Combination of arachidonic acid and guanosine 5'-O-(3-thiotriphosphate) induce translocation of rac p21s to membrane and activation of NADPH oxidase in a cell-free system

The superoxide-generating NADPH oxidase system in phagocytes consists of membrane-associated cytochrome b558 and three cytosolic components named p67-phox, p47-phox, and rac p21s. In a cell-free system consisting of membrane and cytosol, the oxidase can be activated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and an unsaturated fatty acid such as arachidonic acid (AA). Incubation of cytosol and membrane with AA alone caused clear translocation of p47-phox and p67-phox to the membrane, but only slight translocation of rac p21s. GTP gamma S alone did not significantly induce the translocation of rac p21s. However, GTP gamma S in combination with AA markedly augmented rac p21s translocation to the membrane. The translocation of rac p21s is not induced by GDP or GDP beta S. These results indicate that the GTP-bound active form of rac p21s is the entity that is translocated to the membrane by the action of AA.     

Effects of vanadyl sulfate on carbohydrate and lipid metabolism in patients with non-insulin-dependent diabetes mellitus

Mechanisms for the cell-free activation of NADPH oxidase by sodium dodecyl sulfate (SDS) and arachidonate were compared in relation to their responsiveness to short chain diacylglycerols. The plasma membrane and cytosol prepared from guinea pig neutrophils were used for the cell-free system. The activation of NADPH oxidase by SDS was enhanced about 5- to 10-fold by 1,2-dioctanoylglycerol (diC8), but not by either 1,2-dihexanoylglycerol (diC6) or 1,2-didecanoylglycerol (diC10). However, none of these diacylglycerols potentiated the NADPH oxidase activation by arachidonate. The maximal extent of activation by the combination of SDS and diC8 was similar to that by arachidonate alone. In the presence of sufficient amounts of diC8 and SDS, GTP gamma S potentiated the activation of NADPH oxidase. The potentiating activity of diC8 was preserved in the membrane fraction, not in the cytosol fraction. These results suggest that arachidonate may possess the functions of both SDS and diC8 in the activation. In addition, diC8 and GTP gamma S seem to independently enhance the NADPH oxidase activation.     

Possible target components for the inhibitory effect of N-ethylmaleimide on the activation of neutrophil NADPH oxidase

Potential target components for the inhibitory effect of covalent sulfhydryl-modifying reagent N-ethylmaleimide (NEM) on the activation of NADPH oxidase in human neutrophils was studied in a cell-free system. The capacity of both cytosol and membrane fractions to induce the translocation of cytosolic components and O2-generation in the cell-free activation system was affected by NEM. The phosphorylation of p47phox, which mediates the translocation of cytosolic complex, by protein kinase C was not inhibited by NEM and NEM-treated p47phox was as effective as untreated p47phox both in the kinase-dependent and in the amphiphile-dependent cell-free activation systems. In addition, phosphatidic acid-dependent phosphorylation of cytosol including p47phox was not affected by NEM. The inhibition of cytosol's capacity to activate NADPH oxidase was partially reversed by an addition of the fraction containing G-protein rac. Taken together, the data suggest that membrane component cytochrome b558 and cytosolic component rac may be the potential targets for the NEM effect on the activation of NADPH oxidase.     

Ras effector-homologue region on Rac regulates protein associations in the neutrophil respiratory burst oxidase complex

Rac, a small molecular weight GTPase in the Ras superfamily, participates in the activation of the multicomponent superoxide-generating NADPH oxidase of human neutrophils. Rac is 30% identical to Ras overall, but is 75% identical within the sequence corresponding to the effector region of Ras, which regulates mitogenesis through interactions with the protein kinase Raf1. We investigated the role of this region in Rac1 using site-directed mutagenesis. In a cell-free semirecombinant NADPH oxidase system, mutants in the 26, 33, 38, and 45 amino acids showed 20-110-fold reduced binding to the oxidase complex as judged by EC50 values and reduced (44-80%) maximal activities in superoxide generation. Only the GTP gamma S-bound form associated, since the GDP-bound form of Rac neither activated alone nor competed with GTP gamma S-Rac. EC50 values for neither p47-phox nor p67-phox were affected when mutant Racs were used in place of Rac. Data indicate direct binding of the Rac effector region to one or more components of the respiratory burst oxidase. Results indicate a general role for conserved effector-equivalent regions in small GTPases in the regulation of protein-protein interactions.     

