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Molecular and Cellular Biology, July 2006, p. 5190-5200, Vol. 26, No. 13
0270-7306/06/$08.00+0     doi:10.1128/MCB.01979-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Activation State-Dependent Interaction between Gi and p67^phox

Caroline Marty,¹ Tohru Kozasa,¹ Mark T. Quinn,² and Richard D. Ye^1*

Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612,¹ Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717²

Received 10 October 2005/ Returned for modification 3 November 2005/ Accepted 13 April 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 Gi subunit of heterotrimeric G proteins is a novel binding partner for p67^phox in transfected HEK293T cells and peripheral blood polymorphonuclear leukocytes. p67^phox preferably interacted with inactive Gi. Expression of p67^phox caused a dose-dependent decrease in intracellular cyclic AMP concentration, suggesting altered function of Gi. We identified a fragment of p67^phox, consisting of the PB1 domain and the C-terminal SH3 domain, to be critical for the interaction with Gi. Because these domains are involved in the interaction with p47^phox and p40^phox, the relationship between the respective binding events was investigated. Wild-type Gi, but not its QL mutant, could promote the interaction between p67^phox and p47^phox. However, the interaction between p67^phox and p40^phox was not affected by either Gi form. These results provide the first evidence for an interaction between p67^phox and an alpha subunit of heterotrimeric G proteins, suggesting a potential role for Gi in the regulation or activation of NADPH oxidase.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 gp91^phox and p22^phox, which form the redox core flavocytochrome b₅₅₈ of the enzyme, and the cytosolic factors p67^phox, p47^phox, and p40^phox (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).

p67^phox is a multidomain protein implicated in essential NADPH oxidase protein-protein interactions. Its activation domain binds to the catalytic core of gp91^phox 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 p40^phox (12), and the C-terminal Src-homology 3 (SH3) sequence is necessary for association with p47^phox (9). Therefore, p67^phox 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 Gß dimer that regulates a variety of effectors, such as phosphatidylinositol 3-kinase, phospholipase C-ß, and most likely specific Rac guanine nucleotide exchange factors, leading to oxidase activation. The Gi 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 G proteins besides the conventional Gß, 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 Gi/o-Loco (GoLoco)-containing proteins. RGS proteins attenuate G protein signaling by accelerating the intrinsic GTPase activity in G (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 Gi and act as guanine nucleotide dissociation inhibitors while impeding binding of Gß (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 p67^phox could interact with the alpha subunits of heterotrimeric G proteins, because p67^phox was previously shown to bind the small GTPase Rac (19, 22). We show that p67^phox directly interacts with GDP-bound Gi in both transfected cells and human neutrophils. The binding site for Gi was localized to the C-terminal SH3 domain on p67^phox. Overexpression of p67^phox in our system dose dependently decreased basal levels of cyclic AMP (cAMP), a readout for activation of Gi signaling and also a negative regulator of O₂^– production. Furthermore, the association of p47^phox with p67^phox was affected by the Gi activation state, suggesting that Gi not only is a binding partner of p67^phox but also may participate in the regulation of NADPH oxidase activation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibodies. Monoclonal antibody to c-MYC was purchased from Covance (Berkeley, CA) and that to FLAG from Sigma-Aldrich (St. Louis, MO). The anti-p67^phox monoclonal antibody was from BD Transduction Laboratories (Lexington, KY). Antibodies specific to Gi1 and Gi3 were from Santa Cruz Biotechnology (Santa Cruz, CA), as were the anti-p47^phox polyclonal and the anti-ß-actin monoclonal antibodies. The anti-Gi2 serum was produced in a rabbit (with amino acids 213 to 354 as an antigen), as was the anti-p67^phox serum (raised against the purified glutathione S-transferase [GST]-p67^phox fusion protein). The anti-p40^phox 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.

Culture and transient transfection of mammalian cells. 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.

Immunoprecipitation and Western blotting. 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 1x 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-Gi2 (1:250), or anti-p47^phox (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 5x 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 2x 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).

Preparation of human neutrophils. 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 x 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.

