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Pertussis toxin decreases opiate receptor binding and adenylate inhibition in a neuroblastoma x glioma hybrid cell line
Life Sciences, Vol. 33, Sup. I, 1983, pp. 219-222 Printed in the U.S.A.
Pergamon Press
PERTUSSIS TOXIN DECREASES OPIATE RECEPTOR BINDING ANDAD~NYLATE INHIBITION IN A N E U R O B L A S T O M A X G L I O M A H Y B R I D C ~ . L I N F ~ T. Costa, K. Aktories*, G. Schultz* and M. W~ster Department of Neuropharmacology, Max-Planck-Institut f'dr Psychiatrie,' Kraepelinstrasse 2, D-8000 Mitnchen 40, F.R.G. *Pharmakologisches Institut der Universit~t Heidelberg, Im Neuenheimer Feld 366 D-69OO Heidelberg, F.R.G. (Received in final form June 26, 1983) Sun~ Neuroblastcma x glicma hybrid cells NGIO8-15 were treated with a toxin derived frcm Bordetella pertussis. As compared to control cells grown in the absence of toxin, the inhibitory effects of opioid agonists upon cAMP formation were dose-dependently impaired by a nen-competitive mechani~n. Radioligand binding studies revealed that opioid agonist binding was dramatically reduced in toxin-treated membranes when tested in the presence of Na+/Mg++/GMP-PNP. Further, the potencies of guanine nucleotides to decrease opioid agonist binding were differentially modulated. These studies may facilitate our understanding of the mechanisms responsible for acute and chronic opiate effects. An exotoxin produced by Bordetella pertussis (islet-activating protein, IAP) abolishes the ability of hormcnes to inactivate the adenylate cyclase in all, as yet tested, inhibitory systems (I-4). IAP catalyzes the ADP-ribosylation of a specific membrane protein with a molecular weight of 41000 daltons which is different (5,6) from that ADP-ribosylated by cholera toxin and involved in hormone stimulation of adenylate cyclase. It is, thus, likely that hormonal stimulation and inhibition of the enzyme are mediated by two distinct regulatory GTP-binding proteins (5,7,8) , N s and Ni, and t_hat pertussis toxin impairs the signal transmission at any inhibitory receptor by permanently affecting the function of N i. We have shown that in NGIO8-15 cells, in which a single pool of adenylate cyclase is under multireceptor control (9) , opiate induced desensitization is specific: the inhibitory influence of a single type of receptor can be abolished without affecting the ability of the others to inactivate the enzyme (10). Thus, the question occurs which difference exists between the "specific uncoupling" produced by the hormone and the "nonspecific uncoupling" produced by the toxin. Here we present evidence that the two phenomena are qualitatively very similar and that pertussis toxin might be a valuable tool in the evaluation of molecular mechnanisms of acute and chronic effects of inhibitory hormones in NGIO8-15 hybrid cells. Methods NGIO8-15 cells were grown as described (11). The determination of the opioid ability to inhibit cAMP accumulation induced by PGE1 and R020-I 724 was measured as described (10) using cell suspensions. Cells were treated with IAP after reaching confluency: fresh grc~T_h medium containing different concentrations of IAP was added and the cells were further cultured for 24 hours. The 0024-3205/83 $3.00 + .00 Copyright (e) 1983 Pergamon Press Ltd.
220
Pertussis Toxin and Opioid Mechanisms
Vol. 33, Sup. I, 1983
cells were then harvested and the pellet resuspended for studying cAMP accumulation or frozen at -7OOC. Membranes for radioligand binding studies were prepared by homogenization of cells in Tris-buffer (50raM, pH 7.4) containing ~DTA (0.5raM) and centrifugation at 50,000 x g. After resuspension and centrifugation, membranes were suspended in Tris/D3TA buffer at a concentration of 0.5 x 106 cells/ml. Opiate binding was measured in 2ml-aliquots as has been described (12). Results and Discussion I ) I A P effect on opioid inhibition of cAMP accumulation Pretreatment of NGIO8-15 cells with increasing concentratic~s of p@_rtussis toxin produces a progressive reduction of the ability of [D-AIa 2 , D-Leu 5] enkephalin (DADL) to inhibit the intracellular accumulation of cAMP. (Fig. I). As little as .O1 ng-ml -I of IAP reduces the ~ i effect of the pep tide from 75 + 6% in untreated cells to 55 + 4% in treated cells. With I ng.ml -] of toxin, the inhibiton is completely abolished. Computer analysis of the curves shown in fig. I reveals that, while the maximal effects are dose-dependently diminished by the treatments, neither the slopes nor the IC50's are significantly changed. Thus, pertussis toxin abolishes opioid receptor control of adenylate cyclase without altering the potency nor the apparent positive cooperativity (slope larger than unity) of epioid effect. 100(
I.0 ngl rnl
o
Figure I Effect of pertussis toxin treatment upon the inhibition of PGE,-stimulated cAMP accumulation by DADL. Values are expressed as percent of the respective cAMP-content in the absence of DADL. Each point is the mean of ~a~ruplicate samples with S~M generally less than 10%. The experiment was repeated twice.
