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nicotinic α-bungarotoxin binding sites with a derivative of α-bungarotoxin
Molecular Brain Research, 17 (1993) 95-100 © 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00
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BRESM 70548
Photoaffinity labeling of muscle-type nicotinic acetylcholine receptors and neuronal/nicotinic a-bungarotoxin binding sites with a derivative of a-bungarotoxin Anna
M.
Joy *, Hal N. Siegel
**
and Ronald J. Lukas
Division of Neurobiology, Barrow Neurological Institute, Phoenix, AZ 85013 (USA)
(Accepted 1 September 1992)
Key words: Acetylcholine receptor; ot-Bungarotoxin; TE671 cell line; IMR-32 cell line; SH-SY5Y cell line; PC12 cell line
Neuronal/nicotinic a-bungarotoxin binding sites (nBgtS) found in the nervous system are not well characterized. In this study, photolabile toxin derivatives have been used in affinity labeling protocols to investigate the subunit composition of nBgtS expressed by different neuron-like cell lines. Data obtained was compared to the known subunit composition of toxin-binding muscle-type nicotinic acetylcholine receptors (nAChR). Muscle-type nAChR-rich membranes prepared from Torpedo electroplax contain components with corrected apparent molecular sizes of 41, 46, 50, 62 and 66 kDa that are reactive with toxin. The photoaffinity labeling patterns for preparations derived from cells of the TE671 clone, which express muscle-type nAChR, are very similar to that of cells of the IMR-32 or SH-SY5Y clonal lines, which express nBgtS. There is consistent labeling of four polypeptides with corrected apparent molecular weights of 40, 43, 47 and 56 kDa. These results suggest that both mammalian muscle-type nAChR and mammalian nBgtS are similarly composed of at least four kinds of subunits.
INTRODUCTION T h e s n a k e n e u r o t o x i n , a - b u n g a r o t o x i n (Bgt), is a high affinity, specific, a n d f u n c t i o n a l l y - p o t e n t a n t a g o nist u s e d in t h e c h a r a c t e r i z a t i o n o f m u s c l e a n d m u s c l e - t y p e n i c o t i n i c a e e t y l c h o l i n e r e c e p t o r s 2'1°'2° ( n A C h R ) . Bgt also b i n d s to sites in n e u r o n a l tissues, t e r m e d n e u r o n a l / n i c o t i n i c Bgt b i n d i n g sites (nBgtS), with high affinity. H o w e v e r , t h e r e a r e m a n y r e p o r t s ( a n d n o t a b l e e x c e p t i o n s ) t h a t Bgt is f u n c t i o n a l l y i m p o t e n t as a nicotinic a n t a g o n i s t on n o r m a l o r n e o p l a s t i c cells d e r i v e d f r o m t h e a u t o n o m i c o r c e n t r a l n e r v o u s systems ~°. Thus, nBgtS a p p e a r to b e distinct from ' n e u r o n a l ' n A C h R t h a t clearly function as l i g a n d - g a t e d ion c h a n n e l s a n d can be i d e n t i f i e d by a l t e r n a t i v e m e a n s ~°'22. U s i n g o l i g o n u c l e o t i d e p r o b e s c o r r e s p o n d i n g to t h e N - t e r m i n a l a m i n o acid s e q u e n c e o f a nBgtS s u b u n i t i s o l a t e d from chick b r a i n by C o n t i - T r o n c o n i et al. 3, two subunits o f chick n B g t S (now t e r m e d a 7 a n d a 8 ) have