Expression and purification of recombinant GST fusion proteins in Escherichia coli and pulldown assay. The pGEX-2T and pGEX-p67^phox constructs, encoding GST and full-length GST-p67^phox fusion proteins, respectively, were introduced into the E. coli strain DH10B. Protein expression was induced with 0.1 mM isopropyl-1-thio-ß-D-galactopyranoside for 2 h at 30°C for GST-p67^phox 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, 1x 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 x g for 10 min at 4°C, the supernatant was snap-frozen for storage in 10% glycerol.

In vitro binding assay. Rat Gi1 purified from Sf9 cells was preloaded with either 30 µM GDP, 10 µM GTPS (guanosine 5'-[-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 Gi 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 Gi1 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 ß-mercaptoethanol) supplemented with either GDP, GTPS, or AMF where indicated for 2 h at 4°C. Beads were pelleted at 600 x 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 5x SDS-PAGE loading buffer was added to the eluate, and proteins were boiled for 10 min.

cAMP assay. 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).

Measure of NADPH oxidase activity. 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 x 10⁶ to 5 x 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The interaction between Gi2 and p67^phox is direct and depends on the G activation state. To evaluate the potential interaction of p67^phox and Gi, an expression construct was created to produce C-terminal MYC-tagged p67^phox (p67^phox-MYC). Moreover, activity of recombinant p67^phox-MYC was verified in a whole cell-based reconstitution assay, which required exogenous p67^phox for fMLF- and PMA-induced O₂^– generation (11). The p67^phox-MYC DNA construct was transiently cotransfected with vectors encoding each of the three isoforms of Gi into HEK293T cells, which do not express any of the NADPH oxidase components except for Rac1. Both inactive wild-type Gi (Giwt) and the GTPase-deficient (constitutively active) Gi mutants (Q204L for Gi1 and Gi3 and Q205L for Gi2) were examined. Twenty-four hours after transfection, p67^phox-MYC was immunoprecipitated from cell lysates, and the precipitates were analyzed by immunoblotting. Surprisingly, Gi1wt, Gi2wt, and Gi3wt were all detected in the immunoprecipitates (Fig. 1). Additionally, we also observed a modest interaction between p67^phox and the endogenous Gi (vector, mock-transfected cells). Interestingly, the interaction between p67^phox and the active GiQL was significantly reduced to levels as low as or even below those of the vector controls (Fig. 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 Gs could associate with p67^phox (data not shown).

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FIG. 1. In vivo interaction between Gi and p67phox. (A) HEK293T cells were transiently transfected with DNA encoding a C-terminal MYC-tagged p67phox protein either alone (Vector) or together with individual expression vector encoding the inactive form (wt) or GTPase-deficient mutant (QL) of the three isoforms Gi1, Gi2, and Gi3, as indicated. Cell lysates were immunoprecipitated with an anti-MYC MAb. Immunoprecipitates (IP, top panels) and cell homogenates (bottom panels) were analyzed by Western blotting (IB) with the respective specific anti-Gi sera. p67phox-MYC in the IP serves as a loading control (middle panels). In the absence of p67phox-MYC, none of the Gi are detectable in the IP fractions (not shown). (B) Human neutrophils were isolated from peripheral blood as described in Materials and Methods. Cells were treated with 1 µM fMLF for 5 min at 37°C or not treated. Cell lysis and immunoprecipitation with an anti-Gi2 serum were conducted in the absence or presence of AMF. The ability of endogenous p67phox to bind to Gi2 was assessed by blotting (IB) the immunoprecipitates (IP) with an anti-p67phox MAb (top panel). Levels of p67phox and Gi2 expression in neutrophils were detected in homogenates (two bottom panels). Gi2 in the IP serves as a loading control (second panel from top). Data shown are representative of a typical experiment.

