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Neuropeptide y inhibits pertussis toxin-catalyzed ADP-ribosylation in bovine adrenal chromaffin cell membranes
L~fe Scxences, Vol. Printed in the USA
52, pp. PL 285-290
Pergamon
PHARMACOLOGY Accelerated
Press
LETTERS
Communicat£on
N E U R O P E P T I D E Y INHIBITS PERTUSSIS TOXIN-CATALYZED ADPRIBOSYLATION IN BOVINE A D R E N A L CHROMAFFIN CELL MEMBRANES Jianhua Zhu and Terry D. Hexum 1 Department of Pharmacology, UniversityofNebraskaMedical Center, Omaha, Nebraska 68198-6260 (Submxtted March 4, 1993; accepted March 17, received an fxnal form Aprll 13, 1993)
1993;
Abstract: Evidence is presented that the neuropeptide Y receptor is directly coupled to an inhibitory G protein existing in cultured bovine adrenal chromaffin cell membranes Pertussls toxin catalyzes the [32p]ADPribosylation of a 41 kDa plasma membrane protein. 5'-Guanylyhmldodiphosphate inhibited the [32pIADP labelling of this protein in a dose-dependent manner whereas GTP had no effect Premcubatlon of the plasma membranes vnth high concentrations of neuropephde Y followed by a brief exposure to a low concentration of 5'-guanylylim,dodlphosphate sigmficantly inhibited ADP-nbosylation beyond that observed with 5'guanylyhmldodlphosphate alone These results suggest that the neuropeptide Y receptor in bovine adrenal chromaffin cells is directly coupled to a 41 kDa PTX substrate (presumably the a subumt of an intubltory G protem)
Introduction NPY, a 36-amino acid member of the pancreatic polypeptide family of C-terminal amidated peptides, was originally isolated from porcine brain (1). This widely distributed peptide, which has been shown to display a diversity of physiological and pathological roles (2-5), exists in high concentrations in bovine adrenal medulla (6,7). The concentration of NPY-like immunoreactivity m bovine adrenal medulla is much higher than that of either substance P or vasoactive intestinal polypeptide (8,9). Moreover, NPY stored in the chromaffin granules can be released from cultured bovine adrenal chromaffin cells as a result of nicotinic receptor activation (3,10,11). The presence of NPY in chromaffin cell culture medium inhibits catecholamine secretion through an unknown mechanism (5). NPY has been shown to act on various cell types by inhibition of either adenylate cyclase activity or calcium channel function through receptor-G protein coupled processes (12,13). G proteins (a heterotrimer with subunits designated 4, 13 and y) are activated by agonist-receptor binding, the subsequent binding of GTP to the ~ subunit and then dissooation into functional subunits. Hydrolysis of GTP results in the reassociation of the subunits to an inactive G protein. The inhibitory action of NPY probably occurs via the NPY-induced dissociation of a specific inhibitory G protein (G,) into active subunits, one
1To whom correspondence should be addressed.
Copyright
0024-3205/93 $6.00 + .00 © 1993 Pergamon Press Ltd All r~ghts
reserved.
PL-286
NPY Inhibits ADP-r~bosylation
Vol. 52, No. 25, 1993
of which (4 subunit) inhibits adenylate cyclase activity. The dissociation into active subunits can be prevented by PTX-catalyzed ADP-ribosylation of the ~ subunit. Conversely, agonistinduced dissociation into subunits precludes ADP-ribosylation of the G protein e subunit. We have previously demonstrated bovine adrenal chromaffin cells possess an NPY receptor and NPY inhibits forskolin-stimulated cAMP accumulation via a pertussis toxin sensitive process (14). The action of NPY can be blocked by pretreatment of chromaffin cells with PTX. If the NPY receptor is directly coupled to G 1, it should be possible to prevent the PTX-catalyzed /n vitro ADP-ribosylation of G, by performing the PTX incubation in the presence of NPY and the nonhydrolyzable GTP analogue Gpp(NH)p which favors the prolonged existence of the dissociated G protein. Here we use high concentrations of NPY to prevent the PTX catalyzed ADP-ribosylation of a 41 kDa protein. NPY significantly reduced ADP-ribosylation of a 41 kDa protein as demonstrated by a decrease in [32p]NAD labelling of this protein in the presence of Gpp(NH)p. Thus NPY action on bovine adrenal chromaffin cells very likely occurs due to the direct coupling of the NPY receptor to a 41 kDa PTX substrate which is probably the e subunit of G,.