r e c e n t l y b e e n c l o n e d by S c h o e p f e r et al. 23. I n d e p e n dently, C o u t u r i e r et al. 4 used low s t r i n g e n c y h y b r i d i z a tion a n d a p r o b e d e r i v e d f r o m the chick n A C h R a 3 g e n e to clone o n e o f t h e s a m e g e n e s ( a 7 ) . D e d u c e d f e a t u r e s o f the p r o d u c t s o f t h e s e g e n e s i n d i c a t e t h a t nBgtS could, in fact, be m e m b e r s o f the e x t e n d e d gene s u p e r f a m i l y o f l i g a n d - g a t e d ion channels, which inc l u d e s r e c e p t o r s for acetylcholine, glycine a n d g a m m a a m i n o b u t y r i c acid. This i n f e r e n c e has b e e n substantia t e d by the d e m o n s t r a t i o n that a 7 prote~n e x p r e s s e d in Xenopus oocytes can function as a nicotinic l i g a n d - g a t e d ion c h a n n e l sensitive to f u n c t i o n a l b l o c k a d e by Bgt 4. W h e r e a s t h e subunit c o m p o s i t i o n s o f n A C h R f o u n d in v e r t e b r a t e muscle o r o f m u s c l e - t y p e n A C h R from electric tissue o f electric fish have b e e n k n o w n for m o r e t h a n a d e c a d e 2'2°, t h e r e is less c e r t a i n t y a b o u t the c o m p o s i t i o n o f nBgtS. Toxin b i n d i n g m a c r o m o l e c u l e s i s o l a t e d from several tissue sources by various investig a t o r s have b e e n r e p o r t e d to c o m p r i s e b e t w e e n o n e
Correspondence: A.M. Joy, Neuro-Oncology Research, Division of Neurology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013, USA. * Present address: Neuro-oncology Research, Division of Neurology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013, USA. ** Present address: Iatric Corp., 2330 South Industrial Park Drive, Tempe, AZ 85282, USA.
96
nBgtS derived from cells of different clonal lines. Preliminary reports of parts of this work have appeared 25.
and four subunits with apparent molecular sizes ( M app) ranging from 45 to 72 kDa 3'5'8'24'28. This lack of consensus could reflect, among other things, susceptibility to proteolysis of nBgtS, just as there is for nAChR, or use of tissues containing heterogeneous populations of nBgtS. As a complement to affinity purification techniques, and in order to minimize the likelihood that expression of multiple nBgtS subtypes could complicate data interpretation, photolabile, radiolabeled Bgt derivatives were used in affinity labeling protocols to characterize
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MATERIALS AND METHODS
Preparation and characterization of derivatized toxin Preparation and characterization of 1~I-labeled Bgt (I-Bgt) was as described n, except that the final ion exchange chromatography to separate native from mono- and di-iodinated toxin was not performed. The bifunctional photoaffinity reagent, N-5-azido-2-nitrobenzoylaminoacetimidate-HC1 (ANB-AI), was a generous gift of Dr. William Allison of the University of California at San Diego. Photoaffinity derivatized toxin was prepared as described 17. Briefly,
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Fig. 1. Saturation curves for A-IoBgt binding to membranes. Crude membrane fractions were prepared from cell lines as described in the text. Membranes from Torpedo elecfroplax (A), TE671 cells (B), IMR-32 cells (C), or SH-SYSY cells (D) were incubated with the specified concentration of A-I-Bgt for 2 h in the dark. Unbound toxin was removed via two cycles of centrifug~ition at 40,000x g for 10 rain in the dark, and bound toxin was quantitated by counting the pellet on a gamma counter. Non-specific binding was determined in the presence of a 100-fold excess of native, underivatized toxin. Data displayed are representative of a minimum of 3 separate experiments.
97 radioiodinated toxin was incubated overnight at 25°C with excess ANB-AI ( l m g / m l ) in 100 mM Na2CO 3, pH 9. This and all following procedures were carried out in the dark. Unreacted ANB-AI was removed by eluting the reaction mixture from a Sephadex G-10 column with 0.1 mM Na2PO4, pH 7.4. Derivatized and radiolabeled toxin (A-I-Bgt)was characterized as described 11. The derivatized toxin was stored in the dark at - 2 0 ° C and used within 5 days.