Since the resting and activated NADPH oxidase states are tightly regulated by complex protein-protein interactions (35), we examined whether the binding between p67^phox and Gi 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 Gi2 in the absence or presence of AMF, which mimics the transition state of GTP hydrolysis. As expected from the previous observations, an interaction between p67^phox and Gi2 was detectable in nontreated cell lysates and was markedly diminished in the presence of AMF (Fig. 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-Gi2 serum. In this case too, the interaction between the endogenous p67^phox and Gi2 was markedly decreased compared to that observed in unstimulated neutrophils (Fig. 1B, lane 3 versus lane 1). Thus, the binding of p67^phox with Gi2 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-p67^phox and Sf9-expressed Gi1 (Fig. 2). The GST-p67^phox fusion protein coupled to glutathione-Sepharose beads, but not the GST control protein, specifically bound Gi1 in the presence of GDP but not when the Gi was preloaded with either AMF or GTPS. Thus, these data demonstrate a direct interaction between p67^phox and Gi and again show that the interaction is modulated by the Gi activation state.

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FIG. 2. Direct interaction between Gi1 and p67phox in vitro. Binding between 100 pmol of purified bacterially expressed GST or GST-p67phox fusion protein immobilized on glutathione-Sepharose beads and 2.5 pmol of purified Gi1 preloaded with either GDP, AMF, or GTPS was assayed as described in Materials and Methods. Bound proteins were eluted, resolved by 10% polyacrylamide SDS-PAGE, and analyzed by blotting with an anti-Gi1 serum (top panel). Gi1 input was verified in each sample (middle panel), and both GST and GST-p67phox used for the pulldown were detected with Coomassie blue staining and served as a loading control (bottom panel). Blots shown are representative of a typical experiment.

Expression of p67^phox partially inhibits cAMP formation. Activation of the Gi 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 p67^phox in the Gi activation/inactivation cycle in a receptor-independent manner, we examined the effect of p67^phox on the Gi-mediated cAMP basal level. In HEK293T cells transfected to express increasing amounts of untagged p67^phox, cAMP production was dose dependently inhibited (Fig. 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).

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FIG. 3. Dose-dependent effect of p67phox on Gi activity and interaction with Gß. (A) Accumulated cAMP levels were measured in vector- versus p67phox-transfected HEK293T cells. Increasing levels of p67phox expression and equivalent amounts of endogenous Gi2 in each sample were determined by immunoblotting with the respective antisera. Values for cAMP concentration were expressed as a percentage relative to the basal level (vector), which was assigned a value of 100%. The histograms show the means ± standard errors of the means of triplicate experiments (n = 3). Student's t test was used to compare the significance of the decrease to the basal level (*, P < 0.01; **, P < 0.001). (B) FLAG-tagged Gß1 and G2 were coexpressed alone (Vector) and either with Gi2wt or in combination with Gi2wt and two different concentrations of p67phox-MYC as indicated. Cell homogenates were subjected to immunoprecipitation (IP) with an anti-FLAG MAb. The integrity of Gi/Gß interaction was verified by blotting the immunoprecipitates with an anti-Gi2 serum (top panel). The homogenates were analyzed for protein expression (two bottom panels), and total FLAG-Gß1 in the immunoprecipitates serves as a loading control (middle panel). (C) HEK293T cells were transiently transfected to express a C-terminal MYC-tagged p67phox either alone (Vector) or with Gß1 and G2 in the absence or presence of exogenous wild-type Gi2, which directly interacts with Gß. The cell homogenates were subjected to immunoprecipitation using an anti-MYC MAb and analyzed by Western blotting (IB). Blots shown were reproduced in three independent experiments.

Heterotrimeric G proteins are composed of the guanine nucleotide-binding G subunit and the Gß dimer, which are regarded as one functional unit. G proteins are inactive in the GDP-bound state. Since p67^phox preferentially binds to inactive Gi, we expected that it might compete with Gß for association with GDP-bound Gi. Surprisingly, in transiently transfected HEK293T cells, increasing amounts of p67^phox-MYC did not disrupt the interaction between Gß12 and Gi (Fig. 3B). These observations suggest that p67^phox most likely binds to Gi on a site distinct from the Gß contact surface. Indeed, overexpression of p67^phox showed no effect on the basal phosphorylation of Akt, a characterized readout for Gß activation (data not shown). Finally, we verified that the ability of Gß to coimmunoprecipitate p67^phox was mediated by the inactive Gi (Fig. 3C). Indeed, FLAG-tagged Gß1 was found to bind to p67^phox only when Gi2 was overexpressed in the cells. Endogenous Gi appeared to be insufficient to mediate the association between FLAG-Gß1 and p67^phox-MYC, most likely because it already forms heterotrimers with endogenous Gß proteins, which cannot be detected with the anti-FLAG monoclonal antibody (MAb). Based on these data, we concluded that the association between Gß and p67^phox is indirect and mediated through the inactive, GDP-bound Gi.

