oxidase protein-protein interactions. Its activation domain binds to the catalytic core of gp91phox and activates the electron transfer process (7, 27). The stretch of tetratricopeptide repeat (TPR) motifs in its amino terminus is responsible for recruitment of active Rac (19). The phox and bem1 (PB1) domain binds p40phox (12), and the C-terminal Src-homology 3 (SH3) sequence is necessary for association with p47phox (9). Therefore, p67phox appears as a central coordinator for NADPH oxidase assembly.
Chemoattractants such as fMLF stimulate G protein-coupled receptors, leading ultimately to O2− generation. The majority of signals arising from the chemoattractant receptors are pertussis toxin sensitive and therefore mediated by Gi proteins (5, 46). Moreover, it is the Gβγ dimer that regulates a variety of effectors, such as phosphatidylinositol 3-kinase, phospholipase C-β, and most likely specific Rac guanine nucleotide exchange factors, leading to oxidase activation. The Gαi subunit, however, has not been implicated in the assembly or regulation of the NADPH oxidase.
Recent studies have led to the identification of several novel binding partners for Gα proteins besides the conventional Gβγ, downstream effectors, and specific G protein-coupled receptors. With a few exceptions, these proteins fall mainly into two defined groups: the regulators of G protein signaling (RGS) and the Gαi/o-Loco (GoLoco)-containing proteins. RGS proteins attenuate G protein signaling by accelerating the intrinsic GTPase activity in Gα (8, 36). The GoLoco interaction motif is found in a variety of proteins, such as activators of G protein signaling (AGS), Leu-Gly-Asn repeat-enriched protein (LGN), Pcp2, Rap1GAP, and others (20, 25). GoLoco proteins interact with GDP-bound Gαi and act as guanine nucleotide dissociation inhibitors while impeding binding of Gβγ (44). Of particular interest is the R12 class of RGS, comprising RGS10, 12, and 14, which possess both the characteristic RGS box and GoLoco domains in their sequences (16).
In the present study, we investigated whether p67phox could interact with the alpha subunits of heterotrimeric G proteins, because p67phox was previously shown to bind the small GTPase Rac (19, 22). We show that p67phox directly interacts with GDP-bound Gαi in both transfected cells and human neutrophils. The binding site for Gαi was localized to the C-terminal SH3 domain on p67phox. Overexpression of p67phox in our system dose dependently decreased basal levels of cyclic AMP (cAMP), a readout for activation of Gαi signaling and also a negative regulator of O2− production. Furthermore, the association of p47phox with p67phox was affected by the Gαi activation state, suggesting that Gαi not only is a binding partner of p67phox but also may participate in the regulation of NADPH oxidase activation.
Monoclonal antibody to c-MYC was purchased from Covance (Berkeley, CA) and that to FLAG from Sigma-Aldrich (St. Louis, MO). The anti-p67phox monoclonal antibody was from BD Transduction Laboratories (Lexington, KY). Antibodies specific to Gαi1 and Gαi3 were from Santa Cruz Biotechnology (Santa Cruz, CA), as were the anti-p47phox polyclonal and the anti-β-actin monoclonal antibodies. The anti-Gαi2 serum was produced in a rabbit (with amino acids 213 to 354 as an antigen), as was the anti-p67phox serum (raised against the purified glutathione S-transferase [GST]-p67phox fusion protein). The anti-p40phox polyclonal antibody was acquired from Upstate (Lake Placid, NY). Monoclonal antibodies were used at a concentration of 1 μg/ml and sera at a 1:1,000 dilution for Western blotting.
The human embryonic kidney epithelial cell line 293T (HEK293T) was maintained in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS) and antibiotics (2 mM l-glutamine, 100 IU/ml penicillin, and 50 μg/ml streptomycin). Cells were transfected using LipofectAMINE Plus (Life Technologies) according to the manufacturer's instructions. For superoxide generation experiments, the monkey kidney epithelial transgenic COS-phox cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% FBS and in the presence of 0.2 mg/ml hygromycin (Sigma), 0.8 mg/ml neomycin sulfate (Invitrogen), and 1 μg/ml puromycin (Calbiochem). Cells were transiently transfected using LipofectAMINE 2000 reagent (Life Technologies) according to the manufacturer's protocol.