Preparation of membranes from cell lines All cell lines (TE671, PC12, SH-SY5Y, and IMR-32 cells were obtained from Drs. G. Crawford, D. Schubert, J. Biedler, and the American Type Culture Collection, respectively) were grown for 5 - 7 days until confluent, under conditions described previously 12. The TE671 clone was originally thought to derive from a human medulIoblastoma 14, but may be a subclone of the RD human rhabdomyosarcoma cell line 26. The PC12 cell line is derived from a rat pheochromocytoma 6 of presumed neural crest origin. Also of presumed neural crest origin are the SH-SY5Y 21 and IMR-3227 human neuroblastoma cell lines. To prepare crude membrane pellets, cells were harvested in Ringers solution supplemented with 5 mM EDTA, 1 mM EGTA, 0.02% NAN3, 1 /xg/ml pepstatin A, 0.5 /zg/ml leupeptin, 2 mM iodoacetamide, 0.5 mM phenylmethylsulfonyl fluoride, 10 p,M aprotinin, 25/~g/ml soybean trypsin inhibitor and 1 mM benzamidine (solution A) and centrifuged at 40,000× g for 10 min to collect cells and broken processes. To prepare membranez, cells were lysed in hypotonic medium by incubating for 1 h at 4°C in 5 mM Tris pH 7.4 supplemented with the protease inhibitors found in solution A. Cells were then homogenized using a polytron homogenizer at setting 90 for 35 s, and cell debris and the nuclear fraction were removed by centrifugation at 18,000x g for 10 min at 4°C. Subsequent 10 min centrifugation of the supernatant at 40,000× g and 4°C was used to collect the crude plasma membrane fraction. The pellet was resuspended in Ringers solution supplemented with protease inhibitors as listed above and used immediately for photoaffinity labeling.
from Torpedo electroplax or TE671, SH-SY5Y or IMR-32 cells was specific and saturable (Fig. 1) and yielded K a values comparable to those for underivatized I-Bgt. The ratio of total to non-specific binding of A-I-Bgt to PC12 cell or rat brain membranes was very small making it difficult to detect specific binding. Analysis of the kinetics of toxin dissociation showed that a proportion, approximately 20%, of A-I-Bgt was irreversibly bound to Torpedo membranes following irradiation (data not shown). Affinity labeling profiles (representative profiles are shown in Fig. 2) for SDS-PAGE-resolved components from Torpedo preparations indicated that five polypeptides with M app of 49, 54, 58, 70, and 74 kDa were consistently and prominently reactive with A-I-Bgt, whereas an additional band of about 44 kDa was labeled with a varying degree of intensity from experiment to experiment. Assuming that one toxin molecule (8 kDa) was incorporated per labeled band, the corrected M app of polypeptides that were most reactive with A-I-Bgt are 41, 46, 50, 62, and 66 kDa. Labeling of each band was apparently specific, since incubation in the presence of excess unlabeled toxin strongly in-
Preparation of Torpedo and rat brain membranes Torpedo californica electroplax and crude rat brain membranes
>-
n-
were prepared as described 11 except that all buffers contained protease inhibitors (solution A). Some preparations of brain membranes solubilized in 1% Triton X-100 were subjected to affinity purification on agarose columns derivatized with the principal toxin from Naja naja siamensis venom essentially as described 16'~5.
Photoaffinity labeling All steps were carried out in the dark until photolysis of A-I-Bgt in the reaction mixture. The optimal ratio of A-I-Bgt to receptor to achieve the best ratio of specific to non-specific binding was determined prior to photoaffinity labeling by conducting saturation assays 11. Subsequently, membranes were incubated with the determined optimal concentration of A-I-Bgt for 2 h at 25°C. Unbound toxin was removed by three cycles of dilution in Ringers solution and centrifugation at 40,000 x g for 10 rain at 4°C. The toxin-membrane complex was photolyzed by exposure of membranes at 4°C to a sunlamp for 15 min, with shaking. Any remaining unbound toxin was removed by an additional centrifugation for 10 min at 40,000 x g. In parallel, other membrane aliquots were labeled in the presence of a 100-fold excess of native toxin to determine the specificity of the labeling reaction. Labeled membranes were solubilized in sample buffer and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis ( S D S - P A G E ) on a 7.5-15% gradient gel by the method of Laemmli 9. Autoradiographs were obtained by exposing the dried gel to Cronex 4 X-ray film for 2-7 days, and m app of labeled bands were calibrated with reference to standard proteins of known mass visualized on the stained gel. RESULTS
Typically, A-I-Bgt preparations had a specific activity of 30 cpm/fmol. Binding of A-I-Bgt to membranes
97kDa--,
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Fig. 2. S D S - P A G E analysis of membranes photoaffinity labeled with A-I-Bgt. Crude membrane preparations from the specified cell lines or tissue were incubated in the dark with A-I-Bgt for 2 h, and unbound toxin was removed via three cycles of centrifugation at 40,000× g at 5°C for 10 min. Chilled membranes were then irradiated for 15 min, followed by another 40,000× g centrifugation to remove remaining unbound toxin. Labeled membranes were then solubilized in sample buffer, separated on a 7.5-15% gradient gel, and visualized by autoradiography for 3-5 days. Non-specific binding was determined by incubating samples with a 100-fold excess of native, underivatized toxin. The photoaffinity labeling pattern is representative of the results obtained from a minimum of 3 separate experiments.