Characterization of the mechanism of action of tachykinins in rat striatal cholinergic interneurons

Plasma membranes of neutrophil cells contain the redox component of the O2(-)-generating NADPH oxidase complex, namely a heterodimeric flavocytochrome b consisting of an alpha subunit of 22 kDa and a beta subunit of 85-105 kDa of a glycoprotein nature. The NADPH oxidase is dormant in resting neutrophils. When neutrophils are exposed to a variety of particulate or soluble stimuli, the oxidase becomes activated, due to the assembly on the membrane-bound flavocytochrome b of three cytosolic factors, p47phox, p67phox and Rac 2 (or Rac 1). The effect of phenylarsine oxide (PAO), which reacts specifically with vicinal and neighbouring thiol groups in proteins, was assayed on the NADPH oxidase activity of bovine neutrophils, elicited after activation of the oxidase in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils, GTP[S], ATP and arachidonic acid; the effect of PAO on the oxidase activation itself was measured independently. PAO preferentially inhibited oxidase activation rather than the elicited oxidase activity, and inhibition resulted from binding of PAO to the membrane component of the cell-free system. To determine the PAO-binding protein responsible for the loss of oxidase activation, we used photoaffinity labeling with a tritiated azido derivative of PAO, 4-[N-(4-azido-2-nitrophenyl)amino-[3H]acetamido]phenylarsine oxide, ([3H]azido-PAO). Photoirradiation of plasma membranes from resting neutrophils in the presence of [3H]azido-PAO resulted in the prominent labeling of a protein of 85-105 kDa whose migration on SDS/PAGE coincided with that of the beta subunit of flavocytochrome b as identified by immunoreaction. Upon deglycosylation, the photolabeled band at 85-105 kDa was shifted to 50-60 kDa as was the immunodetected beta subunit. Similar results were obtained with isolated flavocytochrome b in liposomes. Photoaffinity labeling of the beta subunit of the membrane-bound flavocytochrome b or the isolated flavocytochrome b in liposomes resulted in abolition of oxidase activation in the reconstituted cell-free system. Incorporation of [3H]azido-PAO into flavocytochrome b was negligible when photoaffinity labeling was performed on neutrophil membranes that had been previously activated. The results suggest that the beta subunit of flavocytochrome contains two target sites for PAO which are accessible in resting neutrophils, but not in activated neutrophils.     

Euthanasia: reconciling culture and human rights

Effects of the farnesylcysteine mimetic, farnesylthiosalicylate on the activation of myeloid cells were studied. In dimethyl-sulfoxide-differentiated HL60 cells and in human neutrophils farnesylthiosalicylate (< or = 20 microM) dose-dependently elevated cytosolic Ca2+ concentrations, suggesting phospholipase-C-mediated release of the ion from intracellular stores. In human neutrophils, in addition to the production of inositol trisphosphate, farnesylthiosalicylate induced activation of the NADPH oxidase and translocation of the cytosolic oxidase components p47-phox and p67-phox to the membrane. The calcium signal, inositol-trisphosphate production and superoxide generation elicited by farnesylthiosalicylate were partially blocked by treatment of the cells with pertussis toxin, consistent with participation of pertussis-toxin-sensitive and pertussis-toxin-resistant elements. In HL60 cells, farnesylthiosalicylate (< or = 20 microM) did not activate NADPH oxidase but dose-dependently augmented PMA-elicited activity of the enzyme. This effect was resistant to pertussis-toxin treatment. In vitro augmentation of PKC-mediated phosphorylation of histone and cytosolic p47-phox by farnesylthiosalicylate and the finding that downregulation of PKC abrogated potentiation of NADPH oxidase activity by farnesylthiosalicylate were compatible with the involvement of PKC in the response of HL60 cells to farnesylthiosalicylate. It is suggested that the effects of farnesylthiosalicylate on myeloid cells reflect interaction of the analog with prenylcysteine-docking sites on cellular signaling elements.     