0li
nglmL
u
0.01 nglmL
~
~J
1
C~'O'-- " C ~
10
control
100
nM
DADL
When we studied the effect of the toxin on adrenergic- or muscarinic-effected inhibiton of cAMP acc~mllation, identical results (data not shown) were obtained, in agreement with Kurose et al. (3). If one cc~pares the kinetics of opioid desensitization induced by IAP (shown in fig. I ) with the kinetics of that produced by chronic opioid treatment (10) , the similarity is striking: in both cases the maximal opioid effect is changed without altering the half-maximal effective concentrations, i.e. both treatments are n o n ~ t i t i v e with respect to the ligand inhibitory effect. This may suggest that, although the two phenomena are induced through different routes and differ in selectivity, they have a mechanistic similarity; a regulatory protein (Ni) with an impaired ability to transfer the receptor input may be responsible for each of them. 2) IAP effect on opioid receptor binding The m e m b r a ~ fraction from vehicle-treated and IAP-treated cells was prepared and the binding of 3H-DADL, 3H-diprenorphine (DIPR) am~ 3H-naloxone (NAL) was measured in Tris-HCl buffer. The three ligands labeled a single class of sites under these conditions; toxin treatment reduced their number by 50 to 70% but did not change their affinities. It is likely that opioid binding in ion and guanine nucleotide-free buffer on isolated membranes involves an uncoupled state of the receptor, since agonist and antagonist are equally affected by the toxin treatment. This is in agreement with our studies of
Vol. 33, Sup. I, 1983
Pertussis Toxin and Opioid Mechanisms
221
opioid binding and effects simultaneously assessed in intact living cells (T. Costa, in preparation). Nevertheless, these results further e ~ s i z e the striking similarity between IAP-induced (non-specific) and opioid-induced (specific) desensitization: when NGIO8-15 cells were chronically treated with DADL and opioid binding was then studied on the isolated membranes in ion-free buffer, a reduction in the maximal number of sites without change in affinity was observed (13). Further, opioid binding under conditions which ensure maximal opioid effect on adenylate cyclase was studied (14): buffer was additionated with Na + (IOOmM), Mg++(5 r~M) and GMP-PNP (10 ~M). The binding of the agonist DADL (Fig.2) and of the antagonist NAL were measured from their competition for the binding sites labeled by 3H-DIPR. It is clear that in IAP treated cells, naloxone binding is little affected. The binding isotherm of the agonist DADL in membranes treated with the toxin is, however, dose-dependently shifted to the right and flatter than in the control. This suggests that a class of very low affinity sites, revealed by the agonist, but not the antagonist, beccmes more abundant after toxin treatment of the membranes.
NAL
DADL 1.0"
B/Bo
OE
/i
,
-9
-g
-~
-g [igand conc.
tog (Pl)
Figure 2 Displacement of specific 3H-DIPR binding by DADL and NAL in control (open) and IAP treated membranes (filled symbols), in Tris buffer (triangles) and in the presence of Na+/ Mg++/(tMP-PNP (circles). Each point is the mean of three experiments performed in triplicate.
3) Guanine nucleotide effects on agonist binding in toxin-treated cells Guanine nucleotides decrease agonist binding for all receptors linked to adenylate cyclase. This is believed to reflect the negative heterotropic effect of the GTP-binding regulatory protein (N) on the affinity of the receptor (R) for its ligand and to be mediated through the physical association of the two components N and R in the cell membrane. We, therefore, have investigated if the ability of guanine nucleotide analogs to decrease agonist binding war altered GDP 8S
100
Inhibition of specific 3H-DADL binding by G T ~ S and GDPBS. Na + (100 mM) and Mg ++ (5 nt4) were added. Data are mean from two experiments assayed in duplicate.