98 hibited formation of covalent toxin-polypeptide complexes (Fig. 2). Membranes prepared from the TE671 cell line consistently displayed incorporation of label into complexes with M ~pp of 47, 50, 55, and 63 kDa and variable degrees of labeling of a band at 42 kDa (Fig. 2). Virtually complete inhibition of labeling occurred for reactions run in the presence of excess native toxin (representative example shown in Fig. 2). Assuming that there is one covalently-attached toxin per polypeptide, corrected m app are 39, 42, 47, and 55 kDa for the prominently labeled bands. Labeling patterns for membranes prepared from IMR-32 or SH-SY5Y cell lines were very similar to those for TE671 cells, although excess native toxin was less effective in inhibiting formation of complexes between polypeptides and A-I-Bgt (Fig. 2). Corrected M ~°° for prominently toxin-reactive components of these neural crest-derived cell lines were about 40, 43, 48, and 57 kDa; there also was a variable degree of labeling of bands with corrected M ~pp of 33, 53 and 60 kDa. A similar labeling pattern was observed for PC12 cell preparations, but specific labeling was difficult to detect. Similar affinity labeling patterns showing four prominently and specifically labeled bands also were obtained for crude rat brain membrane preparations as well as for detergent-solubilized rat brain preparations that had been subjected to affinity purification on Naja naja toxin-agarose columns 25. DISCUSSION Prominent and apparently specific photoaffinity labeling with A-I-Bgt has been found to occur to five components from Torpedo electroplax and four components from TE671 cells, each of which express muscletype nAChR, and to four components from neuron-like cells of the IMR-32 and SH-SY5Y clonal lines, which express nBgtS. The major conclusions of this study are that at least four of the photoaffinity labeled components in each preparation most likely represent subunits of nAChR or nBgtS, and that the similarities in labeling patterns suggest that the subunit composition of mammalian nBgtS is like that for mammalian muscle-type nAChR (as found in TE671 cells). Three technical issues warrant comment. One is that whereas photoaffinity labeling of polypeptide components from SH-SY5Y and IMR-32 cells or from PC-12 or rat brain membrane preparations (data not shown) could be progressively inhibited by increasing the native toxin preincubation period or by increasing the concentration of competing native toxin in the reaction mixture, complete abolition of affinity labeling
was not achieved. This outcome contrasts with the full block by native toxin of affinity labeling of polypeptides from TE671 cell and Torpedo preparations, and occurred even in samples where there was a high ratio of specific to non-specific binding of A-I-Bgt to membrane polypeptides immediately prior to photolysisaided cross-linking. We have no fully satisfactory explanation for these observations. It is possible that a proportion of 'non-specific' A-I-Bgt binding (i.e., binding of A-I-Bgt in the presence of excess native toxin) actually represents binding to 'specific' sites, perhaps due to the irreversible nature of A-I-Bgt binding a n d / o r due to rebinding of A-I-Bgt during sunlamp illumination. This explanation is corroborated by the incidental observation that affinity labeling patterns are perfectly preserved as rat brain preparations are subjected to affinity purification on toxin columns just prior to affinity labeling 25, but that there is little-to-no improvement in the ratio of specific to non-specific labeling as evinced by autoradiographic analysis. A second technical issue is that, despite extensive precautions to limit proteolysis, variability in levels of labeling of lower M app species occurred for all preparations. We suggest that polypeptides with corrected M app of 36 kDa (Torpedo; a similar-sized polypeptide previously has been noted as a major degradation product of muscle-type nAChR subunits), 34 kDa (TE671 cells), and 33 kDa (other cells) represent degradation products of other, larger, toxin-reactive polypeptides. The observation that there was much more consistent labeling of other bands argues against artifacts due to massive proteolytic degradation. A third comment is that labeled polypeptides with M ~pp greater than 70 kDa were observed, however there was no consistent labeling of these bands. It is not possible to establish