Twenty-four hours after transfection, the cells were lysed in buffer containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1× protease inhibitor cocktail set I (Calbiochem). For immunoprecipitation studies, the cleared lysates were incubated overnight at 4°C with either the MYC-specific monoclonal antibody (10 μg/ml), anti-Gαi2 (1:250), or anti-p47phox (1 μg/ml) as indicated. Protein A/G PLUS-agarose (Santa Cruz Biotechnology) was added to the samples, and samples were incubated for 1.5 h at 4°C. The beads were washed and resuspended in 50 μl of 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and boiled for 5 min to release bound proteins. The resolved samples were detected by Western blotting. When working with human neutrophils (107 cells/sample), the same fractionating protocol was followed with minor modifications. Briefly, lysis was achieved in the presence of 2× protease inhibitor cocktail set I and 100 μM E-64 (Sigma). Lysis, immunoprecipitation, and washing were performed in the presence of 10 μM GDP or 10 μM GDP, 30 μM AlCl3, and 10 mM NaF (AMF).
Electrophoresis of the proteins on a 10% SDS-polyacrylamide gel was followed with transfer to a nitrocellulose membrane (Schleicher & Schuell). The blots were blocked with 5% nonfat dry milk in Tris-buffered saline-Tween buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20), washed, and incubated with primary antibodies overnight at 4°C. Anti-rabbit (Bio-Rad) or anti-mouse (Calbiochem) peroxidase-conjugated secondary antibodies were added to the membranes at a 1:3,000 dilution for 1 h at room temperature. The bands on the blots were visualized by chemiluminescence (Pierce).
Blood from healthy donors was collected following a procedure approved by the Institutional Review Board at the University of Illinois at Chicago by using ACD buffer (1.365% citric acid, 2.5% sodium citrate, and 2% dextrose). Erythrocytes were removed by sedimentation with Hespan (6% hetastarch; Abbott Laboratories). Polymorphonuclear leukocytes were further fractionated by centrifugation at 450 × g for 1 h at 12°C on a discontinuous Percoll (Amersham Pharmacia Biotech) gradient (74% and 55%). In a routine preparation, approximately 97% of the cells were neutrophils and the viability was about 98%, as determined by Trypan blue exclusion.
The pGEX-2T and pGEX-p67phox constructs, encoding GST and full-length GST-p67phox fusion proteins, respectively, were introduced into the E. coli strain DH10B. Protein expression was induced with 0.1 mM isopropyl-1-thio-β-d-galactopyranoside for 2 h at 30°C for GST-p67phox and 37°C for GST. The bacterial pellet was resuspended in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mg/ml lysozyme, 1× protease inhibitor cocktail set II (Calbiochem), and 2 mM dithiothreitol and sonicated. The lysate was complemented with 1% Triton X-100 and shaken for 1 h at 4°C. After centrifugation at 12,000× g for 10 min at 4°C, the supernatant was snap-frozen for storage in 10% glycerol.
Rat Gαi1 purified from Sf9 cells was preloaded with either 30 μM GDP, 10 μM GTPγS (guanosine 5′-[γ-thio] triphosphate), or AMF with 30 μM GDP in a buffer containing 20 mM HEPES, pH 8.0, 5 mM MgCl2, 1 mM EDTA, 0.05% Lubrol, and 1 mM dithiothreitol for 1 h at 30°C. The Gαi cycle was stopped by the addition of 10 mM MgCl2. The binding between 1 μM GST fusion proteins coupled to glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) and 0.025 μM preloaded Gαi1 was performed in 100 μl of binding buffer (20 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 10 mM MgCl2, 1 mM EDTA, 0.1% Lubrol, 10 mM β-mercaptoethanol) supplemented with either GDP, GTPγS, or AMF where indicated for 2 h at 4°C. Beads were pelleted at 600 × g for 1 min at 4°C. The “unbound” fraction was recovered from the supernatant. Bound proteins were then eluted in 10 μl of 10 mM glutathione for 15 min at room temperature, 30 μl of 5× SDS-PAGE loading buffer was added to the eluate, and proteins were boiled for 10 min.
Cells cultured in six-well plates were transfected, allowed to recover in 10% FBS-containing culture medium for 2.5 h, and starved overnight in Dulbecco's modified Eagle's medium without serum. cAMP accumulated in the presence of 1 mM isobutylmethylxanthine for 1 h at 37°C was measured using an enzyme-linked immunosorbent assay according to the manufacturer's protocol (Biomol, Pennsylvania).
