Biochimica et Biophysica Acta, 1054 (1990) 237-245
Elsevier BBAMCR 12767
Subcellular distribution and characterization of GTP-binding proteins in human neutrophils * Leili Khachatrian
1,2, Jeffrey B. Rubins 3, Eric C. Manning 3, David Dexter 3, Alfred I. Tauber 1 and Burton F. Dickey 3
i Departments of Medicine and Pathology, 2 Mallory Institute of Pathology and 3 The Pulmonary Center, Boston University School of Medicine, Boston, MA (U.S.A.)
(Received27 October 1989) (Revised manuscript received7 May 1990)
Key words: G-protein; GTP binding protein; Neutrophil; ADP-ribosylation
The subcellular distribution of GTP binding proteins in human neutrophils and their functional coupling to the N-formylmethionylleucylphenylalanine (FMLP) receptor was characterized to provide insight into mechanisms of cellular activation. Human neutrophils were nitrogen cavitated and fractionated on discontinuous Percoli gradients. Four subcellular fractions were obtained: cytosol, light membranes enriched for plasma membranes, specific granules and azurophilic granules. ADP-ribosylation catalyzed by pertussis toxin (PT) revealed a major substrate of 40 kDa only in plasma membrane and cytosol, and antiserum specific for Gia confirmed the presence of neutrophii Gia in plasma membrane and cytosol and its absence from specific granules. The cytosolic PT substrate was shown to be mostly in monomeric form by molecular sieve chromatography. The rate of the ribosyltransferase reaction was several-fold lower in cytosol compared to plasma membranes, and the extent of ADP-ribosylation was greatly augmented by supplementation with fly subunits in cytosoi. ADP-ribosylation catalyzed by cholera toxin (CT) revealed substrates of 52, 43 and 40 kDa in plasma membrane alone. FMLP receptors in plasma membrane were shown to be coupled to the 40 kDa substrate for CT by ligand-modulation of ADP-ribosylation, while FMLP added to specific granules did not induce ribosylation of this substrate even though FMLP receptors were found in high density in this compartment. Both 24 and 26 kDa [32P]GTP binding proteins were found to codistribute with FMLP receptors in specific granules and plasma membranes. Functional evidence for the coupling of GTP binding proteins to the FMLP receptor in specific granules was obtained by modulating [3H]FMLP binding with GTPvS, and by accelerating [3sS]GTPyS binding with FMLP.
Introduction Neutrophil activation with the bacterial chemotactic peptide N-formylmethionylleucylphenylalanine (FMLP) is coupled to numerous effector functions via pertussis toxin (PT)-sensitive [1-5] and PT-insensitive pathways
* This research was presented in part in abstract form: Khachatrian, L. and A.I. Tauber (1988). The presence of G proteins in human neutrophil specificgranules, Clin. Res. 36, 566A. Abbreviations: FMLP, N-formylmethionylleucylphenylalanine; GTPyS, guanosine 5'-(3-O-thio)triphosphate; G-protein, transductional guanine nucleotide-bindingregulatory protein; PT, ADP-ribosyltransferase exotoxin from Bordetella pertussis; CT, ADP-ribosyltransferase exotoxin from Vibrio cholera; SDS-PAGE, sodium dodecylsulfatepolyacrylamidegel electrophoresis. Correspondence: L. Khachatrian, Hematology, Room 311, K600, Boston University School of Medicine, 801 Albany Street, Boston, MA 02118, U.S.A.