at this time whether these complexes represent toxin binding to a single or to more than one polypeptide, toxin labeling of higher molecular weight nAChR subunits or nonspecifically labeled proteins. Corrected M '~pp for four of the A-I-Bgt-reactive components from Torpedo membranes (41, 50, 62, and 66 kDa) are in close agreement with consensus values of M app for subunits of affinity-purified Torpedo nAChR (40, 50, 60, and 65 kDa 2'2°) and derived values of M app for photoaffinity-labeled polypeptides obtained in previous toxin cross-linking studies 1'7'17. This congruence of results coupled with the finding that a nicotinic ligand as small as d-tubocurarine can affinity label y and 6 as well as ~ subunits 19 supports the interpretation that these photoaffinity-labeled peptides are Torpedo nAChR subunits. Whereas it is well established that the 41 kDa a subunit of Torpedo nAChR harbors a binding site for acetylcholine and Bgt 2'1°'2°, it
99 seems that the interaction of Bgt with a subunits places toxin in close proximity to all of the other subunits. An alternative explanation is that some or all of the non-a subunits are labeled through intermolecular rather than intramolecular interactions, nAChR in Torpedo electroplax membranes are known to be densely packed, possibly bringing non-a subunits in close proximity to the a subunit of a neighboring nAChR. Bearing in mind the fact that interpretation of affinity labeling patterns is always complicated by the possibilities, for example, that there are differing degrees of post-translational modification (e.g., phosphorylation, glycosylation, myristylation) of candidate nAChR or nBgtS subunits and that alternative splicing of nAChR-encoding m R N A could give rise to translation products of differing size, it is interesting to note that prominent affinity labeling was obtained of a fifth polypeptide with a corrected m app of about 46 kDa from Torpedo membranes. This band was not reported to be evident in earlier studies 1'v'17, but could represent a major degradation product of a higher m app subunit, a 41 kDa subunit complexed with two toxin molecules, or a non-nAChR protein that is in close proximity to the toxin binding site and is labeled by derivatized toxin. It is reasonable to make a simple interpretation that photoaffinity-labeled bands identified in TE671 cell preparations correspond to subunits of mammalian muscle-type nAChR. The corrected size (39 kDa) of the smallest affinity-labeled band is very similar to that for the smallest photoaffinity-labeled band from Torpedo preparations (41 kDa) and for the TE671 cell nAChR a subunit (42 kDa) identified by Luther et al. ~3. However, corrected M app of the three largest out of the four predominate toxin-reactive polypeptides from TE671 cells (42, 47, and 55 kDa) are at least 10% lower than corrected m app for the three largest photoaffinity-labeled bands from Torpedo membranes (50, 62, and 66 kDa; this study) and for corresponding subunits of affinity-purified nAChR from Torpedo electroplax or from TE671 cells 13 (about 53, 55, and 62 kDa). These results may simply reflect laboratory-tolaboratory variation in apparent sizes of equivalent, SDS-PAGE-resolved species. However, it is also possible that affinity labeling could identify non-nAChR proteins that are in close proximity to nAChR (e.g., the photoaffinity-labeled 47 kDa component of TE671 cells may correspond to the similarly-sized, fifth photoaffinity-labeled band of Torpedo membranes) while failing to engage in intermolecular labeling of authentic components of loosely-packed nAChR that might be labeled intermolecularly in more densely-packed,
nAChR-rich membranes. Thus, whereas there is perfect correspondence between photoaffinity labeling and affinity purification studies in identification of 42 and 55 kDa polypeptides from TE671 cell nAChR ( a and y subunits), the current study might have failed to identify TE671 cell nAChR beta and 6 subunits identified by Luther et al. 13. Moreover, other discrepancies in these data might reflect selective proteolytic degradation or influences of any of a number of factors as discussed above. There was consistent incorporation of affinity label into components with corrected M app of about 40, 43, 48, and 57 kDa from membrane preparations of IMR32 and SH-SY5Y cells. The simplest interpretation of this data is that these polypeptides represent subunits of