Superoxide production was determined using a chemiluminescence (CL) assay, as previously described (6). Briefly, COS-phox cells were collected with enzyme-free cell dissociation buffer (Invitrogen) and resuspended in 0.5% bovine serum albumin in Hanks' balanced salt solution with Ca2+ and Mg2+ at 3 × 106 to 5 × 106 cells/ml. Cells were incubated with 100 μM isoluminol (Sigma) and 40 U/ml horseradish peroxidase (Roche), and 200-μl aliquots were transferred into a white 96-well flat-bottom tissue culture plate (E&K Scientific). Chemiluminescence (count per second [cps]) was continuously assayed by using a Wallac 1420 multilabel counter plate reader (PerkinElmer) for 10 min before and 40 min after stimulation with either 1 μM fMLF (Sigma) or 200 ng/ml phorbol myristate acetate (PMA; Sigma). CL was integrated for 30 min after stimulation to show the relative levels of superoxide generated.
To evaluate the potential interaction of p67phox and Gαi, an expression construct was created to produce C-terminal MYC-tagged p67phox (p67phox-MYC). Moreover, activity of recombinant p67phox-MYC was verified in a whole cell-based reconstitution assay, which required exogenous p67phox for fMLF- and PMA-induced O2− generation (11). The p67phox-MYC DNA construct was transiently cotransfected with vectors encoding each of the three isoforms of Gαi into HEK293T cells, which do not express any of the NADPH oxidase components except for Rac1. Both inactive wild-type Gαi (Gαiwt) and the GTPase-deficient (constitutively active) Gαi mutants (Q204L for Gαi1 and Gαi3 and Q205L for Gαi2) were examined. Twenty-four hours after transfection, p67phox-MYC was immunoprecipitated from cell lysates, and the precipitates were analyzed by immunoblotting. Surprisingly, Gαi1wt, Gαi2wt, and Gαi3wt were all detected in the immunoprecipitates (Fig. (Fig.1).1). Additionally, we also observed a modest interaction between p67phox and the endogenous Gαi (vector, mock-transfected cells). Interestingly, the interaction between p67phox and the active GαiQL was significantly reduced to levels as low as or even below those of the vector controls (Fig. (Fig.1A,1A, lanes 3 versus lanes 1). The specificity of this interaction was validated by the observation that neither the GDP- nor the GTP-bound forms of Gαs could associate with p67phox (data not shown).
Since the resting and activated NADPH oxidase states are tightly regulated by complex protein-protein interactions (35), we examined whether the binding between p67phox and Gαi could occur in the presence of the other NADPH components in an environment where the enzyme is fully functional. We isolated neutrophils from human peripheral blood, lysed them, and immunoprecipitated endogenously expressed Gαi2 in the absence or presence of AMF, which mimics the transition state of GTP hydrolysis. As expected from the previous observations, an interaction between p67phox and Gαi2 was detectable in nontreated cell lysates and was markedly diminished in the presence of AMF (Fig. (Fig.1B,1B, lane 2 versus lane 1). Since fMLF stimulates neutrophil O2− production via activation of the Gi proteins, we treated isolated neutrophils with 1 μM fMLF before lysis and immunoprecipitation with an anti-Gαi2 serum. In this case too, the interaction between the endogenous p67phox and Gαi2 was markedly decreased compared to that observed in unstimulated neutrophils (Fig. (Fig.1B,1B, lane 3 versus lane 1). Thus, the binding of p67phox with Gαi2 is dependent on the G protein activation state and is observed in resting neutrophils.
To further investigate whether this interaction was direct, we performed an in vitro binding assay between purified, bacterially expressed GST-p67phox and Sf9-expressed Gαi1 (Fig. (Fig.2).2). The GST-p67phox fusion protein coupled to glutathione-Sepharose beads, but not the GST control protein, specifically bound Gαi1 in the presence of GDP but not when the Gαi was preloaded with either AMF or GTPγS. Thus, these data demonstrate a direct interaction between p67phox and Gαi and again show that the interaction is modulated by the Gαi activation state.