Materials and Methods Materials: NPY was purchased from Peninsula Laboratories, Inc. (Belmont, CA). Pertussis toxin was from List Biological Laboratories, Inc. (Cambell, CA). [32p]NAD (1000 Ci/mmol) was from Amersham (Arlington Heights, IL). DTI', I$-NAD, ATP, GTP, Gpp(NH)p, benzamidine, bacitracin, collagenase, DNAse and antibiotics were obtained from Sigma Chemical Co. (St. Louis, MO). DMEM, fetal bovine serum, Hanks balanced salt solution and cell culture plasticware were from GIBCO (Gaithersburg, MD). Fluorocarbon filters were from Spectrum Medical Industries (Los Angeles, CA). Cell culture: Isolation of bovine adrenal chromaffin cells followed the procedure of Wilson and Kirshner (15) with modifications. Bovine adrenal glands, obtained from a local slaughter house, were transferred to the laboratory at ambient temperature and perfused with 3 ml/gland of sterile buffer A composed of 5 mM HEPES, 150 mM NaCl, 5 mM KCI, 7.8 mM glucose, 200 units-I~g/ml penicillin-streptomycin and 40 I~g/ml gentamicin, pH 7.4 for 3 times at 37°C within 15 rain. The primed glands were perfused 3 times at 15 min intervals with 3 mi/gland of digestion solution consisting of 0.15% collagenase and 30 I~g/ml DNAse (type I) dissolved in buffer A (warmed to 37°C). The medulla was then carefully dissected from the cortex, finely minced and further digested in a 37°C shaking water bath at a setting of 120 oscillations/min for 30 min. Cells were filtered through a single layer of fluorocarbon filter (mesh opening: 70 I~m), washed by centrifugation at 100 x g at room temperature three times (5 min each) in buffer A and once in buffer B (buffer A plus 1 mM CaCI2 and 1 mM MgSO4). The final pellet was resuspended in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 I~g/ml streptomycin, 40 I~g/ml gentamicin, 50 units/ml nystatin and 1 I~M5-fluoro-2'-deoxyuridine 5'-monophosphate. Cells were plated in 100 X 20 mm cell culture plastic dishes at a density of 2 x 107 cells/10 ml/dish in the atmosphere of 7.5% CO 2 at 37°C. Half of the medium was replaced every other day. Cells were usually used on day 4 or 5 after plating. Membrane preparation: Cells were washed twice with cold Hanks' Balanced Salt Solution and scraped from plates using a rubber policeman in the same buffer followed by a 5 min centrifugation at 100 g at 4 °C. The pelleted cells were resuspended in ice-cold lysis buffer consisting of 5 mM Tris-HCl (pH 7.5) containing 1 mM DTI', 1 mM EDTA and 1 mM PMSF. The cell suspension was kept on ice for 10 min and then homogenized i~ a
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Dounce homogenizer (15 strokes). The homogenate was centrifuged at 100 g for 5 min to remove undisrupted cells and nuclei. The supernatant was then centrifuged at 13,600 g for 10 min and the pellet was washed once more. The final pellet was resuspended in NPY binding buffer consisting of 50 mM HEPES, 1 mM MgCI 2, 1 mM CaCI 2, 1 mM PMSF, 1 mM benzamidine and 1 mg/ml bacitracin, pH 7.4.
NPY
Inhlbits
ADP-ribosylatLon
PL-287
W l W l W 200 kDa116 k D a 97 k D a -
66 kDa-
45 kDa-
PTX-catalyzed [32p]ADPribosylation: ADP-ribosylation of bovine adrenal chromaffin cell membranes was carried out 31 k D a according to Yajima et al. (16). Ten ~1 of PTX stock solution (1 I~g/10 ~1 H20) was preactivated PTX 1 3 5 i0 30 60 (30°C for 30 rain) in 50 ¢1 buffer (m/n) containing 100 mM K2HPO 4 (pH Fig 1 8.0), 10 mM thymidine, 1 mM Time-course for PTX-catalyzed ADP-ribosylation. Plasma ATP, 1 mM MgCI 2, 0.5 mM membranes (50/Jg/lane) were incubated without (lane I) or with EDTA, 1 mM PMSF, 5 mM (lane 2 to lane 7) 10/~g/ml PTX in the presence of [32p]NAD(5 H E P E S and 20 mM DTI'. An #Cl/10 /~M) in ribosylation buffer at 30°C for various Ume aliquot of plasma membranes (50 points The autoradiogram shown represents two separate I~g protein/18 I~l/assay) was experiments w~thidentical results incubated in the absence or presence of 2 I~1 of NPY stock solution (100 I~M in H20 ) at 23°C for 90 min to reach equilibrium binding. At the end of NPY binding, a 10 I~! aliquot of [32p]NAD (5 I~Ci [32p]NAD diluted with unlabelled NAD to 10 I~M) was added followed by sequential addition of 10 I~1 of G T P or its nonhydrolyzable analogue Gpp(NH)p and 60 I~1 of preactivated PTX at the intervals indicated in the figure legends. The reaction was terminated by addition of 0.4 ml ice cold NPY binding buffer followed by centrifugation at 13,600 g for 10 min. The pellet was washed once and solubilized in Laemmli's sample buffer by heating at 100°C for 5 min. Proteins were then subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (17,18). The gels were stained, destained, dried and exposed to Kodak X-Omat film at -80°C.
