Endogenous ADP-ribosylation of the G Protein β Subunit Prevents the Inhibition of Type 1 Adenylyl Cyclase
2000; Elsevier BV; Volume: 275; Issue: 13 Linguagem: Inglês
10.1074/jbc.275.13.9418
ISSN1083-351X
AutoresRosita Lupi, Daniela Corda, Maria Di Girolamo,
Tópico(s)Signaling Pathways in Disease
ResumoMono-ADP-ribosylation is a post-translational modification of cellular proteins that has been implicated in the regulation of signal transduction, muscle cell differentiation, protein trafficking, and secretion. In several cell systems we have observed that the major substrate of endogenous mono-ADP-ribosylation is a 36-kDa protein. This ADP-ribosylated protein was both recognized in Western blotting experiments and selectively immunoprecipitated by a G protein β subunit-specific polyclonal antibody, indicating that this protein is the G protein β subunit. The ADP-ribosylation of the β subunit was due to a plasma membrane-associated enzyme, was sensitive to treatment with hydroxylamine, and was inhibited bymeta-iodobenzylguanidine, indicating that the involved enzyme is an arginine-specific mono-ADP-ribosyltransferase. By mutational analysis, the target arginine was located in position 129. The ADP-ribosylated β subunit was also deribosylated by a cytosolic hydrolase. This ADP-ribosylation/deribosylation cycle might be anin vivo modulator of the interaction of βγ with specific effectors. Indeed, we found that the ADP-ribosylated βγ subunit is unable to inhibit calmodulin-stimulated type 1 adenylyl cyclase in cell membranes and that the endogenous ADP-ribosylation of the β subunit occurs in intact Chinese hamster ovary cells, where the NAD+ pool was labeled with [3H]adenine. These results show that the ADP-ribosylation of the βγ subunit could represent a novel cellular mechanism in the regulation of G protein-mediated signal transduction. Mono-ADP-ribosylation is a post-translational modification of cellular proteins that has been implicated in the regulation of signal transduction, muscle cell differentiation, protein trafficking, and secretion. In several cell systems we have observed that the major substrate of endogenous mono-ADP-ribosylation is a 36-kDa protein. This ADP-ribosylated protein was both recognized in Western blotting experiments and selectively immunoprecipitated by a G protein β subunit-specific polyclonal antibody, indicating that this protein is the G protein β subunit. The ADP-ribosylation of the β subunit was due to a plasma membrane-associated enzyme, was sensitive to treatment with hydroxylamine, and was inhibited bymeta-iodobenzylguanidine, indicating that the involved enzyme is an arginine-specific mono-ADP-ribosyltransferase. By mutational analysis, the target arginine was located in position 129. The ADP-ribosylated β subunit was also deribosylated by a cytosolic hydrolase. This ADP-ribosylation/deribosylation cycle might be anin vivo modulator of the interaction of βγ with specific effectors. Indeed, we found that the ADP-ribosylated βγ subunit is unable to inhibit calmodulin-stimulated type 1 adenylyl cyclase in cell membranes and that the endogenous ADP-ribosylation of the β subunit occurs in intact Chinese hamster ovary cells, where the NAD+ pool was labeled with [3H]adenine. These results show that the ADP-ribosylation of the βγ subunit could represent a novel cellular mechanism in the regulation of G protein-mediated signal transduction. GTP-binding protein Chinese hamster ovary cells dithiothreitol hydroxylamine meta-iodobenzylguanidine glycosylphosphatidylinositol adenylyl cyclase types 1 (AC1) and 2 (AC2) Ca2+/calmodulin Dulbecco's modified