Activation of the Gαi family of G proteins is responsible for the inhibition of adenylyl cyclase and subsequent reduction in the basal and inducible levels of cAMP. To gain an insight into a potential role of p67phox in the Gαi activation/inactivation cycle in a receptor-independent manner, we examined the effect of p67phox on the Gαi-mediated cAMP basal level. In HEK293T cells transfected to express increasing amounts of untagged p67phox, cAMP production was dose dependently inhibited (Fig. (Fig.3A),3A), with a 50% reduction at the highest DNA concentration used for transfection. As a control, similar amounts of a vector encoding AGS3 did not influence the cAMP level (data not shown).
Heterotrimeric G proteins are composed of the guanine nucleotide-binding Gα subunit and the Gβγ dimer, which are regarded as one functional unit. G proteins are inactive in the GDP-bound state. Since p67phox preferentially binds to inactive Gαi, we expected that it might compete with Gβγ for association with GDP-bound Gαi. Surprisingly, in transiently transfected HEK293T cells, increasing amounts of p67phox-MYC did not disrupt the interaction between Gβ1γ2 and Gαi (Fig. (Fig.3B).3B). These observations suggest that p67phox most likely binds to Gαi on a site distinct from the Gβγ contact surface. Indeed, overexpression of p67phox showed no effect on the basal phosphorylation of Akt, a characterized readout for Gβγ activation (data not shown). Finally, we verified that the ability of Gβγ to coimmunoprecipitate p67phox was mediated by the inactive Gαi (Fig. (Fig.3C).3C). Indeed, FLAG-tagged Gβ1 was found to bind to p67phox only when Gαi2 was overexpressed in the cells. Endogenous Gαi appeared to be insufficient to mediate the association between FLAG-Gβ1 and p67phox-MYC, most likely because it already forms heterotrimers with endogenous Gβγ proteins, which cannot be detected with the anti-FLAG monoclonal antibody (MAb). Based on these data, we concluded that the association between Gβγ and p67phox is indirect and mediated through the inactive, GDP-bound Gαi.
p67phox is a multidomain protein (Fig. (Fig.4A).4A). The N-terminal TPR domain (amino acids 3 to 154) is composed of four TPR motifs and directly binds Rac-GTP (19, 22). This segment (amino acids 1 to 199) was also demonstrated to mediate direct interaction with gp91phox (7). A short activation domain (amino acids 199 to 210) immediately follows the TPR domain (27), and the C-terminal segment contains two SH3 modules flanking a PB1 domain. The PB1 and C-terminal SH3 domains are involved in the direct interactions with p40phox and p47phox, respectively (9, 12). In an attempt to localize the region of p67phox required for Gαi binding, a series of truncated MYC-tagged p67phox mutants was generated (Fig. (Fig.4A),4A), verified by sequencing, and separately expressed into HEK293T cells together with Gαi2wt (Fig. (Fig.4B).4B). In immunoprecipitation and immunoblotting experiments, the p67phox C-terminal fragment (amino acids 213 to 526) precipitated Gαi2 as effectively as did full-length p67phox, whereas the N-terminal segment (amino acids 1 to 213) did not retain binding to the Gα protein (Fig. (Fig.4B,4B, lanes 2 and 3 versus lane 1). Furthermore, the fragment covering residues 340 to 526 was shown to be sufficient for interaction with Gαi2, whereas the sequence extending from amino acids 303 to 455 did not bind Gαi2. Therefore, at first view the C-terminal SH3 was the most likely candidate for the Gαi binding site, and two additional p67phox mutants that both contained PB1, p67phox(1-430)-MYC and p67phoxΔ(460-515)-MYC, seemed to confirm this conclusion. However, the C-terminal SH3 and its flanking regions (residues 429 to 526) failed to coimmunoprecipitate Gαi2. Therefore, the C-terminal SH3 in p67phox could be necessary but not sufficient for recruitment of Gαi, and perhaps both PB1 and the second SH3 in p67phox contribute to the binding interaction with Gαi.
Since the C-terminal SH3 domain of p67phox can interact directly with p47phox and its PB1 domain associates with the phox and Cdc (PC) domain of p40phox (9, 12), we investigated whether Gαi2 overexpression could influence these binding interactions. p67phox and p47phox were coexpressed in HEK293T cells together with Gαi2 (Fig. (Fig.5A).5A). Interestingly, the wild type but not the GTPase-deficient mutant of Gαi2 increased the interaction between p67phox and p47phox (Fig. (Fig.5A,5A, top panel, lanes 4 and 5 versus lane 3). However, in the presence of p47phox the association between p67phox and Gαi2 was barely detectable (Fig. (Fig.5A,5A, second panel from top, compare lanes 4 and 5 to lanes 1 and 2). This finding suggests that p47phox competes with Gαi for binding to p67phox.