. We  and others [8-10] have previously reported the purification of a 40 kDa G-protein from neutrophil membranes which is ADP-ribosylated in the presence of FT. Immunochemical evidence indicates that this 40 kDa G-protein is Gia2, and there is evidence for the additional presence of the 41 kDa G-protein Gin3 [11-13]. During the fractionation of neutrophils into particulate and soluble components for the purification of G-proteins from membranes, we noted the presence of substantial PT substrate in the soluble fraction. Gierschick et al. also noted PT substrate in neutrophil cytosol . Although transducin is known to reversibly dissociate from the cell membrane after cellular activation , this phenomenon had not been previously described for other transductional G-proteins. We thus sought to characterize the PT substrate present in neutrophil cytosol. Another novel compartment in neutrophils which might be expected to contain transduetional G-proteins
0167-4889/90//$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
238 is the specific granule. This fraction is preferentially mobilized to the cell surface in response to activating stimuli [15-19] and contains large pools of surface active proteins, including the FMLP receptor. The density of FMLP receptors in specific granules is actually three to four times that in plasma membranes . The teleologic role of these granule-associated receptors is presumably to augment the capability for subsequent cellular activation after submaximal degranulation. We sought to determine whether these receptors are recruited to the cell surface precoupled to their transductional elements, or whether they must couple with Gproteins already present on the cell surface. In adrenal chromaffin ceils, PT substrates have indeed been found in granule membranes . Alternatively, GTP-binding proteins which are not substrates for PT might couple the FMLP receptor in specific granules to effector mechanisms. We have determined that the major [35S]GTPyS binding activity of crude neutrophil membranes is associated with a 24/25 kDa protein doublet, and 'Gp', the major GTP-binding protein previously purified from platelets and placenta [22,23] is identified as a component of this doublet. We have also identified 24 and 26 kDa [32p]GTP binding proteins which codistribute with FMLP receptors in plasma membranes and specific granules. The specific binding of [32p]GTP to 26 kDa proteins has been shown to correlate with activation of polyphosphoinositide phospholipases C [24,25], and 24 and 26 kDa GTP binding proteins have been found to co-chromatograph with the FMLP receptor . We provide functional evidence that FMLP receptor in specific granules is coupled to GTP-binding proteins which are not substrates for PT or CT. These GTP-binding proteins may play a role in coupling the FMLP receptor to the generation of second messengers when mobilized to the cell surface, or they may play more novel roles in coupling receptors to effectors such as the N A D P H oxidase  ! Materials and Methods
Neutrophils were isolated by dextran sedimentation and FicoU-Hypaque density gradient centrifugation from freshly drawn blood of normal human donors using 3 mM sodium citrate (pH 5.2) as an anticoagulant, and treated with 3 mM diisopropylfluorophosphate for 10 rain at 4°C. Cells were then washed and disrupted by nitrogen cavitation, followed by fractionation on discontinuous Percoll gradients as described . Four subcellular compartments which have previously been extensively characterized were collected from the Percoll gradients: cytosol, azurophilic granules (et), specific granules (fl) and light membranes enriched for plasma membranes (y). The purity of the respective fractions was determined by measurement of alkaline
phosphatase (7), cytochrome b (fl), and myeloperoxidase (a) . Protein was determined by a modification of the method of Lowry . Cytosol harvested from the Percoll gradients was further centrifuged at 185 000 x g for one hour prior to ADP-ribosylation, Western blotting or column fractionation. Molecular sieve chromatography of cytosol was performed using Ultrogel AcA 34 (IBF) in a 1.6 cm by 100 cm column at a linear flow rate of 0.78 cm/min. The buffer used for column elution consisted of 10 mM Tris-HC1 (pH 7.5), 1 mM EDTA and 1 mM dithiothreitol. Pertussis and cholera toxin-catalyzed ADP-ribosylation was carried out essentially as described [31,32] using toxins purchased from List Biologicals (CA). Subcellular fractions (0.02-0.1 mg protein) were incubated for 1 h at 30°C in 50 #1 of buffer containing 25 mM Tris-HC1 (pH 8.0), 1 mM EDTA, 1 mM ATP, 0.4 mM MgC12, 10 mM thymidine, 10/~g/ml pyruvate kinase, 3 mM phosphoenolpyruvate and 8 /tM [32p]NAD (4 Ci/mmol) and 1 #g of pertussis toxin or 5/~g of cholera toxin. Both