Conformational changes of the leukocyte NADPH oxidase subunit p47(phox) during activation studied through its intrinsic fluorescence

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O-2 from oxygen using NADPH as the electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid, as an activating agent. In whole cells and under certain circumstances in the cell-free system the phosphorylation of p47phox mediates the activation process. It has been proposed that conformational changes in the protein structure of cytosolic factor p47phox may be an important part of the activation mechanism. We show here that the total protein steady-state intrinsic fluorescence (an emission maximum of 338 nm) exhibited by the tryptophan residues of p47phox substantially decreased when p47phox was treated with anionic amphiphiles. A similar decrease in fluorescence was also observed when p47phox was phosphorylated with protein kinase C. Furthermore, a red shift of emission maximum and an increase of quenching by ionic quenchers and acrylamide were observed in the presence of activators. These results indicate the occurrence of a conformational change in the protein structure of p47phox. We propose that this alteration in conformation results in the appearance of a binding site through which p47phox interacts with cytochrome b558 during the activation process.     

Translocation of recombinant p47phox cytosolic component of the phagocyte oxidase by in vitro phosphorylation

Activation of superoxide-generating NADPH oxidase system of human neutrophils involves phosphorylation-dependent translocation of p47phox and other cytosolic components to the plasma membrane. In contrast to the stimulation of the NADPH oxidase in intact cells, however, the activation of cell-free system requires the addition of anionic amphiphiles such as sodium dodecyl sulfate (SDS) and arachidonate. In this system, translocation of p47phox is also an essential step for activation, but phosphorylation is not required. The basis of this difference in oxidase activation is not yet clear. We now report that in a cell-free oxidase system, phosphorylated recombinant p47phox can be translocated to the membrane in the absence of SDS or arachidonate. These findings suggest that both phosphorylation and SDS could cause a common change in conformation or charge of p47phox that may result in the association of p47phox with the plasma membrane.     

Spontaneous congenital hydrocephalus in the mutant mouse hyh. Changes in the ventricular system and the subcommissural organ

Like neutrophils, Epstein-Barr virus (EBV)-immortalized B lymphocytes express all constituents of the NADPH oxidase complex necessary to generate superoxide anion O2-. The NADPH oxidase activity in EBV-B lymphocytes is only 5% of that measured in neutrophils upon PMA stimulation. Cytochrome b558 is the sole redox membrane component of NADPH oxidase; it is the protein core around which cytosolic factors assemble in order to mediate oxidase activity. In the present study, we have compared the structural and functional properties of cytochrome b558 from EBV-B lymphocytes and neutrophils. Cytochrome b558 from EBV-B lymphocyte plasma membrane, like that from neutrophils, is characterized by a heterodimeric structure with a highly glycosylated beta subunit, known as gp91-phox. While the amount of cytochrome b558 recovered after purification from EBV-B lymphocytes (approximately 0.24 nmol from 1010 cells) was low compared to that recovered from neutrophils (approximately 10 nmol), the biochemical properties of purified cytochrome b558 from both EBV-B lymphocytes and neutrophils were quite similar with respect to their differential spectra, redox potential, and FAD binding site. Once cytochrome b558 was extracted from the EBV-B lymphocyte membrane, it was able to mediate, in a reconstituted system of O2- production the same oxidase turnover as that found for cytochrome b558 extracted from neutrophils. A comparison between membrane bound and soluble cytochrome b558 suggested that the weak oxidase activity measured in intact EBV-B cells might be the result not only of the small amount of expressed cytochrome b558, but also of a defect of the activation process in lymphocyte membrane.     

Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences

Neutrophils play an essential role in the cellular defense of the bovine mammary gland and compromised leukocyte function has been linked to the development of bovine mastitis. During mastitis, large numbers of leukocytes migrate into the mammary tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion-generating complex known as the NADPH oxidase. The key membrane-associated component of the NADPH oxidase is flavocytochrome b, which is a heterodimer of p22-phox and gp91-phox. Currently, only the human, porcine, murine, and rattus p22-phox and the human, porcine, and murine gp91-phox gene sequences are known. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and analyze expression of bovine flavocytochrome b. Using polymerase chain reaction cloning techniques and a bovine spleen cDNA library we have cloned both of the bovine flavocytochrome b subunits, p22-phox and gp91-phox. Comparison of the bovine sequences with those of other species also revealed important information regarding key structural features of gp91-phox and p22-phox, including location of putative glycosylation sites. This study greatly contributes to our understanding of the potential functional sites of the flavocytochrome b subunits as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases such as mastitis.     