Similar experiments were conducted in cells expressing p67phox, Gαi2, and p40phox (Fig. (Fig.5B).5B). The interaction between p67phox and p40phox was not affected by the expression of either Gαi2wt or Gαi2QL (Fig. (Fig.5B,5B, top panel, lanes 3, 4, and 5). Surprisingly, p67phox, which preferentially binds to inactive Gαi in the absence of p40phox, switched its affinity for the active form of the Gαi protein when p40phox was present (Fig. (Fig.5B,5B, second panel from top, lanes 1 and 2 compared to lanes 4 and 5). The decreased but still apparent coimmunoprecipitation between p67phox and inactive Gαi in the presence of p40phox suggests that p40phox might only partially displace Gαi from p67phox.
Finally, all four binding partners were coexpressed together (Fig. (Fig.5C).5C). Interestingly, in the presence of both p47phox and p40phox, the characteristic difference in p67phox binding to Gαi2wt versus Gαi2QL remained (Fig. (Fig.5C,5C, top panel, lanes 4 and 5 versus lanes 2 and 3). Taken together, these data confirm the delicate dynamics in protein-protein interactions involved in the formation and regulation of p67phox-containing complexes.
In HEK293T cells and in the absence of p40phox, p47phox was found to completely block the binding of Gαi2 to p67phox (Fig. (Fig.5A).5A). However, when the three phox proteins and Gαi2 were coexpressed, as a closer mimic of human neutrophils, both p47phox and Gαi2 coimmunoprecipitated with MYC-tagged p67phox (Fig. (Fig.5C).5C). Therefore, we tested whether p67phox, p47phox, and Gαi2 would associate in the same large complex or bind differently to form a variety of smaller ones. Human neutrophils were purified from peripheral blood and either unstimulated or stimulated with 1 μM fMLF before lysis and immunoprecipitation with antibodies against p47phox and Gαi2 (Fig. (Fig.6).6). Both Gαi2 and p67phox were detected in the anti-p47phox precipitates in either resting or fMLF-stimulated cells. Similarly, both p47phox and p67phox coimmunoprecipitated with Gαi2. Interestingly, the interaction between p47phox and p67phox increased upon fMLF stimulation, with Gαi2 concomitantly dissociating from the complex (Fig. (Fig.6,6, “IP: p47phox” set of panels). As expected, p67phox interaction with Gαi2 markedly diminished after fMLF treatment and so did the association between p47phox and Gαi2 (Fig. (Fig.6,6, “IP: Gαi2” set of panels). p67phox most likely bridges p47phox and Gαi2, causing them to coimmunoprecipitate. Indeed, we have not observed any interaction between Gαi2 and p47phox in the absence of p67phox in HEK293T cells (data not shown). Taken together, these results support the presence of a multiprotein complex containing p47phox, Gαi, and p67phox.
We took advantage of the COS-phox system, a whole-cell-based reconstitution assay manipulated to generate fMLF-induced O2− (11, 31). The transgenic cells stably expressing gp91phox, p22phox, p67phox, and p47phox were transiently transfected with DNA coding for the formyl peptide receptor (FPR), as well as protein kinase Cδ (PKCδ) and p40phox, both being highly abundant in neutrophils but scarce in COS7 cells. Where indicated, Gαi2 was overexpressed in the cells. We verified by Western blotting that exogenous Gαi2 did not affect the relative levels of expression of any of the three cytosolic phox components (Fig. (Fig.7C).7C). The transfected cells were then challenged with either 1 μM fMLF (Fig. (Fig.7A,7A, left graph) or 200 ng/ml PMA (Fig. (Fig.7A,7A, right graph), and the produced O2− was measured by isoluminol-dependent chemiluminescence. PMA is a PKC agonist that bypasses activation of Gi to stimulate NADPH oxidase activity. Interestingly, overexpression of Gαi2 increased both fMLF- and PMA-induced O2− generation (Fig. 7A and B). Although promotion of fMLF-stimulated O2− production might be due in part to the availability of more Gαi for FPR, the increase in O2− generated by PMA stimulation indicates that Gαi possibly plays a positive regulatory role in NADPH oxidase activation.