Determination of Gi binding site on p67^phox. p67^phox is a multidomain protein (Fig. 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 gp91^phox (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 p40^phox and p47^phox, respectively (9, 12). In an attempt to localize the region of p67^phox required for Gi binding, a series of truncated MYC-tagged p67^phox mutants was generated (Fig. 4A), verified by sequencing, and separately expressed into HEK293T cells together with Gi2wt (Fig. 4B). In immunoprecipitation and immunoblotting experiments, the p67^phox C-terminal fragment (amino acids 213 to 526) precipitated Gi2 as effectively as did full-length p67^phox, whereas the N-terminal segment (amino acids 1 to 213) did not retain binding to the G protein (Fig. 4B, lanes 2 and 3 versus lane 1). Furthermore, the fragment covering residues 340 to 526 was shown to be sufficient for interaction with Gi2, whereas the sequence extending from amino acids 303 to 455 did not bind Gi2. Therefore, at first view the C-terminal SH3 was the most likely candidate for the Gi binding site, and two additional p67^phox mutants that both contained PB1, p67^phox(1-430)-MYC and p67^phox(460-515)-MYC, seemed to confirm this conclusion. However, the C-terminal SH3 and its flanking regions (residues 429 to 526) failed to coimmunoprecipitate Gi2. Therefore, the C-terminal SH3 in p67^phox could be necessary but not sufficient for recruitment of Gi, and perhaps both PB1 and the second SH3 in p67^phox contribute to the binding interaction with Gi.

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FIG. 4. Identification of Gi2 binding site on p67phox. (A) Schematic representation of full-length p67phox and the truncated mutants generated. All proteins were MYC tagged at either the N or the C terminus. AD, activation domain; PR, proline-rich region; SH3, Src-homology 3 domain; PB1, phox and bem1 domain. (B) The full-length p67phox and deletion p67phox mutants were overexpressed in HEK293T cells together with Gi2wt. Cell lysates were precipitated with anti-MYC MAb. Western blotting (IB) of the immunoprecipitates (IP) with an anti-p67phox serum, generated against the full-length protein, identified proper expression of the various constructs and served as a loading control (middle panel). The ability of the mutants to retain Gi2wt binding was examined by blotting the IP with the anti-Gi2 serum (top panel). Equal levels of Gi2 expression were assessed by probing the cell homogenates with the anti-Gi2 serum (bottom panel). Data shown were reproduced in at least three independent experiments.

The interaction between p47^phox and p67^phox but not between p40^phox and p67^phox is dependent on the Gi activation state. Since the C-terminal SH3 domain of p67^phox can interact directly with p47^phox and its PB1 domain associates with the phox and Cdc (PC) domain of p40^phox (9, 12), we investigated whether Gi2 overexpression could influence these binding interactions. p67^phox and p47^phox were coexpressed in HEK293T cells together with Gi2 (Fig. 5A). Interestingly, the wild type but not the GTPase-deficient mutant of Gi2 increased the interaction between p67^phox and p47^phox (Fig. 5A, top panel, lanes 4 and 5 versus lane 3). However, in the presence of p47^phox the association between p67^phox and Gi2 was barely detectable (Fig. 5A, second panel from top, compare lanes 4 and 5 to lanes 1 and 2). This finding suggests that p47^phox competes with Gi for binding to p67^phox.