Eagle's medium Hanks' Balanced Salt Solution 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid polyacrylamide gel electrophoresis high pressure liquid chromatography Several cell proteins can be modified by covalent reactions that affect their function. Whereas the best described modification involves the phosphorylation of specific residues, evidence is also accumulating that endogenous ADP-ribosylation can play a similar role (Ref. 1.Haag F. Koch-Nolte F. Haag F. Koch-Nolte F. ADP-ribosylation in Animal Tissues. Plenum Publishing Corp., New York1997Crossref Google Scholar and references therein). Enzymatic mono-ADP-ribosylation involves the transfer of the ADP-ribose moiety from NAD to a specific amino acid of cellular proteins. The best characterized mono-ADP-ribosylation reactions are those catalyzed by the bacterial toxins, such as pertussis (2.Katada T. Ui M. Proc. Natl. Acad. Sci. U. S. A. 1982; 88: 3129-3133Crossref Scopus (561) Google Scholar), cholera (3.Moss J. Vaughan M. J. Biol. Chem. 1977; 252: 2455-2457Abstract Full Text PDF PubMed Google Scholar), diphtheria (4.Van Ness B.G. Howard J.B. Bodley J.W. J. Biol. Chem. 1980; 255: 10710-10716Abstract Full Text PDF PubMed Google Scholar), and clostridial toxins (5.Sekine A. 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Chem. 1980; 255: 10710-10716Abstract Full Text PDF PubMed Google Scholar), resulting in permanent activation or inactivation of critical cell functions. For example, cholera toxin, an arginine-specific ADP-ribosyltransferase, ADP-ribosylates the α subunit of the stimulatory G protein (Gs) to irreversibly inhibit its GTPase activity (3.Moss J. Vaughan M. J. Biol. Chem. 1977; 252: 2455-2457Abstract Full Text PDF PubMed Google Scholar, 7.Gill D.M. Meren R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3050-3054Crossref PubMed Scopus (478) Google Scholar, 8.Northup J.K. Sternweis P.C. Smigel M.D. Schleifer L.S. Ross E.M. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6516-6520Crossref PubMed Scopus (327) Google Scholar). Endogenous mono-ADP-ribosylation has also been described in eukaryotic cellular systems; ADP-ribosyltransferases that catalyze ADP-ribosylation of arginine residues of G proteins (similar to cholera toxin) have been described in many cells and tissues (9.Zolkiewska A. Okazaki I. Moss J. Mol. Cell. Biochem. 1994; 138: 107-112Crossref PubMed Scopus (28) Google Scholar, 10.Koch-Nolte F. Haag F. Haag F. Koch-Nolte F. ADP-ribosylation in Animal Tissues. Plenum Publishing Corp., New York1997: 1-13Google Scholar, 11.Zolkiewska A. Nightingale M.S. Moss J. Proc. Natl. Acad. Sci. U. S. A. 1994; 89: 11352-11356Crossref Scopus (152) Google Scholar, 12.Tsuchiya M. Hara N. Yamada K. Osago H. Shimoyama M. J. Biol. Chem. 1994; 269 (27457): 27541Google Scholar). The endogenous ADP-ribosylation of cysteine residues of membrane G proteins (similar to pertussis toxin) has also been suggested to occur in erythrocytes (13.Tanuma S. Kawashima K. Endo H. J. Biol. Chem. 1987; 101: 821-824Google Scholar, 14.Saxty B.A. van Heyningen S. Biochem. J. 1995; 310: 931-937Crossref PubMed Scopus (22) Google Scholar). Thus, some of these enzymes are able to modify G proteins (15.Watkins P.A. Kanaho Y. Moss J. Biochem. J. 1987; 248: 749-754Crossref PubMed Scopus (12) Google Scholar, 16.Ehret-Hilberer S. Nullans G. Aunis D. Virmaux N. FEBS Lett. 1992; 309: 394-398Crossref PubMed Scopus (31) Google Scholar, 17.Quist E.E. Coyle D.L. Vasan R. Satumtira N. Jacobson E.L. Jacobson M.K. J. Mol. Cell. Cardiol. 