toxins were freshly activated with 33 mM dithiothreitol for 30 rain at 37 o C. Guanine nucleotides were added as noted in the figure legends. Incubation was terminated by the addition of Laemmli sample buffer, followed by heating of samples for 2 min at 100°C. The consumption of [32p]NAD during the ribosylation reaction was monitored by thin layer chromatography of 2/~l of the reaction mixture at the end of the incubation . SDS-PAGE of samples was performed according to Laemmli  using 11% polyacrylamide gels. Molecular weight standards were rabbit muscle phosphorylase b (97.4 kDa), bovine serum albumin kDa (66.2 kDa), ovalbumin (42.7 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa) and hen egg lysozyme (14.4 kDa) (Bio-Rad). Quantitation of labelling with [32p]ADP-ribose was by densitometry of autoradiograms using a computer-assisted Helena Laboratories Model EDC Densitometer, or by scintillation counting of excised bands of dried gels after swelling in Liquiscint scintillation fluid (National Diagnostics). Western transfer of Laemmli gel-electrophoresed samples to BA85 nitrocellulose (Schleicher and Shuell) was accomplished by electrophoresis at 2000 mAh in 25 mM Tris, 192 mM glycine (pH 8.3) and 20% methanol. The nitrocellulose transfers were probed with [32p]GTP (see below), or with antisera raised in rabbits against G-protein subunits. The fl subunit used for immunization was from bovine brain, purified as described previously , and used as antigen in Freund's adjuvant. This antiserum reacts with a doublet of M r 35 000 and 36 000 in rat heart, liver, lung and brain and reacts with as little as 5 ng of purified brain fly subunits when used at a dilution of 1:500. The peptide CGAGESGKSTIVKQMK, which is shared among Gil-3 and Go a subunits, was used to generate an anti-peptide anti-
239 serum after conjugation to keyhold limpet hemocyanin, essentially as described . This antiserum reacts against major bands of M r 40000 and 41000 in rat heart, liver and lung and of 39 000, 40 000 and 41 000 in brain. It also reacts with 39, 40 and 41 kDa G protein ct subunits purified from bovine brain, and used at a dilution of 1 : 500 detects as little as 500 pg of purified ot subunit. Unlike the antiserum A-259 developed by Mumby et al. , our antiserum did not react with the 45 or 52 kDa Gsa subunits which differ at a single amino acid in this peptide sequence. Antibody binding was detected by immunoperoxidase staining using either a biotinylated second antibody and horseradish peroxidase-conjugated streptavidin or a ]25I-labelled donkey anti-rabbit IgG (Amersham) with autoradiography. Bovine brain G i / G o a and fl subunits, purified and resolved as described by Sternweis et al. , were used as standards for quantitation. Both anti-sera were used at a dilution of 1 : 500. Polyclonal rabbit antiserum to the low molecular mass GTP-binding protein 'Gp', purified from placenta , was provided by John Northup and used at a dilution of 1:1000. This antiserum does not cross react with other known GTP-binding proteins. The purification of [35S]GTP~/S-binding proteins from solubilized crude rabbit neutrophil membranes by sequential ion exchange, molecular sieve and hydrophobic chromatographies was carried out as described previously by us . [3sS]GTP~,S binding capacity was detected by a filtration assay , as was the quantitation of pertussis toxin-catalyzed [32p]ADP-ribosylation . The Western blotting of neutrophil fractions for detection of low molecular weight GTP binding proteins by [32p]GTP binding was carried out exactly as for immunoblotting, except that SDS-PAGE was through 14% polyacrylamide gels. Binding of [32P]GTP to blotted proteins was accomplished after first blocking the nitrocellulose filters for 60 min with 5% milk solids in Tris-buffered saline and 0.05% Tween 20. Blots were then rinsed in binding buffer consisting of 50 mM Tris (pH 7.4), 0.3% Tween-20, 5 mM MgC12 and 1 mM EGTA, and incubating the blots for 1 h at 21°C in binding buffer containing 2 /~Ci/ml of [a-32p]GTP (3000 Ci/mmol, NEN). Blots were then washed three times for 20 min each in binding buffer, dried, and exposed to Kodak XAR5 film. High affinity [3H]FMLP binding was determined by incubating 30/~g of membranes with 5 nM [3H]FMLP for 30 min at 4 ° C. Nonspecific binding was determined in the presence of a 500-fold excess of unlabelled FMLP. Bound ligand was separated from free by rapid vacuum filtration at - 5 mm Hg pressure through Whatman G F / F glass fiber filters, followed by scintillation counting of the dried filters. Specific binding was calculated to be the difference between total and nonspecific binding, and nonspecific binding always accounted for less