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FIG. 5. Effect of Gi2 activation state on p47phox and p40phox interactions with p67phox. (A) The p67phox-MYC protein was transiently expressed in HEK293T cells in combination with the NADPH oxidase component p47phox and/or Gi2 as indicated. The cell homogenates were subjected to immunoprecipitation with anti-MYC MAb and analyzed by Western blotting. Total p67phox-MYC in the precipitates (IP) was shown by probing the blot (IB) with an anti-MYC MAb (middle panel). The IP (two upper panels) and cell homogenates (two lower panels) were probed with an anti-p47phox polyclonal Ab and an anti-Gi2 serum for the ability to coimmunoprecipitate with p67phox-MYC and for their expression in the cells. The same experiment was performed with p40phox instead of p47phox (B) and with both p47phox and p40phox together (C). Results are representative of at least three independent experiments.

Similar experiments were conducted in cells expressing p67^phox, Gi2, and p40^phox (Fig. 5B). The interaction between p67^phox and p40^phox was not affected by the expression of either Gi2wt or Gi2QL (Fig. 5B, top panel, lanes 3, 4, and 5). Surprisingly, p67^phox, which preferentially binds to inactive Gi in the absence of p40^phox, switched its affinity for the active form of the Gi protein when p40^phox was present (Fig. 5B, second panel from top, lanes 1 and 2 compared to lanes 4 and 5). The decreased but still apparent coimmunoprecipitation between p67^phox and inactive Gi in the presence of p40^phox suggests that p40^phox might only partially displace Gi from p67^phox.

Finally, all four binding partners were coexpressed together (Fig. 5C). Interestingly, in the presence of both p47^phox and p40^phox, the characteristic difference in p67^phox binding to Gi2wt versus Gi2QL remained (Fig. 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 p67^phox-containing complexes.

The phox components p67^phox and p47^phox associate with Gi in one large complex. In HEK293T cells and in the absence of p40^phox, p47^phox was found to completely block the binding of Gi2 to p67^phox (Fig. 5A). However, when the three phox proteins and Gi2 were coexpressed, as a closer mimic of human neutrophils, both p47^phox and Gi2 coimmunoprecipitated with MYC-tagged p67^phox (Fig. 5C). Therefore, we tested whether p67^phox, p47^phox, and Gi2 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 p47^phox and Gi2 (Fig. 6). Both Gi2 and p67^phox were detected in the anti-p47^phox precipitates in either resting or fMLF-stimulated cells. Similarly, both p47^phox and p67^phox coimmunoprecipitated with Gi2. Interestingly, the interaction between p47^phox and p67^phox increased upon fMLF stimulation, with Gi2 concomitantly dissociating from the complex (Fig. 6, "IP: p47^phox" set of panels). As expected, p67^phox interaction with Gi2 markedly diminished after fMLF treatment and so did the association between p47^phox and Gi2 (Fig. 6, "IP: Gi2" set of panels). p67^phox most likely bridges p47^phox and Gi2, causing them to coimmunoprecipitate. Indeed, we have not observed any interaction between Gi2 and p47^phox in the absence of p67^phox in HEK293T cells (data not shown). Taken together, these results support the presence of a multiprotein complex containing p47^phox, Gi, and p67^phox.

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FIG. 6. Association of Gi2, p67phox, and p47phox in one large complex. Purified human neutrophils were either unstimulated or stimulated with 1 µM fMLF for 5 min and lysed. Proteins from cell homogenates were precipitated (IP) with either an anti-p47phox polyclonal antibody or an anti-Gi2 serum, followed by Western blotting (IB) using specific antibodies against each component as indicated. The expression levels in neutrophils of the three proteins were detected by probing the homogenates with the respective antibodies (panels on the right side). Data are representative of similar results obtained in three independent experiments.

Functional implications of Gi interaction with p67^phox on NADPH oxidase activity. 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 gp91^phox, p22^phox, p67^phox, and p47^phox were transiently transfected with DNA coding for the formyl peptide receptor (FPR), as well as protein kinase C (PKC) and p40^phox, both being highly abundant in neutrophils but scarce in COS7 cells. Where indicated, Gi2 was overexpressed in the cells. We verified by Western blotting that exogenous Gi2 did not affect the relative levels of expression of any of the three cytosolic phox components (Fig. 7C). The transfected cells were then challenged with either 1 µM fMLF (Fig. 7A, left graph) or 200 ng/ml PMA (Fig. 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 Gi2 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 Gi for FPR, the increase in O₂^– generated by PMA stimulation indicates that Gi possibly plays a positive regulatory role in NADPH oxidase activation.