1994; 26: 251-260Abstract Full Text PDF PubMed Scopus (24) Google Scholar) and presumably play a role in signal transduction, although their substrates have been poorly characterized and their functional significance is even less understood. It has also been proposed that an ADP-ribosyltransferase may be coupled to an ADP-ribosylarginine hydrolase that is able to remove the ADP-ribose group and hence regenerate free arginine, completing an ADP-ribosylation cycle that can reversibly regulate the functions of substrate proteins (18.Moss J. Zolkiewska A. Okazaki I. Haag F. Koch-Nolte F. ADP-ribosylation in Animal Tissues. Plenum Publishing Corp., New York1997: 25-33Google Scholar, 19.Moss J. Jacobson M.K. Syanley S.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5603-5607Crossref PubMed Scopus (79) Google Scholar). An example of this cycle in eukaryotes is given by desmin, the muscle-specific intermediate filament protein as follows: ADP-ribosylation blocks the assembly of desmin into 10-nm filaments in vitro, and an incubation with ADP-ribosylarginine hydrolase restores the self-assembly properties of desmin (20.Soman G. Mickelson J.R. Louis C.F. Graves D.J. Biochem. Biophys. Res. Commun. 1984; 120: 973-980Crossref PubMed Scopus (61) Google Scholar, 21.Chang Y. Soman G. Graves D.J. Biochem. Biophys. Res. Commun. 1986; 139: 932-939Crossref PubMed Scopus (20) Google Scholar, 22.Huang H.-Y. Graves D.J. Robson R.M. Huiatt T.W. Biochem. Biophys. Res. Commun. 1993; 197: 570-577Crossref PubMed Scopus (30) Google Scholar). Here we report the direct demonstration of endogenous mono-ADP-ribosylation of the G protein β subunit, and we provide evidence that this modification can modulate βγ activity in a similar way to the regulation of some G protein α subunits. Thus we propose that the ADP-ribosylation/deribosylation cycle of the βγ subunit might represent a novel cellular mechanism to regulate G protein-mediated signal transduction. Dulbecco's modified Eagle's medium (DMEM) and Hanks' Balanced Salt Solution (HBSS) were purchased from Life Technologies, Inc.; [32P]NAD was from Amersham Pharmacia Biotech, and [2,8-3H]adenine was from NEN Life Science Products. Cholera toxin was from Calbiochem, and pertussis toxin was a generous gift of Dr. R. Rappuoli (Chiron Vaccines, Siena, Italy). Tosylphenylalanyl chloromethyl ketone-treated trypsin was from Worthington. Other chemicals used were obtained from Sigma at the highest available purities. Part of the purified bovine brain βγ and the antibodies raised against the carboxyl terminus and the amino terminus of β subunit were generously supplied by Dr. W. F. Simond (National Institutes of Health, Bethesda). Baculovirus encoding His6-αi1 was a generous gift of Dr. P. Gierschik (University of Ulm, Germany). Chinese hamster ovary (CHO) cells were grown in monolayers at 37 °C in 95% air, 5% CO2 in DMEM supplemented with 10% fetal calf serum, 34 μg/ml proline, 100 units/ml penicillin, 100 mg/ml streptomycin. Plasma membranes were prepared as described previously (23.Gettys T.W. Sheriff-Carter K. Moomaw J. Taylor I.J. Raymond J.R. Anal. Biochem. 1994; 220: 82-91Crossref PubMed Scopus (44) Google Scholar), with the following modification: cells (6 × 108 cells for each preparation) were washed with HBSS, detached with hypotonic buffer containing 10 mm TES (pH 7.0) and 1 mm EDTA, and then lysed with a Teflon/glass Potter homogenizer. CHO cytosol was prepared as described previously (24.Di Girolamo M. Silletta M.G. De Matteis M.A. Braca A. Colanzi A. Pawlak P. Rasenick M.M. Luini A. Corda D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7065-7069Crossref PubMed Scopus (41) Google Scholar), with the following modification: CHO cells (detached in HBSS