GTPyS
eA O~
control pertussis toxin treated 3
10
30
100
3~ nu¢leotide
1000
(nM)
222
Pertussis Toxin and Opioid Mechanisms
Vol. 33, Sup. I, 1983
by IAP treatment of the cells. Fig. 3 shows that the effect of G ~ S , a potent and stable analog of GTP, in decreasing 3H-DADL binding in the presence of Na + (1OO n~4) is little affected by IAP pretreatment. This is in line with the finding that in the fat cell adenylate cyclase system (4) IAP treatment abolishes the inhibitory effect of GTP but does not change the one of GTPyS. In contrast, GDPBS was dramatically more potent in IAP-treated than in control cells: its po tency in decreasing agonist binding was enhanced 40 times by the toxin. Other nucleotides exhibited shifts in their potencies intermediate between the two extremes shown in fig. 3; GMP-PNP, 8 times, and GMP-PCP 14 times. Thus, while the effect of guanine nucleotide is still present in membranes desensitized by IAP, the specificity of the guanine nucleotide binding site which mediated the negative heterotropic effect cn the receptor, appears profoundly altered. Conclusions I. Desensitization induced by pertussis-toxin differs in some aspects from that induced by chronic opiate action: site of induction, specificity and reversibility of effect. Phenomenologically, however, both treatments appear strikingly similar; is highly suggestive that in both cases the correct functioning of N i is impaired. This irmplies that an increasing amount of receptors are uncoupl ed from the catalytic unit of cyclase. Since the pertussis-toxin impaired N i is still able to tran~nitt GTPyS-induced inhibition upon adenylate cyclase (4), the defect may rather be attributed to a disturbed receptor-N i interaction. 2. Radioligand binding studies reveal the critical significance of N i for the control of receptor affinities. In contrast to a notion forwarded recently (3) we conclude that such input is maintained in toxin-treated membrane, even though profound differences with respect to control preparations emerge. Thus, IAP alters the character of the nucleotide binding site on N i which might impede any opioid-induced GDP-GTP exchange. Such reasoning would be supported by the observation that pertussis toxin abolishes enkephalin stimulated GTPase activity in these membranes (15). However, any final definition of the mechanis~n underlying the IAP-induced impairment of adenylate cyclase inhibition must await the clarification of other phencrnena, such as alterations in the ioneffected control or receptor affinities. References I. KATADA, T. and UI, M., J. Biol. Chem. 256, 8310-8317 (1981). 2. HAZEKI, O. and UI, M., J. Biol. Chem. 256, 2856-2862 (1981) 3. KUROSE, H., KATADA, T. AMANO, T. and UI, M. J. Biol. Chem. 258, 4870-4875 (1982). 4. AKTORIES, K., SCHULTZ, G. and JACOBS, K.H., Febs. Lett. in press (1983). 5. HILDEBRANDT, J.D., SEKURA, R.D., CODINA, J., IYENGAR, R., MANCLARK, C.R. and BIRNBAUMER, L. Nature 302, 706-709 (1983) 6. KATADA, T. and UI, M. Proc. Natl. Acad. Sci. 79, 3129-3133 (1982) 7. RODBELT., M. Nature 284, 17-22 (1980) 8. JAKOBS, K.H., AKTORIES, K. and SCHULTZ, G., Nature 303, 177-178 (1983). 9. SABOL, S.L. and NIR/~kU3ERG, M., J. Biol. Chem. 245, I ~ 3 - 1 9 2 0 (1979). 10. W~STER, M., COSTA, T. and GRAMSCH, CH. Life Sci. this volume (1983). 11. HAMPRECHT, B. Int. Rev. Cytol. 49 99-170. 12. CHANG, K.J., MILLER, R.J. and CUATRECASAS, P. Mol. Phalqnacol. 14, 961970 (1978). 13. CHANG, K.J., ECKEL, R.W. and BLANCHARD, S.G. Nature 296, 446-448 (1982). 14. BLUME, A.J., LICHTSHTEIN, D. and BOONE, G. Proc. Natl. Acad. Sci. 76, 5626-5630 (1979) . 15. BbThNS, D.L., HEWLETT, E.L., MOSS, J. and VAUGHAN, M. J. Biol. Chem. 258, 1435-1438 (1983). Supported by Deutsche Forschungsgemeinschaft, Bonn.