Results T i m e c o u r s e o f PTX-catalyzed ADP-ribosylation: PTX-catalyzed /n v/tro ADPribosylation is usually carried out for a period of 30-60 min (16,19,20). Two faint bands, a 29 kDa and a 32 kDa species, were observed after 1 h incubation with [32p]NAD in the absence of PTX (Fig. 1). These bands probably represent auto-ribosylated proteins. A major band with apparent molecular mass of 41 kDa appeared after incubation with PTX
PL-288
NPY
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ADP-ribosylation
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(10 pg/ml). ADP-ribosylation increased with incubation time for at least 1 h (Fig. 1). Sufficient incorporation of radioactivity into m e m b r a n e protein was observed after only 1 min incubation.
1
;I
3
4
5
6
7
8
116 kDa97 kDa-
116 kDa97 PDa-
66 kDa-
66 kDa-
45 kDa-
45 kDa-
Ii11!
31 kDa31 kDa-
PTX
-
+
+
+
+
+
+
+
C p p (NH) p o "1~
o
~:
o
o o
o o
X
~
o
B
A Fig. 2.
Effect of GTP and Gpp(NH)p on PTX-catalyzedADP-ribosylation. Plasma membranes (50/Jg/lane) were premcubated w3th different concentrations of GTP (A) or Gpp(NH)p (B) at 30°C for 3 mm followed by 1 mm incubation with [32p]NAD and PTX at 30°C. The autoradiograms shown are the representative of 3 (Fig 2A) and 4 (Fig 2B) individual experiments with similar results Effect o f GTP and G p p ( N H ) p on PTX-catalyzed ADP-ribosylation: Membranes were incubated with either G T P or its nonhydrolyzable analogue G p p ( N H ) p for 3 rain followed by a i min incubation with PTX. A short incubation time was chosen for PTX incubation for two reasons: I) one minute is sufficient to allow PTX-catalyzed ADP-ribosylation to occur, and 2) longer incubation times could reduce labelling since either G T P or G p p ( N H ) p causes G protein dissociation and hence disappearance of PTX substrate. Pretreatment of membranes for 3 rain with G T P (0 to 300 pM) had no effect on the labelling of the 41 kDa P T X substrate (Fig. 2A). However, preincubation of the membranes with the nonhydrolyzable analogue, G p p ( N H ) p , for 3 rain inhibited the ADP-ribosylation in a dosedependent manner (Fig. 2B). The inhibition was first observed at a G p p ( N H ) p concentration of i0 pM.
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Effect of NPY on PTXcatalyzed ADP-ribosylation: Preincubation of plasma membranes with 10 tiM NPY had no effect on A D P ribosylation (Fig. 3, lane 3). However, addition of 30 ~M G p p ( N H ) p inhibited the [32p]NAD labelling of the 41 kDa protein (lane 4)(p < 0.05 compared to lane 3). When membranes were pre-incubated in the presence of 10 I~M NPY followed by 30 ttM G p p ( N H ) p , a further reduction in labelling was seen (lane 5)(p < 0.05 compared to lane 4).
Discussion VI'X has been frequently used as a tool to identify and investigate the functional roles of various G proteins in biological systems. The enzymatically active component of the toxin possesses both N A D + glycohydrolase and ADP-ribosyltransferase activities. In the presence of DTI" and ATP, P T X catalyzes the transfer of the [32p]ADP moiety of N A D onto the e~ subunit of G, and thus blocks the interaction of G, with the associated receptor. Activation of a G protein requires the binding of G T P to the • subunit (accelerated by hormone binding to the G protein coupled receptor), which dissociates the ec subunit from the I~7 subunits (21). PTX-catalyzed ADP-ribosylation depends on the integrity of the G protein heterotrimer (22).