without Ca2+ and Mg2+, plus 5 mm EGTA), were broken (109 cells/ml) by sonication in 5 mm Tris-HCl (pH 8.0), 1 mmEGTA, and protease inhibitors. Samples (4-μg plasma membranes) were incubated at 37 °C for 60 min in 50 μl of buffer containing 50 mm potassium phosphate (pH 7.4), 0.5 mmMgCl2, 4 mm dithiothreitol (DTT), 5 mm thymidine, 30 μm β-NAD, 1–2 μCi of [32P]NAD (specific activity, 1000 Ci/mmol), and (where specified) 0.05–0.5 μg of purified bovine brain βγ (either generously provided by Dr. W. F. Simonds, National Institutes of Health, Bethesda, or purified as described previously (25.Sternweis P.C. Pang I. Hulme E.C. Receptor-Effector Coupling. Oxford University Press, Oxford1990: 1-30Google Scholar)). Pertussis and cholera toxin ADP-ribosylations were performed as described previously (26.Di Girolamo M. D'Arcangelo D. Cacciamani T. Gierschik P. Corda D. J. Biol. Chem. 1992; 267: 17397-17403Abstract Full Text PDF PubMed Google Scholar). The reactions were terminated by diluting the samples with 50 μl of Laemmli sample buffer, followed by 2 min boiling, and analysis by 10% SDS-PAGE without or with 4 m urea. Proteins were electroblotted (4 h at 500 mA) onto nitrocellulose membranes, and the filters were exposed for about 12 h to Kodak X-Omat film using an intensifying screen. For quantitative analysis an Instantimager (Packard Instrument Co.) was employed. Following the ADP-ribosylation assay,32P-labeled plasma membranes were washed twice with 5 mm Tris-HCl (pH 8.0) and incubated for 30 min at 37 °C with 50 μg of CHO cell cytosol, without or with 10 mmMgCl2 (in 50 μl of 5 mm Tris-HCl (pH 8.0) and protease inhibitors). The analysis of protein samples was performed as described above for the ADP-ribosylation assay, whereas the labeled compounds released in the supernatant were separated by HPLC with a Partisil 10 SAX column (4.6 mm × 25 cm; Whatman) using a non-linear gradient of 0–1 m ammonium phosphate (pH 3.35) at a flow rate of 1 ml/min (a linear gradient of 0–15 mmammonium phosphate for the first 45 min, 15–24 mm for 1 min, 24–45 mm from 46 to 80 min, and 45 mmto 1 m for the last min). Fractions of 1 ml were collected, and the 32P radioactivity was evaluated by scintillation counting. The ADP-ribosylated β subunit was immunoprecipitated as described previously (27.Morris D. McHugh-Sutkowski E. Moos M. Simonds W.F. Spiegel A.M. Seamon K.B. Biochemistry. 1990; 29: 9079-9084Crossref PubMed Scopus (18) Google Scholar), with the difference that plasma membranes were solubilized with 1% sodium cholate for 1 h at 4 °C. For immunoblot analysis, antisera raised against the amino-terminal decapeptide of the β subunit (MSELDQLRQE, the MS 1 antiserum; Ref.28.Murakami T. Simonds W. Spiegel A.M. Biochemistry. 1992; 31: 2905-2911Crossref PubMed Scopus (28) Google Scholar) and the carboxyl-terminal peptide (which includes residues 330–340, GSWDSFLKIWN, the SW28 antiserum; Ref. 28.Murakami T. Simonds W. Spiegel A.M. Biochemistry. 1992; 31: 2905-2911Crossref PubMed Scopus (28) Google Scholar) were employed. Antibody-antigen complexes were visualized using a peroxidase-conjugated secondary antibody (26.Di Girolamo M. D'Arcangelo D. Cacciamani T. Gierschik P. Corda D. J. Biol. Chem. 1992; 267: 17397-17403Abstract Full Text PDF PubMed Google Scholar). Membrane proteins were ADP-ribosylated for 2 h at 37 °C. Trypsin, dissolved in 1 mm HCl, was added at a concentration of 0.01 mg/ml (the final HCl concentration of 0.1 mm did not affect the ADP-ribosylation). The samples were incubated in the presence of trypsin or HCl for 30 min at 37 °C, as described previously (29.Thomas T.C. Sladek T. Yi F. Smith T. Neer E.J. Biochemistry. 