than 10% of total binding. Assays were performed in quadruplicate for each neutrophil preparation, and the statistical evaluation of multiple determinations was performed by a computer assisted two-way analysis of variance using the Statistical Analysis System (SAS Institute, Inc., Cary, North Carolina). The modulation of [35S]GTP-/S binding by 100 nM FMLP was also measured by a vacuum filtration binding assay, as noted above. The reaction was initiated by the addition of membranes to an aliquot of solution containing all the other reagents. Assays were carried out in triplicate for each neutrophil preparation at times of 5, 7.5, 11 and 15 min after the addition of FMLP. Statistical evaluation was by a two-way analysis of variance. Results
Three distinct particulate fractions were visible on the Percoll gradients, and these have been labelled from most to least dense: a (primary granules), fl (specific granules) and ~/ (light membrane fraction, enriched for plasma membranes). If the specific activity of myeloperoxidase in the alpha fraction, cytochrome b in the fl fraction, and alkaline phosphatase in the 7 fraction are each taken as 100%, then relative specific activities in other fractions averaged as follows: myeloperoxidase, 7% in fl and 0% in "t; cytochrome b, 0% in a and 22% in 3'; and alkaline phosphatase, 2.6% in a- and 8.6% in B, values closely approximating those of our previous reports . These fractions were ADP-ribosylated in the presence of [32p]NAD and either PT or CT; representative autoradiograms of these experiments are shown in Fig. 1. CT substrates were seen only in the plasma membrane fraction, and these were of M r 52 000, 43 000 and 40000. ADP-ribosylation of the 52 and 43 kDa substrates by CT was greater in the presence of GTP3,S, while the 40 kDa substrate was more heavily labelled in 7
40 K D ~
Fig. 1. Bacterial toxin-catalyzed [32p]ADP-ribosylation of proteins in neutrophil subceUular fractions. Plasma membranes (3' fraction) or cytosol (cyto), 87 #g each, were incubated with [32p]NAD and pertussis (20 ttg/ml) or cholera toxin (100/~g/ml) as described in Materials and Methods. Samples were then electrophoresed on SDS-PAGE gels and autoradiograms developed. The autoradiograms were deliberately overdeveloped to convicingly demonstrate the absence of CT substrate in cytosol. Added nucleotides were GTP at 2 ~M or GTP3'S at 100 jaM.
Fig. 2. Identification of Gi subunits in subcellular fractions of neutrophils by immunoblots. 20 pg of each fraction were run on 11% SDS-PAGE gels, transferred to nitrocellulose, then probed with antisera. (A) Antiserum against a Gi/Goor-common peptide used in a 1: 500 dilution, with antibody detected by immunoperoxidase. (B) Antiserum against purified brain B subunits used in a 1: 500dilution.
the absence of added guanine nucleotides. Pertussis toxin substrates of Mr 40000-41000 were consistently detected in the plasma membrane fraction and in cytosol (Fig. l), and rarely in specific granules (not shown). Anti-Giar antiserum was immunoreactive with plasma membrane and cytosol, but anti-p antiserum detected no substrate in cytosol (Fig. 2). These antisera did not detect cuor jI subunits in either granule fraction. The cytosolic PT substrate was further characterized by analysis of the PT-catalyzed ADP-ribosyltransferase reaction and by molecular sieve chromatography. The time course of ADP-ribosylation of cytosolic PT substrate was dramatically different from that of plasma membrane substrate. Equilibrium of the PT-catalyzed ribosyltransferase reaction was reached within 1 h in plasma membranes, but was essentially linear through 4 h in cytosol. In addition, the incorporation of [32P]ADP-ribose was augmented 12- to 22-fold at one hour in cytosol after addition of 1.2 PM /3y subunits purified from bovine brain, but only l.Zfold in plasma membranes (not shown). In the presence of added j?y subunits, maximal ribosylation was reached in cytosol by one hour. There were 3.7 pmol [32P]/mg protein incorporated into cytosolic PT substrate at equilibrium after supplementation with 1.2 PM py subunits, and 17 pmol/mg incorporated into plasma membrane substrate. These observations may reflect a relative deficiency of /?y subunits in cytosol as compared to plasma membranes. Molecular sieve chromatography of the cytosolic PT substrate is illustrated in Fig. 3. Column fractions were assayed by PT-catalyzed ADP-ribosylation for 1 h after supplementation with 1.2 PM fiy
Fig. 3. Ultrogel AcA-34 chromatography of human neutrophil cytosol. Aliquots (40 ~1) of indicated fractions were supplemented with 1.2 PM By. assayed for pertussis toxin ADP-ribosylation as in Fig. 1, and quantitated by densitometric scanning of autoradiographs. Molecular weight standards used were: blue dextran (2000 kDa), yeast alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and cytochrome c (13 kDa).