NPY
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ADP-ribosylation
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116 k D a 97 k D a -
66 k D a -
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31 k D a -
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Fig. 3 Autoradiogram of PTX-catalyzedADP-ribosylation. Membranes were preincubated without or with 10/JM NPY at 23°C for 90 rain in NPY binding buffer followedby 3 mm incubation with 30 ~M Gpp(NH)p at 30°C in the ribosylaUon buffer Incubauon with 10 ~g/ml PTX and 5 pCi [32p]NAD was carned out as described m Fig. 2 Lane 1 without PTX; lane 2" control (ROD = 1 000 + 0.000), lane 3 10 ~M NPY (ROD = 1.009 + 0 003), lane 4 30 ~M Gpp(NI-I)p (ROD = 0.826 + 0 044); lane 5 10 /~M NPY and 30 ~M Gpp(NH)p ROD = 0 678 + 0.027) The autora&ogram shown is representaUveof 4 separate experiments with ulentical results. Relative opucal density (ROD) was obtained by scamung the 41 kDa bands of the autoradtograms using the MCID image analysis system. Results are presented as mean__+ s.d from the 4 autoradlograms
Our results demonstrate that cultured bovine adrenal chromaffin cells possess a 41 k D a P T X substrate. This 41 kDa protein is most likely the e~ subunit of the inhibitory G protein, G, (14). Activated PTX effectively catalyzed ADP-ribosylation in 1 min. ADPribosylation was inhibited by G p p ( N H ) p but not the natural ligand for G protein, GTP. This is because when hydrolysis is at a steady-state, the GDP-bound form of a G protein constitutes the majority of the G protein pool since kcat exceeds koff for G D P by an order
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NPY Inhiblts ADP-ribosylation
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of magnitude (23). The nonhydrolyzable G T P analogue Gpp(NH)p binds the ~ subunit tightly and this complex accumulates, resulting in reduction of the PTX sensitive pool. H o r m o n e binding to the G protein-coupled receptor promotes the exchange of GTP (or its analogues) for G D P on the ~ subunit (21). The binding of high concentrations of NPY to its receptors which is coupled to G, (14) would result in the acceleration of G D P dissociation and Gpp(NH)p association. To obtain the lowest possible G protein subunit dissociation rate to facilitate a demonstration of NPY action, we used 30 ~M Gpp(NH)p, which is slightly above the minimum concentration capable of bringing about inhibition of ADP-ribosylation. NPY along with Gpp(NH)p significantly inhibited ADP-ribosylation compared to Gpp(NH)p alone. The fact that the inhibition is not complete may be explained by the assumption that only a small fraction of the total PTX-sensitive pool is normally coupled to NPY receptors. Full activation of this fraction would thus only result in a partial inhibition of ADP-ribosylation of the PTX-sensitive pool. Nevertheless, the partial inhibition of PTX-catalyzed ADP-ribosylation by NPY reveals a direct interaction between NPY receptors and inhibitory G proteins in bovine adrenal chromaffin cells. This study provides a useful approach to establish the relationship between a receptor population and inhibitory G protein should functional assays be unavailable.
Acknowledgements This work was supported in part by grants from the National Institutes of Health # NS26479 and the American Heart Association, Nebraska Affiliate # NE-91-G-10. We thank Susan Moore for excellent technical assistance and Lori Swigart for skillful secretarial assistance. References 1.
2 3. 4
5. 6 7 8.
9. 10 11. 12. 13 14. 15. 16. 17. 18. 19. 20 21 22. 23