1993; 321: 8628-8635Crossref Scopus (33) Google Scholar). Proteolysis was terminated by the addition of Laemmli sample buffer. 200 μg of washed, [32P]ADP-ribosylated plasma membranes were stirred at 4 °C for 60 min in 0.6 ml of PBS buffer (pH 7.4) containing 1% sodium cholate and 1 mm DTT. The samples were centrifuged at 100,000 × g for 60 min, and 0.2 ml of the cholate extract (supernatant) were applied to a Sepharose 12 HR 10/30 size exclusion column (Amersham Pharmacia Biotech) at a flow rate of 0.4 ml/min. The column was equilibrated and eluted with PBS buffer containing 1% cholate and 1 mm DTT. Fractions of 0.2 ml were collected, with the protein elution pattern being evaluated by the absorbance at 254 nm. The column was calibrated with the following molecular mass standard proteins (Amersham Pharmacia Biotech): aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease (13.7 kDa). Confluent CHO cells (2.5 × 106 cells/35 mm dish) were metabolically labeled as described previously (30.Staddon J.M. Bouzyk M.M. Rozengurt E. J. Cell Biol. 1991; 115: 949-957Crossref PubMed Scopus (15) Google Scholar, 31.Frieden P.J. Gaut R.J. Hendershot L.M. EMBO J. 1992; 11: 63-70Crossref PubMed Scopus (152) Google Scholar), with the following modifications: the cells in each well were incubated for 16 h at 37 °C with 100 μCi of [3H]adenine (specific activity, 20–40 Ci/mmol) in 1 ml of DMEM without fetal calf serum, containing 20 μg/ml actinomycin D to avoid [3H]adenine incorporation into RNA. The labeled medium was replaced with fresh medium, and the incubation was carried out for an additional 4 h in the absence or in the presence of pertussis toxin (100 ng/ml). Then the supernatant was removed, and the incubation was continued for 10 min at 37 °C with RNase (100 μg/ml) in 100 μl of 20 mm Tris-HCl (pH 7.5). The reaction was terminated by diluting the samples with 50 μl of Laemmli sample buffer, followed by 2 min boiling and analysis by 10% SDS-PAGE. The protein-associated radioactivity was evaluated by a Bio-Imaging Analyzer (FUJI Film) or by fluorography with the gels exposed for at least 20 and 90 days, respectively. To verify the [3H]adenine incorporation into the cellular NAD+ pool, radiolabeled CHO cells were extracted with methanol/chloroform/water/12 n HCl (1:1:0.5:0.01) and analyzed by HPLC as described above, with the following modifications: H2O for the first 5 min, followed by a linear gradient of 0–30 mm ammonium phosphate (5–55 min), and of 30 mm to 1 m (55–115 min). [3H]NAD represented ∼8% of the total [3H]adenine metabolites. Moreover, the CHO intracellular NAD concentration was determined, considering that 106 cells have a volume of 1.2 μl (as measured by a Coulter Counter ZM linked to a Coulter Channelizer 256), and was found to be 785 ± 10 μm. This allows the calculation of the specific radioactivity of the NAD+ pool as 380 μCi/mmol, the value used to estimate the amount of the endogenously mono-ADP-ribosylated β subunit. [3H]Adenine-labeled CHO cells (6 × 107 for each experiment) were washed with HBSS and then broken with a Teflon/glass Potter homogenizer in hypotonic buffer containing 5 mm Tris-HCl (pH 8.0), 1 mmEGTA, and protease inhibitors. Unbroken cells and nuclei were removed by low speed centrifugation (10 min at 600 × g), and the crude membranes (300 μg) were collected by centrifuging the supernatant for 15 min at 25,000 × g. The3H-labeled βγ subunit were extracted and analyzed as described previously (32.Tanaka T. Kubota M. Samizo K. Nakajima Y. Hoshino M. Kohno T. Wakamatsu K. Protein Expression Purif. 