Yersinia-hsp60-reactive T cells are efficiently stimulated by peptides of 12 and 13 amino acid residues in a MHC class II (I-Ab)-restricted manner

Generation of the microbicidal oxidative burst in human neutrophils requires participation of four proteins, a membrane bound flavocytochrome beta-558, two soluble proteins termed p47-phox and p67-phox, and the Ras-related GTPase Rac. Because plant cells exposed to pathogens produce a similar oxidative burst, we have looked for similarities between the oxidase complexes of the two systems. Antibodies against human neutrophil p47-phox and p67-phox were used to immunoblot cell extracts from several plant cell lines and were found to cross-react with proteins of the same molecular weight. Furthermore, plant cell lines not previously shown to produce an oxidative burst, yet found to express these immunoreactive proteins, rapidly generated hydrogen peroxide in response to elicitation. Finally, diphenylene iodonium (DPI) and alpha-naphthol, known specific inhibitors of the NADPH oxidase in neutrophils, also inhibited the oxidative burst in soybean cell suspensions with similar Ki values (about 15 microM and 30 microM respectively). These results provide evidence for involvement of proteins related to the neutrophil oxidase complex in the defense-related oxidative burst of plants.     

Guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system

BACKGROUND & AIMS: Superoxide anion (O2-) plays an important role in gastric pathophysiology. The aims of this study were to identify O2--producing activity in gastric mucosal cells and to elucidate its possible roles in inflammatory responses of the cells. METHODS: The amount of O2- was measured by the reduction of cytochrome c, and O2--producing cells were visualized by nitroblue tetrazolium reaction. Cytosolic components of the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were detected by immunoblotting and immunocytochemical analyses with antibodies against p47-phox and p67-phox. RESULTS: Gastric pit cells, but not parietal cells, spontaneously released O2- at 50 nmol . mg protein-1 . h-1. NADPH or guanosine 5'-O-(3-thiotriphosphate) increased the release more than threefold, whereas diphenylene iodonium inhibited it. A reconstituted cell-free system showed that both membrane fraction and neutrophil-related cytosolic components were required for the activity. p47-phox and p67-phox were expressed in the cells. Live Helicobacter pylori organisms and their culture supernatants significantly increased the O2- release. Furthermore, H. pylori lipopolysaccharide could enhance the release more effectively than Escherichia coli lipopolysaccharide. The O2--dependent activation of nuclear factor kappaB occurred in these primed cells. CONCLUSIONS: Gastric pit cells may actively regulate inflammatory responses of gastric mucosa through a phagocyte NADPH oxidase-like activity.     

Purification and mass spectrometric analysis of ADP-ribosylation factor proteins from Xenopus egg cytosol

The GTP analog GTP gamma S potently inhibits nuclear envelope assembly in cell-free Xenopus egg extracts. GTP gamma S does not affect vesicle binding to chromatin but blocks vesicle fusion. Fusion inhibition by GTP gamma S is mediated by a soluble factor, initially named GSF (GTP gamma S-dependent soluble factor). We previously showed that vesicles pretreated with GTP gamma S plus recombinant mammalian ARF1 were inhibited for fusion, suggesting that "GSF activity" was due to the ARF (ADP-ribosylation factor) family of small GTP-binding proteins. To ask if any soluble proteins other than ARF also inhibited vesicle fusion in the pretreatment assay, we purified GSF activity from Xenopus egg cytosol. At all steps in the purification, fractions containing ARF, but no other fractions, showed GSF activity. The purified GSF was identified as Xenopus ARF by immunoblotting and peptide sequence analysis. Reverse phase HPLC and mass spectrometry revealed that GSF contained at least three distinct ARF proteins, all of which copurified through three chromatography steps. The most abundant isoform was identified as ARF1 (62% of the total GSF), because its experimentally determined mass of 20 791 Da matched within experimental error that predicted by the sequence of the Xenopus ARF1 cDNA, which is reported here. The second-most abundant isoform (25% of GSF activity) was identified as ARF3. We concluded that ARF is most likely the only soluble protein that inhibits nuclear vesicle fusion after pretreatment with GTP gamma S.     