K. TATEMOTO, Proc. Natl. Acad. So. USA 79, 5485-5489 (1982) T.E. ADRIAN, J.M. ALLEN, S R. BLOOM, M.A. GHATEI, M N. ROSSER, G.W. ROBERTS, TJ. CROW, K. TATEMOTO, and J.M POLAK, Nature ( L o n d ) ~ 584-586 (1983). E.A MAJANE, H ALHO, Y. KATAOKA, C.H. LEE, and H.-Y.T YANG, Endocrinology117, 1162-1168 (1985) W.R. CROWLEY and S.P KALRA, Synapse2, 276-281 (1988). H. HIGUCHI, E. COSTA, and H -Y.T. YANG, J. Pharmacol Exp Ther 244, 468-474 (1988) J M. ALLEN, T E. ADRIAN, J.M POLAK,and S.R. BLOOM, J Auton Nerv Syst.9_,559-563 (1983). I.M. VARDELL, J.M. POLAKM, J.M. ALLEN, G. TERENGHI, and S R BLOOM, Endocrinology 114, 1460-1462 (1984) B.G. LIVETT, Cell Biology of the Secretory Process, M Cantm (Ed.), 30%358, Karger, Basel (1984) L.E. EIDEN, R.L ESKAY,J. SCOTT, H POLLARD, and AJ HOTCHKISS,Life Sci. 33, 687-693 (1983). Y. KATAOKA,E.A MAJANE, and H -Y.T YANG, Neuropharma~ology24, 693-695 (1985). T.D. HEXUM, E.A. MAJANE, L R. RUSSETT, and H-Y T YANG, J Pharmacol Exp Ther. 927-930 (1987). LA LOBAUGH and P.J BLACKSHEAR,Am. J Phystol 258, (Cell Physlol 27). C913-C922(1990) D.A EWALD, P C STERNWEIS,and R.J MILLER, Proc. Natl. Acad. Sci USA~_~,3633-3637(1988). J. ZHU, W. LI, M.L TOEWS, and T D. HEXUM, J Pharmacol Exp Ther 263, 1479-1486 (1992). S P WILSONand N. KIRSHNER, Methods Enzymol. 103, 305-312 (1983). Y YAJIMA, Y AKITA, and T SAITO, J Blol Chem 261, 2684-2689 (1986) W LI, R G. MACDONALD,and T.D HEXUM, J Biol. Chem. 267, 7570-7575 (1992). U K. LAEMMLI, Nature 227, 680.685 (1970). G.M. BOKOCH, T KATADA,J K NORTHUP, E L. HEWLETT, and A G. GILMAN,J. Biol Chem 258, 2072-2075 (1983) S.B JONES and D B. BYLUND, J Blol Chem 263, 14236-14244 (1988) L. BIRNBAUMER, Annu Rev. Pharmacol. Toxacol 30, 675-705 (1990) E.J NEER, J M LOK, and L G. WOLF, J Biol. Chem. 259, 14222-14229 (1984). A.G. GILMAN, Annu. Rev Biochem. 56, 615-649 (1987)
52, pp. PL 285-290
Pergamon
PHARMACOLOGY Accelerated
Press
LETTERS
Communicat£on
N E U R O P E P T I D E Y INHIBITS PERTUSSIS TOXIN-CATALYZED ADPRIBOSYLATION IN BOVINE A D R E N A L CHROMAFFIN CELL MEMBRANES Jianhua Zhu and Terry D. Hexum 1 Department of Pharmacology, UniversityofNebraskaMedical Center, Omaha, Nebraska 68198-6260 (Submxtted March 4, 1993; accepted March 17, received an fxnal form Aprll 13, 1993)
1993;
Abstract: Evidence is presented that the neuropeptide Y receptor is directly coupled to an inhibitory G protein existing in cultured bovine adrenal chromaffin cell membranes Pertussls toxin catalyzes the [32p]ADPribosylation of a 41 kDa plasma membrane protein. 5'-Guanylyhmldodiphosphate inhibited the [32pIADP labelling of this protein in a dose-dependent manner whereas GTP had no effect Premcubatlon of the plasma membranes vnth high concentrations of neuropephde Y followed by a brief exposure to a low concentration of 5'-guanylylim,dodlphosphate sigmficantly inhibited ADP-nbosylation beyond that observed with 5'guanylyhmldodlphosphate alone These results suggest that the neuropeptide Y receptor in bovine adrenal chromaffin cells is directly coupled to a 41 kDa PTX substrate (presumably the a subumt of an intubltory G protem)
Introduction NPY, a 36-amino acid member of the pancreatic polypeptide family of C-terminal amidated peptides, was originally isolated from porcine brain (1). This widely distributed peptide, which has been shown to display a diversity of physiological and pathological roles (2-5), exists in high concentrations in bovine adrenal medulla (6,7). The concentration of NPY-like immunoreactivity m bovine adrenal medulla is much higher than that of either substance P or vasoactive intestinal polypeptide (8,9). Moreover, NPY stored in the chromaffin granules can be released from cultured bovine adrenal chromaffin cells as a result of nicotinic receptor activation (3,10,11). The presence of NPY in chromaffin cell culture medium inhibits catecholamine secretion through an unknown mechanism (5). NPY has been shown to act on various cell types by inhibition of either adenylate cyclase activity or calcium channel function through receptor-G protein coupled processes (12,13). G proteins (a heterotrimer with subunits designated 4, 13 and y) are activated by agonist-receptor binding, the subsequent binding of GTP to the ~ subunit and then dissooation into functional subunits. Hydrolysis of GTP results in the reassociation of the subunits to an inactive G protein. The inhibitory action of NPY probably occurs via the NPY-induced dissociation of a specific inhibitory G protein (G,) into active subunits, one
1To whom correspondence should be addressed.
Copyright
0024-3205/93 $6.00 + .00 © 1993 Pergamon Press Ltd All r~ghts
reserved.