Pergamon Press
PERTUSSIS TOXIN DECREASES OPIATE RECEPTOR BINDING ANDAD~NYLATE INHIBITION IN A N E U R O B L A S T O M A X G L I O M A H Y B R I D C ~ . L I N F ~ T. Costa, K. Aktories*, G. Schultz* and M. W~ster Department of Neuropharmacology, Max-Planck-Institut f'dr Psychiatrie,' Kraepelinstrasse 2, D-8000 Mitnchen 40, F.R.G. *Pharmakologisches Institut der Universit~t Heidelberg, Im Neuenheimer Feld 366 D-69OO Heidelberg, F.R.G. (Received in final form June 26, 1983) Sun~ Neuroblastcma x glicma hybrid cells NGIO8-15 were treated with a toxin derived frcm Bordetella pertussis. As compared to control cells grown in the absence of toxin, the inhibitory effects of opioid agonists upon cAMP formation were dose-dependently impaired by a nen-competitive mechani~n. Radioligand binding studies revealed that opioid agonist binding was dramatically reduced in toxin-treated membranes when tested in the presence of Na+/Mg++/GMP-PNP. Further, the potencies of guanine nucleotides to decrease opioid agonist binding were differentially modulated. These studies may facilitate our understanding of the mechanisms responsible for acute and chronic opiate effects. An exotoxin produced by Bordetella pertussis (islet-activating protein, IAP) abolishes the ability of hormcnes to inactivate the adenylate cyclase in all, as yet tested, inhibitory systems (I-4). IAP catalyzes the ADP-ribosylation of a specific membrane protein with a molecular weight of 41000 daltons which is different (5,6) from that ADP-ribosylated by cholera toxin and involved in hormone stimulation of adenylate cyclase. It is, thus, likely that hormonal stimulation and inhibition of the enzyme are mediated by two distinct regulatory GTP-binding proteins (5,7,8) , N s and Ni, and t_hat pertussis toxin impairs the signal transmission at any inhibitory receptor by permanently affecting the function of N i. We have shown that in NGIO8-15 cells, in which a single pool of adenylate cyclase is under multireceptor control (9) , opiate induced desensitization is specific: the inhibitory influence of a single type of receptor can be abolished without affecting the ability of the others to inactivate the enzyme (10). Thus, the question occurs which difference exists between the "specific uncoupling" produced by the hormone and the "nonspecific uncoupling" produced by the toxin. Here we present evidence that the two phenomena are qualitatively very similar and that pertussis toxin might be a valuable tool in the evaluation of molecular mechnanisms of acute and chronic effects of inhibitory hormones in NGIO8-15 hybrid cells. Methods NGIO8-15 cells were grown as described (11). The determination of the opioid ability to inhibit cAMP accumulation induced by PGE1 and R020-I 724 was measured as described (10) using cell suspensions. Cells were treated with IAP after reaching confluency: fresh grc~T_h medium containing different concentrations of IAP was added and the cells were further cultured for 24 hours. The 0024-3205/83 $3.00 + .00 Copyright (e) 1983 Pergamon Press Ltd.
220
Pertussis Toxin and Opioid Mechanisms
Vol. 33, Sup. I, 1983
cells were then harvested and the pellet resuspended for studying cAMP accumulation or frozen at -7OOC. Membranes for radioligand binding studies were prepared by homogenization of cells in Tris-buffer (50raM, pH 7.4) containing ~DTA (0.5raM) and centrifugation at 50,000 x g. After resuspension and centrifugation, membranes were suspended in Tris/D3TA buffer at a concentration of 0.5 x 106 cells/ml. Opiate binding was measured in 2ml-aliquots as has been described (12). Results and Discussion I ) I A P effect on opioid inhibition of cAMP accumulation Pretreatment of NGIO8-15 cells with increasing concentratic~s of p@_rtussis toxin produces a progressive reduction of the ability of [D-AIa 2 , D-Leu 5] enkephalin (DADL) to inhibit the intracellular accumulation of cAMP. (Fig. I). As little as .O1 ng-ml -I of IAP reduces the ~ i effect of the pep tide from 75 + 6% in untreated cells to 55 + 4% in treated cells. With I ng.ml -] of toxin, the inhibiton is completely abolished. Computer analysis of the curves shown in fig. I reveals that, while the maximal effects are dose-dependently diminished by the treatments, neither the slopes nor the IC50's are significantly changed. Thus, pertussis toxin abolishes opioid receptor control of adenylate cyclase without altering the potency nor the apparent positive cooperativity (slope larger than unity) of epioid effect. 100(
I.0 ngl rnl
o
Figure I Effect of pertussis toxin treatment upon the inhibition of PGE,-stimulated cAMP accumulation by DADL. Values are expressed as percent of the respective cAMP-content in the absence of DADL. Each point is the mean of ~a~ruplicate samples with S~M generally less than 10%. The experiment was repeated twice.