In phagocytic cells, functional assembly of the NADPH oxidase in the phagosome and plasma membrane is a rapid and complex process and is essential for host defense against pathogens (32). Chemoattractant-induced O2− generation is mediated through Gβγ originating from activation of heterotrimeric Gi proteins (5). However, it is unclear whether the Gαi proteins also play a role in NADPH oxidase activation through a different mechanism. A major finding of this study is the identification of GDP-bound Gαi as a direct p67phox binding partner. To our knowledge, this is the first report that suggests a potential role for Gαi in oxidase enzyme assembly and raises the possibility that Gαi may be a distinct player in NADPH oxidase regulation.
Using coimmunoprecipitation and in vitro binding analysis, we observed an interaction between the inactive, GDP-bound Gαi and the cytosolic factor p67phox. This binding exists not only in transiently transfected cells (Fig. (Fig.1A)1A) but also, and more importantly, in human neutrophils (Fig. (Fig.1B).1B). The interaction significantly decreases upon activation of Gαi, suggesting that p67phox can recognize the conformational changes associated with Gαi activation. Since p67phox does not appear to compete with Gβγ for association with GDP-bound Gαi (Fig. (Fig.3B),3B), the binding site for p67phox on Gαi must be different from the Gαi/Gβγ interface. Furthermore, there is no observable difference in Gαi2 and p67phox coimmunoprecipitation between untreated and pertussis toxin-treated (500 ng/ml, 4 h) cells (data not shown), which irreversibly ADP-ribosylates a specific C-terminal cysteine residue on the Gαi subunit that leads to uncoupling from the receptor. Thus, the C-terminal sequence of Gαi does not appear to be involved in the interaction with p67phox.
The possible relationship between Gαi and the three cytosolic factors of NADPH oxidase is depicted in Fig. Fig.8.8. Analysis of the deletion mutants of p67phox revealed that the C-terminal SH3 motif of p67phox (Fig. (Fig.8A),8A), previously characterized as a direct binding site for p47phox (9), also contributes to the recruitment of Gαi (Fig. (Fig.4).4). Indeed, p47phox can fully compete inactive Gαi off of p67phox (Fig. (Fig.5A).5A). However, in neutrophils p67phox clearly binds better with inactive Gαi (Fig. (Fig.1B),1B), even in the presence of endogenous p40phox and p47phox. Moreover, Gαi2, p47phox, and p67phox were found to associate in the same complex (Fig. (Fig.6).6). Therefore, we tested whether coexpression of p67phox, p47phox, and p40phox in HEK293T cells, together with Gαi2, would mimic what we observed in neutrophils (Fig. (Fig.5C).5C). Indeed, the preference of p67phox for inactive Gαi2 is maintained in the presence of both p47phox and p40phox. In addition, the binding of p47phox and Gαi2 to p67phox is no longer mutually exclusive in the presence of p40phox. Coimmunoprecipitation assays performed in neutrophils have confirmed the presence of a multisubunit complex containing p47phox, p67phox, and Gαi2 (Fig. (Fig.6).6). However, it is possible that only subpopulations of p67phox and Gαi are associated at a given time, and it is also likely that a variety of multimers coexists in the cell. Additionally, evidence suggesting that p67phox can homodimerize may support the observation that p47phox and Gαi are both present in the same large complex with p67phox and most likely have overlapping binding sites on p67phox. Although the intriguing prospect that p67phox can homodimerize has been approached in only a few biochemical studies drawing contradictory conclusions (10, 21), we have observed homodimerization of p67phox in transfected HEK293T cells (data not shown). Taken together, these findings suggest the possibility that the p47phox- and Gαi-containing complex may comprise two or more copies of p67phox.