Biochemical effects of retinoic acid on GTP-binding Protein/Transglutaminases in HeLa cells. Stimulation of GTP-binding and transglutaminase activity, membrane association, and phosphatidylinositol lipid turnover

Treatment of HeLa cells with retinoic acid (RA) gives rise to a marked stimulation in the incorporation of [alpha-32P]GTP into an approximately 87-kDa cytosolic protein that cross-reacts with a monoclonal antibody raised against tissue transglutaminases. In the absence of RA treatment, the transglutaminase immunoreactivity elutes from a gel filtration column with an apparent size of approximately 600 kDa (designated TGa), whereas following RA treatment, a second peak of transglutaminase immunoreactivity (designated TGb) is detected with an apparent size of approximately 150 kDa. The TGa fractions show little or no GTP-binding or GTP hydrolytic activity and very little transglutaminase activity. However, the TGb fractions show all three activities. Retinoic acid treatment also promotes the association of the GTP-binding protein/transglutaminase with membrane fractions, as detected by Western blotting and photoaffinity cross-linking with [alpha-32P]GTP. In addition, the TGb fraction shows a markedly enhanced ability (relative to TGa) to associate with membranes from control (non-RA-treated) cells. The ability of the GTP-binding protein/transglutaminase to bind to membranes is correlated with the stimulation of a membrane-associated phospholipase C activity. Thus, these findings indicate that RA treatment results in a number of changes in the biochemical properties of a GTP-binding protein/transglutaminase which strongly enhance its ability to bind GTP, associate with plasma membranes, and stimulate phosphoinositide lipid turnover.     

Protein kinases potentially capable of catalyzing the phosphorylation of p47-phox in normal neutrophils and neutrophils of patients with chronic granulomatous disease

A procedure for uncovering novel protein kinases was used to search for enzymes in neutrophils that may catalyze the phosphorylation of the 47-Kd subunit of the NADPH oxidase system (p47-phox). This component of the oxidase can undergo phosphorylation on multiple sites. The method is based on the ability of renatured kinases to recognize exogenous substrates fixed in gels. We report that neutrophils contain several uncharacterized protein kinases that catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 through 331 of p47-phox. Some of these enzymes are strongly activated on stimulation of the cells with phorbol 12-myristate 13-acetate (PMA). The results indicate that the phosphorylation of p47-phox in neutrophils may be more complicated than previously appreciated and may involve multiple protein kinases. In addition, we have examined both the renaturable protein kinases and the properties of protein kinase C (PKC) in neutrophils from patients with chronic granulomatous disease (CGD) who are deficient in cytochrome b558. Previous studies have shown that these cells exhibit incomplete phosphorylation of p47-phox on stimulation. In this study, we were unable to detect any alterations in the renaturable protein kinases or PKC in CGD neutrophils that could explain these defects in the phosphorylation of p47-phox.     

Binding of guanine nucleotides to bronchial membranes: effect of maturation

Guanosine 5-[y-thio]triphosphate ([35S]GTP gamma S) binding to guinea pig bronchial membranes from immature and mature guinea pigs was rapid (Kon: 3.8 x 10(5) mol-1 min-1), saturable (Bmax: 160 pmoles/mg protein) and of high affinity (Kd: 0.6 microM). [35S]GTP gamma S rapidly dissociated in the absence of magnesium (Koff: 0.06 min-1), but 50 mM magnesium inhibited the dissociation. Maturation did not alter the affinity of the ligand, but Bmax (pmoles/mg DNA) was greater in preparations from mature animals (929 +/- 16 vs. 620 +/- 64). [35S]GTP gamma S was displaced by guanine nucleotides with a rank order of potency of GDP beta S = Gpp(NH)p > GDP > GTP, but not by ATP. We conclude that [35S]GTP gamma S is a specific and useful method to quantitate bronchial membrane-bound GTP-binding proteins. The technique shows that there is a significant increase in the cellular content of G-proteins during maturation.     

GDP dissociation inhibitor prevents intrinsic and GTPase activating protein-stimulated GTP hydrolysis by the Rac GTP-binding protein