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FIG. 7. Functional relevance of Gi overexpression in NADPH oxidase activity. COS-phox cells were transiently transfected with vectors coding for FPR, PKC, and p40phox in the absence or presence of overexpressed Gi2. (A) Cells were stimulated with either 1 µM fMLF (left panel) or 200 ng/ml PMA (right panel), as indicated by the black arrow, and monitored for O2– generation. (B) Histograms showing CL integrated for 30 min after stimulation as means ± standard deviations of two independent experiments performed in duplicate. (C) Western blots indicating expression of Gi2, p40phox, and the stably expressed p67phox and p47phox in COS-phox cells transfected with DNA encoding Gi2 or not transfected, using specific antibodies. ß-Actin serves as a loading control.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 Gß originating from activation of heterotrimeric Gi proteins (5). However, it is unclear whether the Gi 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 Gi as a direct p67^phox binding partner. To our knowledge, this is the first report that suggests a potential role for Gi in oxidase enzyme assembly and raises the possibility that Gi 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 Gi and the cytosolic factor p67^phox. This binding exists not only in transiently transfected cells (Fig. 1A) but also, and more importantly, in human neutrophils (Fig. 1B). The interaction significantly decreases upon activation of Gi, suggesting that p67^phox can recognize the conformational changes associated with Gi activation. Since p67^phox does not appear to compete with Gß for association with GDP-bound Gi (Fig. 3B), the binding site for p67^phox on Gi must be different from the Gi/Gß interface. Furthermore, there is no observable difference in Gi2 and p67^phox 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 Gi subunit that leads to uncoupling from the receptor. Thus, the C-terminal sequence of Gi does not appear to be involved in the interaction with p67^phox.

The possible relationship between Gi and the three cytosolic factors of NADPH oxidase is depicted in Fig. 8. Analysis of the deletion mutants of p67^phox revealed that the C-terminal SH3 motif of p67^phox (Fig. 8A), previously characterized as a direct binding site for p47^phox (9), also contributes to the recruitment of Gi (Fig. 4). Indeed, p47^phox can fully compete inactive Gi off of p67^phox (Fig. 5A). However, in neutrophils p67^phox clearly binds better with inactive Gi (Fig. 1B), even in the presence of endogenous p40^phox and p47^phox. Moreover, Gi2, p47^phox, and p67^phox were found to associate in the same complex (Fig. 6). Therefore, we tested whether coexpression of p67^phox, p47^phox, and p40^phox in HEK293T cells, together with Gi2, would mimic what we observed in neutrophils (Fig. 5C). Indeed, the preference of p67^phox for inactive Gi2 is maintained in the presence of both p47^phox and p40^phox. In addition, the binding of p47^phox and Gi2 to p67^phox is no longer mutually exclusive in the presence of p40^phox. Coimmunoprecipitation assays performed in neutrophils have confirmed the presence of a multisubunit complex containing p47^phox, p67^phox, and Gi2 (Fig. 6). However, it is possible that only subpopulations of p67^phox and Gi are associated at a given time, and it is also likely that a variety of multimers coexists in the cell. Additionally, evidence suggesting that p67^phox can homodimerize may support the observation that p47^phox and Gi are both present in the same large complex with p67^phox and most likely have overlapping binding sites on p67^phox. Although the intriguing prospect that p67^phox can homodimerize has been approached in only a few biochemical studies drawing contradictory conclusions (10, 21), we have observed homodimerization of p67^phox in transfected HEK293T cells (data not shown). Taken together, these findings suggest the possibility that the p47^phox- and Gi-containing complex may comprise two or more copies of p67^phox.