nBgtS. Previously, Whiting and Lindstrom 2s used Bgt affinity chromatography to isolate a macromolecufar complex from rat brain consisting of four subunits with M app of 45, 52, 57 and 65 kDa. Kemp et al. 8 used a similar approach to isolate three subunits, also from rat brain, with M app of 49, 53 and 55 kDa. Gotti et al. 5 used toxin columns to isolate nBgtS from the IMR-32 cell line and reported candidate subunit m app of 52, 60, and 67 kDa, sometimes along with a band of 48 kDa. The total number of presently-described photoaffinity-labeled peptides and some of the deduced sizes of those polypeptides most closely correlate with the results of Whiting and Lindstrom, but all of these results could be rationalized based on many of the arguments advanced above. Recent insight into the make-up of nBgtS has been obtained from a study demonstrating that peptides based on the sequence of the nAChR a5 gene are capable of specifically binding Bgt ~5. Since, PC12, SHSY5Y, and IMR-32 cells express mRNA that hybridizes under high stringency conditions with rat a5 cDNA probes TM (Lukas, Norman and Lucero; unpublished results), these results suggest that an a5 gene product could be a component of nBgtS identified in the current study. Antibodies raised against unique sequences of a 7 and a8 gene products recognize 57 and 60 kDa polypeptides, respectively, on Western blots of nBgtS purified on toxin columns 23. The 57 kDa polypeptide identified in the current study may correspond to a product of the a 7 gene. Alpha7 gene products appear to be capable of forming homooligomers that can function as Bgt-sensitive nicotinic agonist-gated ion channels 4, but the current results suggest that nBgtS have more than 1 subunit and are similar in subunit make-up to muscle-type nAChR. The current results also suggest that nBgtS derived from IMR-32 or SH-SY5Y and possibly the PC12 cell lines are very similar, and that these clonal cell lines
100 may prove useful in the further elucidation of relationships between nAChR subunit genes, proteins, and functional characteristics. Acknowledgments. We thank Mary Jane Cullen, Donna Kucharski and Linda Lucero for maintenance of cell lines, Merouane Bencherif for helpful comments, and David Schubert, Garrett Crawford and June Biedler for cell stocks. This work was supported by capitalization and faculty endowment funds from the Men's and Women's Boards of the Barrow Neurological Foundation and by grants from the National Institutes of Health (NS16821) and the Council for Tobacco Research, USA, Inc.
REFERENCES 1 Chatrenet, B., Tremeau, O., Bontems, F., Goeldner, M.P., Hirth, C.G. and Menez, A., Topography of toxin-acetylcholine receptor complexes by using photoactivatable toxin derivatives, Proc. Natl. Acad. Sci. USA, 87 (1990) 3378-3382. 2 Conti-Tronconi, B.M. and Raftery, M.A., The nicotinic cholinergic receptor: correlation of molecular stucture with functional properties, Annu. Rev. Biochem., 51 (1982) 491-530. 3 Conti-Tronconi, B.M., Dunn, S.M.J., Barnard, E.A., Dolly, J.O., Lai, F.A., Ray, N. and Raftery, M.A., Brain and muscle nicotinic acetylcholine receptors are different but homologous proteins, Proc. Natl. Acad. Sci. USA, 82 (1985) 5208-5212. 4 Couturier, S., Bertrand, D., Matter, J.-M., Hernandez, M.-C., Bertrand, S., Millar, N., Valera, S., Barkas, T. and Ballivet, M., A neuronal nicotinic acetylcholine receptor subunit (o~7) is developmentally regulated and forms a homo-oligomeric channel blocked by a-BTX, Neuron, 5 (1990) 847-856. 5 Gotti, C., Ogando, A.E. and Clementi, F., The a-bungarotoxin receptor purified from a human neuroblastoma cell line: biochemical and immunological characterization, Neuroscience, 32 (1989) 759-767. 6 Greene, L.A. and Tischler, A.S., Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma, Proc. NatL Acad. Sci. USA, 73 (1976) 2424-2428. 7 Hucho, F., Photoaffinity derivatives of a-bungarotoxin and a-Naja naja siamensis toxin, FEBS Lett., 103 (1979) 27-32. 8 Kemp, G., Bentley, L., McNamee, M.G. and Morley, B.J., Purification and characterization of the a-bungarotoxin binding protein from rat brain, Brain Res., 347 (1985) 274-283. 9 Laemmli, U.K., Cleavage of stuctural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. 10 Lukas, R.J. and Bencherif, M., Int. Rev. Neurobiol., in press. 11 Lukas, R.J., Detection of low-affinity a-bungarotoxin binding sites in the rat central nervous system, Biochemistry, 23 (1984) 1152-1160.