It is presently unclear how the binding of Gαi to p67phox facilitates or influences assembly of the enzyme complex. One possibility is that this association promotes colocalization of the relevant interacting proteins in specific subcellular compartments, most likely in the cytosol or in the cytoskeletal fraction. Indeed, members of the large TPR-containing proteins, such as AGS3 (14, 30) and TPR1 (24), are emerging as novel adaptors and scaffolds for G protein signaling. A possible explanation for the increased interaction between p67phox and p47phox in the presence of the GDP-bound Gαi is that this G protein, through its association with p67phox, positions p67phox in close proximity to p47phox, thereby facilitating p47phox-p67phox interaction (Fig. (Fig.8B).8B). These interactions most likely occur within the cell, as both p67phox and p47phox are cytosolic factors, and their association precedes membrane translocation of the complex. Moreover, several groups have confirmed the presence of two distinct pools of Gαi2, which are located in the plasma membrane and the cytosol fractions of unstimulated neutrophils (4, 15, 37, 38). Studies have also shown Gαi binding to F-actin and tubulin in cytoplasmic structures and at the plasma membrane (34). The cytoskeleton provides a dynamic network between cellular structures and a docking surface for various signaling proteins, including Gαi, in response to cell activation (13). Consistent with these observations, p67phox and p47phox are principally recovered in the cytoskeletal fraction of unstimulated and stimulated neutrophils (26, 41, 45). Thus, reorganized cytoskeleton may provide a scaffold for activation of the NADPH oxidase components in response to cell stimulation (33). Taken together with these previous observations, our findings support the hypothesis that the association between Gαi and p67phox occurs in the cytosol of resting cells to favor the interaction between p67phox and p47phox, presumably involving the cytoskeleton. Upon stimulation, the readily mobilizable, preformed cytosolic phox complex can then translocate to the membrane for full assembly and activation of NADPH oxidase (18). Exactly how fMLF stimulation triggers activation of an intracellularly localized Gαi has not been investigated. The availability of Gαi as well as its activation state may also be influenced by multiple factors and regulated by fMLF. Indeed, Sarndahl et al. (39) showed activation and dissociation of cytoskeleton-bound Gαi upon fMLF stimulation.
In addition to p67phox and p47phox, neutrophils contain p40phox, a cytosolic factor copurified with p67phox (40). The exact function of p40phox in promoting or inhibiting neutrophil NADPH oxidase assembly remains debatable. Interestingly, the addition of p40phox to our system causes a change in preference of p67phox from the inactive to the active form of Gαi (Fig. (Fig.5B).5B). However, in the presence of p47phox, this change is reverted (Fig. (Fig.5C).5C). We speculate that under these conditions, p67phox, p47phox, and p40phox cooperate in a temporal and spatial manner to favor formation of the p67phox/GDP-bound Gαi. The mechanism underlying this event is currently unknown and is a subject of our ongoing research. Based on data obtained from the in vitro binding assay with GTPγS-loaded Gαi (Fig. (Fig.2),2), the coimmunoprecipitation between p67phox and the active form of Gαi2 in the presence of p40phox is most likely indirect and may require an unidentified factor (Fig. (Fig.8C).8C). Thus, the prospect of an additional cofactor(s) raises the order of complexity in the sequence of interactions that takes place in NADPH oxidase assembly.
The interaction between p67phox and Gαi may functionally impact NADPH oxidase assembly and activation. In the intact COS-phox cell-based assay, overexpression of Gαi2 promoted not only fMLF- but also PMA-stimulated O2− generation (Fig. (Fig.7).7). This observation supports the possibility that Gαi interaction with p67phox facilitates the formation of the p67phox-p47phox cytosolic complex, which favors NADPH oxidase activation. Stimulation of the Gαi-coupled FPR by fMLF not only activates Gα but also makes Gβγ available for its downstream effectors. Along with these events, the GTP-bound Gαi can dissociate from p67phox (Fig. (Fig.6).6). Therefore, in activated cells, the increased binding between p47phox and p67phox and enhanced NADPH oxidase activity can be attributed to a combination of factors, including signals arising from Gβγ. To our surprise, exogenous expression of p67phox dose dependently reduced cAMP basal levels in HEK293T cells, appearing as a positive regulator of the Gαi signaling pathway (Fig. (Fig.3A).3A). The cAMP-dependent protein kinase A is a characterized inhibitor of O2− production in inflammatory cells (3, 28). Although it is still unclear whether p67phox has an effect on cAMP in neutrophils, it is conceivable that a dampening of cAMP levels in neutrophils may contribute to priming for O2− generation. Given the ability of Gαi-coupled receptors, such as the interleukin-8 receptor, to potentiate NADPH oxidase activation, the direct and activation state-dependent interaction between p67phox and Gαi may represent another regulatory mechanism for NADPH oxidase activation.
We thank Mary Dinauer for providing the COS-phox cells and Rong He for helpful discussions.
This work was supported by NIH grants AI033503, HL077806, GM066182, and AR042426.