PL-286
NPY Inhibits ADP-r~bosylation
Vol. 52, No. 25, 1993
of which (4 subunit) inhibits adenylate cyclase activity. The dissociation into active subunits can be prevented by PTX-catalyzed ADP-ribosylation of the ~ subunit. Conversely, agonistinduced dissociation into subunits precludes ADP-ribosylation of the G protein e subunit. We have previously demonstrated bovine adrenal chromaffin cells possess an NPY receptor and NPY inhibits forskolin-stimulated cAMP accumulation via a pertussis toxin sensitive process (14). The action of NPY can be blocked by pretreatment of chromaffin cells with PTX. If the NPY receptor is directly coupled to G 1, it should be possible to prevent the PTX-catalyzed /n vitro ADP-ribosylation of G, by performing the PTX incubation in the presence of NPY and the nonhydrolyzable GTP analogue Gpp(NH)p which favors the prolonged existence of the dissociated G protein. Here we use high concentrations of NPY to prevent the PTX catalyzed ADP-ribosylation of a 41 kDa protein. NPY significantly reduced ADP-ribosylation of a 41 kDa protein as demonstrated by a decrease in [32p]NAD labelling of this protein in the presence of Gpp(NH)p. Thus NPY action on bovine adrenal chromaffin cells very likely occurs due to the direct coupling of the NPY receptor to a 41 kDa PTX substrate which is probably the e subunit of G,.
Materials and Methods Materials: NPY was purchased from Peninsula Laboratories, Inc. (Belmont, CA). Pertussis toxin was from List Biological Laboratories, Inc. (Cambell, CA). [32p]NAD (1000 Ci/mmol) was from Amersham (Arlington Heights, IL). DTI', I$-NAD, ATP, GTP, Gpp(NH)p, benzamidine, bacitracin, collagenase, DNAse and antibiotics were obtained from Sigma Chemical Co. (St. Louis, MO). DMEM, fetal bovine serum, Hanks balanced salt solution and cell culture plasticware were from GIBCO (Gaithersburg, MD). Fluorocarbon filters were from Spectrum Medical Industries (Los Angeles, CA). Cell culture: Isolation of bovine adrenal chromaffin cells followed the procedure of Wilson and Kirshner (15) with modifications. Bovine adrenal glands, obtained from a local slaughter house, were transferred to the laboratory at ambient temperature and perfused with 3 ml/gland of sterile buffer A composed of 5 mM HEPES, 150 mM NaCl, 5 mM KCI, 7.8 mM glucose, 200 units-I~g/ml penicillin-streptomycin and 40 I~g/ml gentamicin, pH 7.4 for 3 times at 37°C within 15 rain. The primed glands were perfused 3 times at 15 min intervals with 3 mi/gland of digestion solution consisting of 0.15% collagenase and 30 I~g/ml DNAse (type I) dissolved in buffer A (warmed to 37°C). The medulla was then carefully dissected from the cortex, finely minced and further digested in a 37°C shaking water bath at a setting of 120 oscillations/min for 30 min. Cells were filtered through a single layer of fluorocarbon filter (mesh opening: 70 I~m), washed by centrifugation at 100 x g at room temperature three times (5 min each) in buffer A and once in buffer B (buffer A plus 1 mM CaCI2 and 1 mM MgSO4). The final pellet was resuspended in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 I~g/ml streptomycin, 40 I~g/ml gentamicin, 50 units/ml nystatin and 1 I~M5-fluoro-2'-deoxyuridine 5'-monophosphate. Cells were plated in 100 X 20 mm cell culture plastic dishes at a density of 2 x 107 cells/10 ml/dish in the atmosphere of 7.5% CO 2 at 37°C. Half of the medium was replaced every other day. Cells were usually used on day 4 or 5 after plating. Membrane preparation: Cells were washed twice with cold Hanks' Balanced Salt Solution and scraped from plates using a rubber policeman in the same buffer followed by a 5 min centrifugation at 100 g at 4 °C. The pelleted cells were resuspended in ice-cold lysis buffer consisting of 5 mM Tris-HCl (pH 7.5) containing 1 mM DTI', 1 mM EDTA and 1 mM PMSF. The cell suspension was kept on ice for 10 min and then homogenized i~ a
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Dounce homogenizer (15 strokes). The homogenate was centrifuged at 100 g for 5 min to remove undisrupted cells and nuclei. The supernatant was then centrifuged at 13,600 g for 10 min and the pellet was washed once more. The final pellet was resuspended in NPY binding buffer consisting of 50 mM HEPES, 1 mM MgCI 2, 1 mM CaCI 2, 1 mM PMSF, 1 mM benzamidine and 1 mg/ml bacitracin, pH 7.4.