0li
nglmL
u
0.01 nglmL
~
~J
1
C~'O'-- " C ~
10
control
100
nM
DADL
When we studied the effect of the toxin on adrenergic- or muscarinic-effected inhibiton of cAMP acc~mllation, identical results (data not shown) were obtained, in agreement with Kurose et al. (3). If one cc~pares the kinetics of opioid desensitization induced by IAP (shown in fig. I ) with the kinetics of that produced by chronic opioid treatment (10) , the similarity is striking: in both cases the maximal opioid effect is changed without altering the half-maximal effective concentrations, i.e. both treatments are n o n ~ t i t i v e with respect to the ligand inhibitory effect. This may suggest that, although the two phenomena are induced through different routes and differ in selectivity, they have a mechanistic similarity; a regulatory protein (Ni) with an impaired ability to transfer the receptor input may be responsible for each of them. 2) IAP effect on opioid receptor binding The m e m b r a ~ fraction from vehicle-treated and IAP-treated cells was prepared and the binding of 3H-DADL, 3H-diprenorphine (DIPR) am~ 3H-naloxone (NAL) was measured in Tris-HCl buffer. The three ligands labeled a single class of sites under these conditions; toxin treatment reduced their number by 50 to 70% but did not change their affinities. It is likely that opioid binding in ion and guanine nucleotide-free buffer on isolated membranes involves an uncoupled state of the receptor, since agonist and antagonist are equally affected by the toxin treatment. This is in agreement with our studies of
Vol. 33, Sup. I, 1983
Pertussis Toxin and Opioid Mechanisms
221
opioid binding and effects simultaneously assessed in intact living cells (T. Costa, in preparation). Nevertheless, these results further e ~ s i z e the striking similarity between IAP-induced (non-specific) and opioid-induced (specific) desensitization: when NGIO8-15 cells were chronically treated with DADL and opioid binding was then studied on the isolated membranes in ion-free buffer, a reduction in the maximal number of sites without change in affinity was observed (13). Further, opioid binding under conditions which ensure maximal opioid effect on adenylate cyclase was studied (14): buffer was additionated with Na + (IOOmM), Mg++(5 r~M) and GMP-PNP (10 ~M). The binding of the agonist DADL (Fig.2) and of the antagonist NAL were measured from their competition for the binding sites labeled by 3H-DIPR. It is clear that in IAP treated cells, naloxone binding is little affected. The binding isotherm of the agonist DADL in membranes treated with the toxin is, however, dose-dependently shifted to the right and flatter than in the control. This suggests that a class of very low affinity sites, revealed by the agonist, but not the antagonist, beccmes more abundant after toxin treatment of the membranes.
NAL
DADL 1.0"
B/Bo
OE
/i
,
-9
-g
-~
-g [igand conc.
tog (Pl)
Figure 2 Displacement of specific 3H-DIPR binding by DADL and NAL in control (open) and IAP treated membranes (filled symbols), in Tris buffer (triangles) and in the presence of Na+/ Mg++/(tMP-PNP (circles). Each point is the mean of three experiments performed in triplicate.
3) Guanine nucleotide effects on agonist binding in toxin-treated cells Guanine nucleotides decrease agonist binding for all receptors linked to adenylate cyclase. This is believed to reflect the negative heterotropic effect of the GTP-binding regulatory protein (N) on the affinity of the receptor (R) for its ligand and to be mediated through the physical association of the two components N and R in the cell membrane. We, therefore, have investigated if the ability of guanine nucleotide analogs to decrease agonist binding war altered GDP 8S
100
Inhibition of specific 3H-DADL binding by G T ~ S and GDPBS. Na + (100 mM) and Mg ++ (5 nt4) were added. Data are mean from two experiments assayed in duplicate.