The majority of the GTP-binding proteins of the Ras superfamily hydrolyze GTP to GDP very slowly. A notable exception to this are the Rac proteins, which have intrinsic GTPase rates at least 50-fold those of Ras or Rho. A protein (or proteins) capable of inhibiting this GTPase activity exists in human neutrophil cytosol. Since Rac appears to exist normally in neutrophils as a cytosolic protein complexed to (Rho)GDI, we examined the ability of (Rho)GDI to inhibit GTP hydrolysis by Rac. (Rho)GDI produced a concentration-dependent inhibition of GTP hydrolysis by Rac1 that paralleled its ability to inhibit GDP dissociation from the Rac protein. Maximal inhibition occurred at or near equimolar concentrations of the GDI and the Rac substrate. The ability of two molecules exhibiting GTPase activating protein (GAP) activity toward Rac to stimulate GTP hydrolysis was also inhibited by the presence of (Rho)GDI. The inhibitory effect of the GDI could be overcome by increasing the GAP concentration to levels equal to that of the GDI. (Rho)GDI weakly, but consistently, inhibited GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) dissociation from Rac1, confirming an interaction of (Rho)GDI with the GTP-bound form of the protein. These data describe an additional activity of (Rho)GDI and suggest a mechanism by which Rac might be maintained in an active form in vivo in the presence of regulatory GAPs.     

The biochemical basis of the NADPH oxidase of phagocytes

The NADPH oxidase is an electron transport chain found in lymphocytes and in the wall of the endocytic vacuole of 'professional' phagocytic cells. It is so called because NADPH is used as an electron donor to reduce oxygen to superoxide and hydrogen peroxide. The redox components are provided by a very unusual flavocytochrome b from the membrane, which is dependent upon cytosolic factors (including two specialized proteins, p47phox and p67phox) for activation. The small GTP-binding protein, p21rac, is also implicated in this system, possibly as the switch that triggers electron transport. This system provides a key to our understanding of the way in which these GTP-binding proteins function.     

Phosphatidyl inositol-phospholipase C in squid photoreceptor membrane is activated by stable metarhodopsin via GTP-binding protein, Gq

Phosphatidyl inositol-phospholipase C (PI-PLC) in squid retina was studied by immunoblotting and its activities were determined using [3H]phosphatidyl inositol bisphosphate ([3H]PIP2) as substrate. PI-PLC activity was found mostly in soluble fraction when the retina homogenate was treated with 400 mM KCl, but was associated with rhabdomal membranes under low salt conditions (20 mM Hepes). A protein with apparent molecular mass of 130kD was recognized by an antibody against PLC beta 4/norp A in both 400 mM KCl soluble and rhabdomal membrane fractions. A 42 kD protein recognized by antibody against the C-terminus of Gq alpha was also present in these two fractions. GTP gamma S stimulated only the PI-PLC activity associated with membrane and was magnesium dependent. PI-PLC activity was found to be (i) highly dependent upon calcium concentrations, (ii) enhanced by GTP but not by other nucleotides, and (iii) significantly stimulated by light at lower concentrations of GTP gamma S. The stimulation by light was still observed when irradiated membrane was incubated at 10 degrees C for 10 min and then mixed with GTP gamma S. These results suggest that stable metarhodopsin stimulates a PLC beta 4/norp A-like enzyme via a G-protein, Gq.     

The chemokine monocyte chemotactic protein 1 triggers Janus kinase 2 activation and tyrosine phosphorylation of the CCR2B receptor

Gelsolin is an actin filament-capping protein that has been shown to play a key role in cell migration. Here we have studied the involvement of phosphoinositide 3-kinase (PI 3-kinase) and GTP-binding proteins (G-proteins) in the regulation of gelsolin-actin interactions in neutrophils. Inhibition of PI 3-kinase activity in vivo by wortmannin did not affect the dissociation of actin-gelsolin (1:1) complexes induced by neutrophil stimulation with N-formyl-Met-Leu-Phe. Guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) indirectly promoted the dissociation of actin-gelsolin complexes in a cell-free system using neutrophil cytosol, and this effect was blocked by the GDP dissociation inhibitor for Rho (Rho-GDI). The GTPgammaS-loaded ialpha2 and the beta1gamma2 subunits of heterotrimeric G-proteins (Gialpha2 and Gbeta1gamma2) also triggered actin-gelsolin dissociation in a Rho-GDI-sensitive manner. GTP-loaded activated Rac, but not activated Rho, induced the dissociation of cytosolic actin-gelsolin complexes. The guanine nucleotide exchange on Rac was increased by addition of GTPgammaS-loaded Gialpha2 or Gbeta1gamma2 to neutrophil cytosol. These findings suggest that activation of Rac by G-protein-coupled receptors in neutrophils triggers uncapping of actin filaments, independently of PI 3-kinase.