12 Lukas, R.J., Characterization of curaremimetic neurotoxin binding sites on membrane fractions derived from the human medulloblastoma clonal line, TE671, J~ Neurochem., 47 (1986) 17681773. 13 Luther, M.A., Schoepfer, R., Whiting, P., Casey, B., Blatt, Y., Montal, M.S., Montal, M. and Lindstrom, J., A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671, J. Neurosci., 9 (1989) 1082-1096. 14 McAllister, R.M., lsaacs, H., Rongey, R., Peer, M., Au, W., Soukup, S.W. and Gardner, M.B., Establishment of a human medulloblastoma cell line, Int. J. Cancer, 20 (1977) 206-212. 15 McLane, K.E., Wu, X. and Conti-Tronconi, B.M., Identification of a brain acetylcholine receptor a subunit able to bind abungarotoxin, .L Biol. Chem., 265 (1990)9816-9824. 16 Morley, B.J. and Kemp, G.E., Characterization of a putative nicotinic acetylcholine receptor in mammalian brain, Brain Res. Ret'., 3 (1981) 81-104. 17 Nathanson, N.M. and Hall, Z.W., In situ labeling of Torpedo and rat muscle acetylcholine receptor by a photoaffinity derivative of a-bungarotoxin, J. Biol. Chem., 255 (1980) 1698-1703. 18 Norman, S., Lucero, L. and Lukas, R.J., Soc. Neurosci. Abstr., 16 (1990) 681. 19 Pedersen, S.E. and Cohen, J.B., d-Tubocurarine binding sites are located at a-3, and a-6 subunit interfaces of the nicotinic acetylcholine receptor, Proc. Natl. Acad. Sci. USA, 87 (1990) 2785-2789. 20 Popot, J.L. and Changeux, J.P., Nicotinic receptor of acetylcholine: structure of an oligomeric integral membrane protein, Physiol. Rev., 64 (1984) 1162-1239. 21 Ross, R.A., Spengler, B.A. and Biedler, J.L., Coordinate morphological and biochemical interconversion of human neuroblastoma cells, JNCI, 71 (1983) 741-749. 22 Schmidt, J., Biochemistry of nicotinic acetylcholine receptors in the vertebrate brain, Int. Retd. Neurobiol., 30 (1988) 1-38. 23 Schoepfer, R., Conroy, W.G., Whiting, P., Gore, M. and Lindstrom, J., Brain a-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily, Neuron, 5 (1990) 35-48. 24 Seto, A., Arimatsu, Y. and Amano, T., Subunit stucture of a-bungarotoxin binding component in mouse brain, J. Neurochem., 37 (1981) 210-216. 25 Siegel, H.N. and Lukas. R.J., Soc. Neurosci. Abstr., 11 (1985) 1042. 26 Stratton, M.R., Darling, J., Pilkington, G.J., Lantos, P.L., Reeves, B.R. and Cooper, C.S., Characterization of the human cell line TE671, Carcinogenesis, 10 (1989) 899-905. 27 Tumilowicz, J.J., Nichols, W.W., Cholon, J.J. and Greene, A.E., Definition of a continuous human cell line derived from neuroblastoma, Cancer Res., 30 (1970) 2110-2118. 28 Whiting, P. and Lindstrom, J., Purification and characterization of a nicotinic acetylcholine receptor from rat brain, Proc. Natl. Acad. Sci. USA, 84 (1987) 595-599.