1999; 15: 207-212Crossref PubMed Scopus (5) Google Scholar), with the difference that His6-αi1 was employed to reassociate with βγ. After washing with NaSCN, the bound βγ subunits were solubilized in Laemmli sample buffer and analyzed as described above. About 2 μg of βγ were recovered from this affinity purification (as estimated by densitometric analysis of the immunoblot) with ∼0.2% being modified in intact cells. Membranes (25 μg/25 μl) prepared from freshly dissected rat brain (33.De Matteis M.A. Di Girolamo M. Colanzi A. Pallas M. Di Tullio G. McDonald L.J. Moss J. Santini G. Bannykh S. Corda D. Luini A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1114-1118Crossref PubMed Scopus (69) Google Scholar, 34.Mironov A. Colanzi A. Silletta M.G. Fiucci G. Flati S. Fusella A. Polishchuk R. Mironov Jr., A. Di Tullio G. Weigert R. Malhotra V. Corda D. De Matteis M.A. Luini A. J. Cell Biol. 1997; 139: 1-10Crossref PubMed Scopus (43) Google Scholar) were assayed for adenylyl cyclase activity as described previously (35.Chen J. De Vivo M. Dingus J. Harry A. Li J. Sui J. Carty D.J. Blank J.L. Exton J.H. Stoffel R.H. Inglese J. Lefkowitz R.J. Logothetis D.E. Hildebrandt J.D. Iyengar R. Science. 1995; 268: 1166-1169Crossref PubMed Scopus (236) Google Scholar), with the exception that cAMP was separated by thin layer chromatography using silica gel G plates (Kieselgel 60 F254, Merck) pretreated with 1% potassium oxalate and 2 mm EDTA, with chloroform/methanol/4m NH4OH (54:42:12) as the solvent system. The effect of the ADP-ribosylated βγ subunit was evaluated by adding to the reaction mixture 7 μg of plasma membranes from CHO cells preincubated for 6 h at 37 °C with different concentrations of purified bovine brain βγ subunit (100–500 nm), in the absence ("unmodified") or presence ("modified") of 1 mm NAD+ (under these experimental conditions about 60–80% of the added βγ subunit was ADP-ribosylated). After the incubation, CHO membranes containing either unmodified or modified βγ subunit were centrifuged (15 min at 12,000 × g) and resuspended in 5 μl of 10 mm HEPES (pH 8.0) and 0.1% Lubrol, and then added to the adenylyl cyclase assay mixture. Point mutations were generated using the Quickchange, site-directed mutagenesis kit (Stratagene), and the sequence of all constructs was confirmed by automated DNA sequencing. Snake venom phosphodiesterase digestion of ADP-ribosylated proteins (33.De Matteis M.A. Di Girolamo M. Colanzi A. Pallas M. Di Tullio G. McDonald L.J. Moss J. Santini G. Bannykh S. Corda D. Luini A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1114-1118Crossref PubMed Scopus (69) Google Scholar, 36.Kots A.Y. Skurat A.V. Sergienko E.A. Bulargina T.V. Severin E.S. FEBS Lett. 1992; 300: 9-12Crossref PubMed Scopus (83) Google Scholar), production of [32P]ADP-ribose (36.Kots A.Y. Skurat A.V. Sergienko E.A. Bulargina T.V. Severin E.S. FEBS Lett. 1992; 300: 9-12Crossref PubMed Scopus (83) Google Scholar), sensitivity of the ADP-ribosylated protein to hydroxylamine (NH2OH) and HgCl2 (36.Kots A.Y. Skurat A.V. Sergienko E.A. Bulargina T.V. Severin E.S. FEBS Lett. 1992; 300: 9-12Crossref PubMed Scopus (83) Google Scholar), and phosphoinositide-specific phospholipase C assay (37.Okazaki I.J. Kim H.