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FIG. 8. Model for the participation of Gi in interactions between NADPH oxidase components. (A) Linear representations of p47phox, p67phox, and p40phox. The C-terminal SH3 domain of p67phox interacts with the proline-rich (PR) region of p47phox, and the PB1 domain binds to the phox and cdc motif (PC) of p40phox. AD, activation domain; SH3, Src-homology 3 domain; PB1, phox and bem1 domain; PX, phox domain. (B) A potential role of Gi in targeting p67phox to specific subcellular compartments. Inactive Gi, tethered to either the membrane or the cytoskeleton, may position p67phox in close proximity to p47phox and promote their interaction. The association between the two phox proteins then competes Gi off of p67phox. (C) The recruitment of p40phox to p67phox is not significantly influenced by Gi. p40phox may only partially displace Gi from p67phox, allowing the formation of various complexes which may or may not include Gi. Based on our experimental results, we hypothesize that upon activation of the G protein an additional partner may be involved in the association between the active Gi and p67phox.

It is presently unclear how the binding of Gi to p67^phox 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 p67^phox and p47^phox in the presence of the GDP-bound Gi is that this G protein, through its association with p67^phox, positions p67^phox in close proximity to p47^phox, thereby facilitating p47^phox-p67^phox interaction (Fig. 8B). These interactions most likely occur within the cell, as both p67^phox and p47^phox are cytosolic factors, and their association precedes membrane translocation of the complex. Moreover, several groups have confirmed the presence of two distinct pools of Gi2, which are located in the plasma membrane and the cytosol fractions of unstimulated neutrophils (4, 15, 37, 38). Studies have also shown Gi 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 Gi, in response to cell activation (13). Consistent with these observations, p67^phox and p47^phox 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 Gi and p67^phox occurs in the cytosol of resting cells to favor the interaction between p67^phox and p47^phox, 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 Gi has not been investigated. The availability of Gi 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 Gi upon fMLF stimulation.

In addition to p67^phox and p47^phox, neutrophils contain p40^phox, a cytosolic factor copurified with p67^phox (40). The exact function of p40^phox in promoting or inhibiting neutrophil NADPH oxidase assembly remains debatable. Interestingly, the addition of p40^phox to our system causes a change in preference of p67^phox from the inactive to the active form of Gi (Fig. 5B). However, in the presence of p47^phox, this change is reverted (Fig. 5C). We speculate that under these conditions, p67^phox, p47^phox, and p40^phox cooperate in a temporal and spatial manner to favor formation of the p67^phox/GDP-bound Gi. 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 GTPS-loaded Gi (Fig. 2), the coimmunoprecipitation between p67^phox and the active form of Gi2 in the presence of p40^phox is most likely indirect and may require an unidentified factor (Fig. 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 p67^phox and Gi may functionally impact NADPH oxidase assembly and activation. In the intact COS-phox cell-based assay, overexpression of Gi2 promoted not only fMLF- but also PMA-stimulated O₂^– generation (Fig. 7). This observation supports the possibility that Gi interaction with p67^phox facilitates the formation of the p67^phox-p47^phox cytosolic complex, which favors NADPH oxidase activation. Stimulation of the Gi-coupled FPR by fMLF not only activates G but also makes Gß available for its downstream effectors. Along with these events, the GTP-bound Gi can dissociate from p67^phox (Fig. 6). Therefore, in activated cells, the increased binding between p47^phox and p67^phox and enhanced NADPH oxidase activity can be attributed to a combination of factors, including signals arising from Gß. To our surprise, exogenous expression of p67^phox dose dependently reduced cAMP basal levels in HEK293T cells, appearing as a positive regulator of the Gi signaling pathway (Fig. 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 p67^phox 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 Gi-coupled receptors, such as the interleukin-8 receptor, to potentiate NADPH oxidase activation, the direct and activation state-dependent interaction between p67^phox and Gi 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.

 
* Corresponding author. Mailing address: Department of Pharmacology, University of Illinois at Chicago, 835 S. Wolcott Avenue, Chicago, IL 60612. Phone: (312) 996-5087. Fax: (312) 996-7857. E-mail: yer{at}uic.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Molecular and Cellular Biology, July 2006, p. 5190-5200, Vol. 26, No. 13
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