GTPyS
eA O~
control pertussis toxin treated 3
10
30
100
3~ nu¢leotide
1000
(nM)
222
Pertussis Toxin and Opioid Mechanisms
Vol. 33, Sup. I, 1983
by IAP treatment of the cells. Fig. 3 shows that the effect of G ~ S , a potent and stable analog of GTP, in decreasing 3H-DADL binding in the presence of Na + (1OO n~4) is little affected by IAP pretreatment. This is in line with the finding that in the fat cell adenylate cyclase system (4) IAP treatment abolishes the inhibitory effect of GTP but does not change the one of GTPyS. In contrast, GDPBS was dramatically more potent in IAP-treated than in control cells: its po tency in decreasing agonist binding was enhanced 40 times by the toxin. Other nucleotides exhibited shifts in their potencies intermediate between the two extremes shown in fig. 3; GMP-PNP, 8 times, and GMP-PCP 14 times. Thus, while the effect of guanine nucleotide is still present in membranes desensitized by IAP, the specificity of the guanine nucleotide binding site which mediated the negative heterotropic effect cn the receptor, appears profoundly altered. Conclusions I. Desensitization induced by pertussis-toxin differs in some aspects from that induced by chronic opiate action: site of induction, specificity and reversibility of effect. Phenomenologically, however, both treatments appear strikingly similar; is highly suggestive that in both cases the correct functioning of N i is impaired. This irmplies that an increasing amount of receptors are uncoupl ed from the catalytic unit of cyclase. Since the pertussis-toxin impaired N i is still able to tran~nitt GTPyS-induced inhibition upon adenylate cyclase (4), the defect may rather be attributed to a disturbed receptor-N i interaction. 2. Radioligand binding studies reveal the critical significance of N i for the control of receptor affinities. In contrast to a notion forwarded recently (3) we conclude that such input is maintained in toxin-treated membrane, even though profound differences with respect to control preparations emerge. Thus, IAP alters the character of the nucleotide binding site on N i which might impede any opioid-induced GDP-GTP exchange. Such reasoning would be supported by the observation that pertussis toxin abolishes enkephalin stimulated GTPase activity in these membranes (15). However, any final definition of the mechanis~n underlying the IAP-induced impairment of adenylate cyclase inhibition must await the clarification of other phencrnena, such as alterations in the ioneffected control or receptor affinities. References I. KATADA, T. and UI, M., J. Biol. Chem. 256, 8310-8317 (1981). 2. HAZEKI, O. and UI, M., J. Biol. Chem. 256, 2856-2862 (1981) 3. KUROSE, H., KATADA, T. AMANO, T. and UI, M. J. Biol. Chem. 258, 4870-4875 (1982). 4. AKTORIES, K., SCHULTZ, G. and JACOBS, K.H., Febs. Lett. in press (1983). 5. HILDEBRANDT, J.D., SEKURA, R.D., CODINA, J., IYENGAR, R., MANCLARK, C.R. and BIRNBAUMER, L. Nature 302, 706-709 (1983) 6. KATADA, T. and UI, M. Proc. Natl. Acad. Sci. 79, 3129-3133 (1982) 7. RODBELT., M. Nature 284, 17-22 (1980) 8. JAKOBS, K.H., AKTORIES, K. and SCHULTZ, G., Nature 303, 177-178 (1983). 9. SABOL, S.L. and NIR/~kU3ERG, M., J. Biol. Chem. 245, I ~ 3 - 1 9 2 0 (1979). 10. W~STER, M., COSTA, T. and GRAMSCH, CH. Life Sci. this volume (1983). 11. HAMPRECHT, B. Int. Rev. Cytol. 49 99-170. 12. CHANG, K.J., MILLER, R.J. and CUATRECASAS, P. Mol. Phalqnacol. 14, 961970 (1978). 13. CHANG, K.J., ECKEL, R.W. and BLANCHARD, S.G. Nature 296, 446-448 (1982). 14. BLUME, A.J., LICHTSHTEIN, D. and BOONE, G. Proc. Natl. Acad. Sci. 76, 5626-5630 (1979) . 15. BbThNS, D.L., HEWLETT, E.L., MOSS, J. and VAUGHAN, M. J. Biol. Chem. 258, 1435-1438 (1983). Supported by Deutsche Forschungsgemeinschaft, Bonn.