-J. Moss J. J. Biol. Chem. 1996; 271: 22052-22057Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) were performed as described previously. Substrates of endogenous ADP-ribosylation can be identified by supplying cell extracts with radiolabeled NAD+. In enriched plasma membrane preparations from different cell lines, including Swiss 3T3, CHO and HL60 cells, NAD+ prominently labeled a 36-kDa protein (Fig.1 A). In the CHO preparation, the labeled protein co-migrated with the purified G protein β subunit on SDS-PAGE and was recognized on Western blots by a polyclonal antibody raised against the carboxyl-terminal decapeptide of the β subunit (SW28, which stains a doublet representing the β1and β2 isoforms) (Fig. 1, B and C). Purified bovine brain βγ subunit added to the assay mixture was also ADP-ribosylated (Fig. 1 A, lanes 2, 4, 6, 8, and 10 and Fig. 1, B and C, lane 2) and precisely co-migrated with the ADP-ribosylated 36-kDa endogenous protein on SDS-PAGE (Fig. 1 A, lanes 1, 3, 5,7, and 9 and Fig. 1, B andC, lanes 1, and 3–6). The labeled 36-kDa band contained both the ADP-ribosylated β1 and β2 subunits, and the doublet could be fully resolved employing extra-long gels for the protein separation (Fig. 1,B and C). It has been reported that the electrophoretic mobility of the G protein β subunit is decreased when urea (4 m) is introduced into the SDS-PAGE (38.Wieland T. Nurnberg B. Ulibarri I. Kaldenberg-Stasch S. Schultz G. Jakobs K.H. J. Biol. Chem. 1993; 268: 18111-18118Abstract Full Text PDF PubMed Google Scholar). Under these conditions, the ADP-ribosylated protein recognized by the SW28 antibody had a higher apparent molecular mass (39 kDa) and, again, precisely co-migrated with pure β subunit (Fig. 1 C), whereas the mobility of other proteins, such as the G protein α subunit (αi, ADP-ribosylated by pertussis toxin,lane 4, and αs, ADP-ribosylated by cholera toxin, lane 6, of Fig. 1, B and C), remained unchanged. Thus the major substrate of endogenous ADP-ribosylation in these enriched plasma membrane preparations appears to be the G protein β subunit. Moreover, to exclude the possibility that this is another ADP-ribosylated protein that can co-migrate on SDS-PAGE (plus or minus urea) with the β subunit, we immunoprecipitated the solubilized, ADP-ribosylated CHO membranes with the SW28 antibody. Both the labeled endogenous 36-kDa protein (Fig.2 A, lane 3) and the labeled purified β subunit (Fig. 2 A, lane 2) were immunoprecipitated by the SW28 antibody; as a control, non-immune rabbit serum did not precipitate any radioactivity (Fig. 2 A, lane 1). Moreover, when solubilized from membranes and analyzed by gel filtration chromatography, the ADP-ribosylated 36-kDa protein eluted as a single peak of ∼50-kDa (Fig. 2 B), which completely overlapped with the peak of the β subunit as revealed by the SW28 antibody (Fig. 2 C). Thus we provide compelling evidence indicating that the substrate of endogenous ADP-ribosylation is the β subunit of the heterotrimeric G protein.Figure 2Immunoprecipitation of the ADP-ribosylated G protein β subunit. A, CHO plasma membrane proteins ADP-ribosylated in the absence (lanes 1 and 3) and in the presence (lane 2) of purified bovine brain βγ subunit (2.5 μg/ml) were solubilized and subjected to immunoprecipitation using a non-immune serum (lane 1) or the β-specific SW28 antiserum (lanes 2 and3). B and C, gel filtration of the ADP-ribosylated G protein β subunit. B, autoradiography of the nitrocellulose filter containing the indicated fractions.C, immunoblot of the same fractions with the SW28 polyclonal antibody. Fractions 33–35 correspond to a molecular mass of 53 kDa. The data shown are from a single experiment performed in duplicate, which is representative of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To verify that the modification of the β subunit is indeed enzymatic mono-ADP-ribosylation, the labeled protein was digested with snake venom phosphodiesterase. This caused the release of radiolabeled 5′-AMP (∼70% of total radioactivity, as analyzed