subunits. The peak of PT substrate eluted with an estimated M, of approx. 40000, consistent with its presence as an alpha monomer. By SDS-PAGE this peak of cytosolic PT substrate also had an M, of 40000. Bands approx. 2 kDa above and below the 40 kDa band were also seen (not shown), but these bands were present without PT and were actually more intense in the absence of PT. Similar labelling of proteins by [ 32P]NAD which is not dependent on toxin has been noted by others [38-401. Evidence of guanine nucleotide-dependent coupling of the FMLP receptor to a 40 kDa protein which appears to be a substrate for both PT and CT in plasma membranes is shown in Fig. 4. Incubation of plasma membranes with 1 PM FMLP caused a mean decrease
Fig. 4. Autoradiogram showing the effect of FMLP on [32P]ADPribosylation of 40 kDa proteins by pertussis and cholera toxins. Plasma membranes were incubated in the presence of [32P]NAD with pertussis toxin and 100 pM GTP, or with cholera toxin and no guanine nucleotide added. The reaction was performed in the presence or absence of 1 CM FMLP, and was terminated after 1 h incubation at 30’ C by the addition of Laemmh sample buffer and boiling. Samples were then subjected to SDS-PAGE and autoradiography.
241 in PT-catalyzed ADP-ribosylation in the presence of GTP of 25%, and an increase of 370% in CT-catalyzed ribosylation of the 40 kDa G-protein in the absence of added nucleotides. There was no labelling of the 40 kDa G-protein by CT in other fractions in the presence or absence of added FMLP, with or without added guanine nucleotides. Since FMLP receptors are known to be present in neutrophil specific granules [20,38,41,42] as well as in plasma membranes, but only plasma membranes and not specific granules contained PT and CT substrate or immunoreactive Gi, we sought evidence for other transductional proteins which might couple the FMLP receptor to effector proteins. During our previously reported purification of the PT substrate from crude rabbit neutrophil membranes , we noted that the major [35S]GTPyS binding activity actually is associated with a 24/25 kDa protein doublet which copurified through three steps of chromatography. Fig. 5 shows a silverstained gel of serial fractions eluted from a phenylSepharose column, following sequential anion exchange and molecular sieve chromatography of [35S]GTPySbinding activity. The peak of binding activity (fraction 22) is seen to correspond more closely to the peak of the 24/25 kDa silver stained doublet than to the peak of 40/41 kDa PT substrate (fraction 18). The 24 kDa silver stained protein was identified as a GTP-binding protein by its ability to bind [32p]GTP after transfer to nitrocellulose, and is shown to have an identical mobility to 24 kDa [32p]GTP-binding protein(s) found in specific granules and plasma membrane (Fig. 6A). The 25 kDa silver stained protein band was immunochemically shown to include 'Gp', the major GTP binding protein of platelets and placenta [23,24], by use of a polyclonal antiserum specific for 'Gp' (Fig. 6B). This antiserum, however, did not allow immunolocalization of 'Gp' since it did not specifically react with unpurified proteins from any subcellular fraction or from crude memrbanes, as is the case with this antiserum in other tissues (John Northup, personal communication). In addition to the 24 kDa [32p]GTP-binding protein, we were able to detect six other bands of [3zP]GTP-binding activity ranging in M r from 19 000 to 26 000 on Western blots (Fig. 6C). Several of these proteins will be further characterized in another manuscript. Of note, strong [3zp]GTP-binding activity of identical electrophoretic mobility was detected in both plasma membranes and specific granules at M r 26 000 and 24000 (see Discussion). All [3zP]GTP-binding to blotted proteins was completely blocked when a 1000-fold excess of unlabelled GTP was included in the incubation, whereas none of these bands showed diminished [32P]GTP-binding with a 1000-fold excess of ATP (not shown). To determine if the FMLP receptor in specific granules coupled with GTP-binding proteins which are not substrates for PT or CT, we sought evidence for recep-
tor G-protein coupling in these fractions by criteria other than ligand-dependent changes in ADP-ribosylation. Modulation of FMLP binding by guanine nucleotides is presumptive evidence of functional coupling of the FMLP receptor to a G-protein [43,44]. Freezethawed membranes from each fraction were incubated with 50 nM [3H]FMLP in the presence or absence of 100/xM GTPyS. There was a highly significant decrease in specific [3H]FMLP equilibrium binding to plasma membranes in the presence of GTPyS (mean decrease of 72%, n = 9, P = 0.013 by analysis of variance). There was also a less dramatic but highly significant effect (mean decrease of 40%, P = 0 . 0 0 7 ) of GTP'yS on [3H]FMLP binding to specific granule membranes. Secondly, the association rate of [35S]GTPyS with GTP-binding proteins in specific granule membranes determined at 5, 7.5, 11 and 15 min was significantly increased by 100 nM FMLP (n = four neutrophil pre-
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