NPY
Inhlbits
ADP-ribosylatLon
PL-287
W l W l W 200 kDa116 k D a 97 k D a -
66 kDa-
45 kDa-
PTX-catalyzed [32p]ADPribosylation: ADP-ribosylation of bovine adrenal chromaffin cell membranes was carried out 31 k D a according to Yajima et al. (16). Ten ~1 of PTX stock solution (1 I~g/10 ~1 H20) was preactivated PTX 1 3 5 i0 30 60 (30°C for 30 rain) in 50 ¢1 buffer (m/n) containing 100 mM K2HPO 4 (pH Fig 1 8.0), 10 mM thymidine, 1 mM Time-course for PTX-catalyzed ADP-ribosylation. Plasma ATP, 1 mM MgCI 2, 0.5 mM membranes (50/Jg/lane) were incubated without (lane I) or with EDTA, 1 mM PMSF, 5 mM (lane 2 to lane 7) 10/~g/ml PTX in the presence of [32p]NAD(5 H E P E S and 20 mM DTI'. An #Cl/10 /~M) in ribosylation buffer at 30°C for various Ume aliquot of plasma membranes (50 points The autoradiogram shown represents two separate I~g protein/18 I~l/assay) was experiments w~thidentical results incubated in the absence or presence of 2 I~1 of NPY stock solution (100 I~M in H20 ) at 23°C for 90 min to reach equilibrium binding. At the end of NPY binding, a 10 I~! aliquot of [32p]NAD (5 I~Ci [32p]NAD diluted with unlabelled NAD to 10 I~M) was added followed by sequential addition of 10 I~1 of G T P or its nonhydrolyzable analogue Gpp(NH)p and 60 I~1 of preactivated PTX at the intervals indicated in the figure legends. The reaction was terminated by addition of 0.4 ml ice cold NPY binding buffer followed by centrifugation at 13,600 g for 10 min. The pellet was washed once and solubilized in Laemmli's sample buffer by heating at 100°C for 5 min. Proteins were then subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (17,18). The gels were stained, destained, dried and exposed to Kodak X-Omat film at -80°C.
Results T i m e c o u r s e o f PTX-catalyzed ADP-ribosylation: PTX-catalyzed /n v/tro ADPribosylation is usually carried out for a period of 30-60 min (16,19,20). Two faint bands, a 29 kDa and a 32 kDa species, were observed after 1 h incubation with [32p]NAD in the absence of PTX (Fig. 1). These bands probably represent auto-ribosylated proteins. A major band with apparent molecular mass of 41 kDa appeared after incubation with PTX
PL-288
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(10 pg/ml). ADP-ribosylation increased with incubation time for at least 1 h (Fig. 1). Sufficient incorporation of radioactivity into m e m b r a n e protein was observed after only 1 min incubation.
1
;I
3
4
5
6
7
8
116 kDa97 kDa-
116 kDa97 PDa-
66 kDa-
66 kDa-
45 kDa-
45 kDa-
Ii11!
31 kDa31 kDa-
PTX
-
+
+
+
+
+
+
+
C p p (NH) p o "1~
o
~:
o
o o
o o
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~
o
B
A Fig. 2.
Effect of GTP and Gpp(NH)p on PTX-catalyzedADP-ribosylation. Plasma membranes (50/Jg/lane) were premcubated w3th different concentrations of GTP (A) or Gpp(NH)p (B) at 30°C for 3 mm followed by 1 mm incubation with [32p]NAD and PTX at 30°C. The autoradiograms shown are the representative of 3 (Fig 2A) and 4 (Fig 2B) individual experiments with similar results Effect o f GTP and G p p ( N H ) p on PTX-catalyzed ADP-ribosylation: Membranes were incubated with either G T P or its nonhydrolyzable analogue G p p ( N H ) p for 3 rain followed by a i min incubation with PTX. A short incubation time was chosen for PTX incubation for two reasons: I) one minute is sufficient to allow PTX-catalyzed ADP-ribosylation to occur, and 2) longer incubation times could reduce labelling since either G T P or G p p ( N H ) p causes G protein dissociation and hence disappearance of PTX substrate. Pretreatment of membranes for 3 rain with G T P (0 to 300 pM) had no effect on the labelling of the 41 kDa P T X substrate (Fig. 2A). However, preincubation of the membranes with the nonhydrolyzable analogue, G p p ( N H ) p , for 3 rain inhibited the ADP-ribosylation in a dosedependent manner (Fig. 2B). The inhibition was first observed at a G p p ( N H ) p concentration of i0 pM.