by HPLC; data not shown), which is considered diagnostic of mono-ADP-ribosylation (33.De Matteis M.A. Di Girolamo M. Colanzi A. Pallas M. Di Tullio G. McDonald L.J. Moss J. Santini G. Bannykh S. Corda D. Luini A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1114-1118Crossref PubMed Scopus (69) Google Scholar, 36.Kots A.Y. Skurat A.V. Sergienko E.A. Bulargina T.V. Severin E.S. FEBS Lett. 1992; 300: 9-12Crossref PubMed Scopus (83) Google Scholar). Non-enzymatic mono-ADP-ribosylation, which might be caused by the formation of adducts with ADP-ribose generated from NAD+ by cellular NAD-glycohydrolases (NADases) (39.Hiltz H. Koch R. Fanick W. Klapproth K. Adamietz P. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3929-3933Crossref PubMed Scopus (74) Google Scholar), was ruled out because the β subunit was not labeled by free ADP-ribose (Fig.3 A, lane 2). The modified amino acid of the β subunit was further investigated by means of a characterization of the chemical stability of the ADP-ribosyl linkage. Treatment of the [32P]ADP-ribosylated β subunit for 12 h with NH2OH (which hydrolyzes the ADP-ribose of arginine), as opposed to HgCl2 or HCl (which act on ADP-ribosylated cysteine and serine/threonine residues, respectively), completely removed the ADP-ribose bound to the β subunit (Fig. 3 B, lane 2), as we also observed with the [32P]ADP-ribosylated α subunit of Gsinduced by cholera toxin (Fig. 3 B, lane 4). The possibility that the ADP-ribose is linked to glutamate could be ruled out since a 20-min treatment with NH2OH (a time sufficient to hydrolyze the ADP-ribose linked to glutamate) removed only 22% of the label from the ADP-ribosylated β subunit (a 55% loss of label was observed after 2 h and, as mentioned above, the complete loss of label occurred at 12 h). These results indicate that the mono-ADP-ribosylation occurs at an arginine residue of the β subunit. In line with these data, the mono-ADP-ribosylation of the β subunit was inhibited by agmatine (data not shown) and bymeta-iodobenzylguanidine (MIBG) in a dose-dependent manner (Fig. 3 C), both well characterized inhibitors of arginine-specific mono-ADP-ribosyltransferases (40.Loesberg C. van Rooij H. Smets L.A. Biochim. Biophys. Acta. 1990; 1037: 92-99Crossref PubMed Scopus (41) Google Scholar, 41.Smets L.A. Loesberg C. Janssen M. Van Rooij H. Biochim. Biophys. Acta. 1990; 1054: 49-55Crossref PubMed Scopus (51) Google Scholar). Thus, a plasma membrane-associated, arginine-specific, mono-ADP-ribosyltransferase can ADP-ribosylate the β subunit of heterotrimeric G proteins. Notably, the enzyme displayed a good degree of specificity for the β subunit, indicated by the fact that this protein is the most intensely labeled in membrane preparations (as seen in Fig. 1, B andC). A kinetic investigation of the ADP-ribosylation assays employing purified bovine brain βγ subunit and enriched plasma membranes as the enzyme source gave a K m value for NAD+ of 350 ± 20 μm, a value compatible with the physiological concentrations of NAD+ (see "Experimental Procedures" for calculation of the CHO intracellular NAD concentration and Ref. 42.Lee A.-C. Zizi M. Colombini M. J. Biol. Chem. 1994; 269: 30974-30980Abstract Full Text PDF PubMed Google Scholar). The V max was 500 ± 80 pmol/h/mg membrane protein. This rate might conceivably be regulated by activators (and/or co-factors) yet to be identified and, hence, might well be faster in the natural intracellular milieu. Few enzymes that catalyze ADP-ribosylation reactions have been purified and characterized, but among these the best
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