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Effect of NPY on PTXcatalyzed ADP-ribosylation: Preincubation of plasma membranes with 10 tiM NPY had no effect on A D P ribosylation (Fig. 3, lane 3). However, addition of 30 ~M G p p ( N H ) p inhibited the [32p]NAD labelling of the 41 kDa protein (lane 4)(p < 0.05 compared to lane 3). When membranes were pre-incubated in the presence of 10 I~M NPY followed by 30 ttM G p p ( N H ) p , a further reduction in labelling was seen (lane 5)(p < 0.05 compared to lane 4).
Discussion VI'X has been frequently used as a tool to identify and investigate the functional roles of various G proteins in biological systems. The enzymatically active component of the toxin possesses both N A D + glycohydrolase and ADP-ribosyltransferase activities. In the presence of DTI" and ATP, P T X catalyzes the transfer of the [32p]ADP moiety of N A D onto the e~ subunit of G, and thus blocks the interaction of G, with the associated receptor. Activation of a G protein requires the binding of G T P to the • subunit (accelerated by hormone binding to the G protein coupled receptor), which dissociates the ec subunit from the I~7 subunits (21). PTX-catalyzed ADP-ribosylation depends on the integrity of the G protein heterotrimer (22).
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116 k D a 97 k D a -
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Fig. 3 Autoradiogram of PTX-catalyzedADP-ribosylation. Membranes were preincubated without or with 10/JM NPY at 23°C for 90 rain in NPY binding buffer followedby 3 mm incubation with 30 ~M Gpp(NH)p at 30°C in the ribosylaUon buffer Incubauon with 10 ~g/ml PTX and 5 pCi [32p]NAD was carned out as described m Fig. 2 Lane 1 without PTX; lane 2" control (ROD = 1 000 + 0.000), lane 3 10 ~M NPY (ROD = 1.009 + 0 003), lane 4 30 ~M Gpp(NI-I)p (ROD = 0.826 + 0 044); lane 5 10 /~M NPY and 30 ~M Gpp(NH)p ROD = 0 678 + 0.027) The autora&ogram shown is representaUveof 4 separate experiments with ulentical results. Relative opucal density (ROD) was obtained by scamung the 41 kDa bands of the autoradtograms using the MCID image analysis system. Results are presented as mean__+ s.d from the 4 autoradlograms
Our results demonstrate that cultured bovine adrenal chromaffin cells possess a 41 k D a P T X substrate. This 41 kDa protein is most likely the e~ subunit of the inhibitory G protein, G, (14). Activated PTX effectively catalyzed ADP-ribosylation in 1 min. ADPribosylation was inhibited by G p p ( N H ) p but not the natural ligand for G protein, GTP. This is because when hydrolysis is at a steady-state, the GDP-bound form of a G protein constitutes the majority of the G protein pool since kcat exceeds koff for G D P by an order
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of magnitude (23). The nonhydrolyzable G T P analogue Gpp(NH)p binds the ~ subunit tightly and this complex accumulates, resulting in reduction of the PTX sensitive pool. H o r m o n e binding to the G protein-coupled receptor promotes the exchange of GTP (or its analogues) for G D P on the ~ subunit (21). The binding of high concentrations of NPY to its receptors which is coupled to G, (14) would result in the acceleration of G D P dissociation and Gpp(NH)p association. To obtain the lowest possible G protein subunit dissociation rate to facilitate a demonstration of NPY action, we used 30 ~M Gpp(NH)p, which is slightly above the minimum concentration capable of bringing about inhibition of ADP-ribosylation. NPY along with Gpp(NH)p significantly inhibited ADP-ribosylation compared to Gpp(NH)p alone. The fact that the inhibition is not complete may be explained by the assumption that only a small fraction of the total PTX-sensitive pool is normally coupled to NPY receptors. Full activation of this fraction would thus only result in a partial inhibition of ADP-ribosylation of the PTX-sensitive pool. Nevertheless, the partial inhibition of PTX-catalyzed ADP-ribosylation by NPY reveals a direct interaction between NPY receptors and inhibitory G proteins in bovine adrenal chromaffin cells. This study provides a useful approach to establish the relationship between a receptor population and inhibitory G protein should functional assays be unavailable.
Acknowledgements This work was supported in part by grants from the National Institutes of Health # NS26479 and the American Heart Association, Nebraska Affiliate # NE-91-G-10. We thank Susan Moore for excellent technical assistance and Lori Swigart for skillful secretarial assistance. References 1.
2 3. 4
5. 6 7 8.
9. 10 11. 12. 13 14. 15. 16. 17. 18. 19. 20 21 22. 23
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