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BRESM 70548
Photoaffinity labeling of muscle-type nicotinic acetylcholine receptors and neuronal/nicotinic a-bungarotoxin binding sites with a derivative of a-bungarotoxin Anna
M.
Joy *, Hal N. Siegel
**
and Ronald J. Lukas
Division of Neurobiology, Barrow Neurological Institute, Phoenix, AZ 85013 (USA)
(Accepted 1 September 1992)
Key words: Acetylcholine receptor; ot-Bungarotoxin; TE671 cell line; IMR-32 cell line; SH-SY5Y cell line; PC12 cell line
Neuronal/nicotinic a-bungarotoxin binding sites (nBgtS) found in the nervous system are not well characterized. In this study, photolabile toxin derivatives have been used in affinity labeling protocols to investigate the subunit composition of nBgtS expressed by different neuron-like cell lines. Data obtained was compared to the known subunit composition of toxin-binding muscle-type nicotinic acetylcholine receptors (nAChR). Muscle-type nAChR-rich membranes prepared from Torpedo electroplax contain components with corrected apparent molecular sizes of 41, 46, 50, 62 and 66 kDa that are reactive with toxin. The photoaffinity labeling patterns for preparations derived from cells of the TE671 clone, which express muscle-type nAChR, are very similar to that of cells of the IMR-32 or SH-SY5Y clonal lines, which express nBgtS. There is consistent labeling of four polypeptides with corrected apparent molecular weights of 40, 43, 47 and 56 kDa. These results suggest that both mammalian muscle-type nAChR and mammalian nBgtS are similarly composed of at least four kinds of subunits.
INTRODUCTION T h e s n a k e n e u r o t o x i n , a - b u n g a r o t o x i n (Bgt), is a high affinity, specific, a n d f u n c t i o n a l l y - p o t e n t a n t a g o nist u s e d in t h e c h a r a c t e r i z a t i o n o f m u s c l e a n d m u s c l e - t y p e n i c o t i n i c a e e t y l c h o l i n e r e c e p t o r s 2'1°'2° ( n A C h R ) . Bgt also b i n d s to sites in n e u r o n a l tissues, t e r m e d n e u r o n a l / n i c o t i n i c Bgt b i n d i n g sites (nBgtS), with high affinity. H o w e v e r , t h e r e a r e m a n y r e p o r t s ( a n d n o t a b l e e x c e p t i o n s ) t h a t Bgt is f u n c t i o n a l l y i m p o t e n t as a nicotinic a n t a g o n i s t on n o r m a l o r n e o p l a s t i c cells d e r i v e d f r o m t h e a u t o n o m i c o r c e n t r a l n e r v o u s systems ~°. Thus, nBgtS a p p e a r to b e distinct from ' n e u r o n a l ' n A C h R t h a t clearly function as l i g a n d - g a t e d ion c h a n n e l s a n d can be i d e n t i f i e d by a l t e r n a t i v e m e a n s ~°'22. U s i n g o l i g o n u c l e o t i d e p r o b e s c o r r e s p o n d i n g to t h e N - t e r m i n a l a m i n o acid s e q u e n c e o f a nBgtS s u b u n i t i s o l a t e d from chick b r a i n by C o n t i - T r o n c o n i et al. 3, two subunits o f chick n B g t S (now t e r m e d a 7 a n d a 8 ) have
r e c e n t l y b e e n c l o n e d by S c h o e p f e r et al. 23. I n d e p e n dently, C o u t u r i e r et al. 4 used low s t r i n g e n c y h y b r i d i z a tion a n d a p r o b e d e r i v e d f r o m the chick n A C h R a 3 g e n e to clone o n e o f t h e s a m e g e n e s ( a 7 ) . D e d u c e d f e a t u r e s o f the p r o d u c t s o f t h e s e g e n e s i n d i c a t e t h a t nBgtS could, in fact, be m e m b e r s o f the e x t e n d e d gene s u p e r f a m i l y o f l i g a n d - g a t e d ion channels, which inc l u d e s r e c e p t o r s for acetylcholine, glycine a n d g a m m a a m i n o b u t y r i c acid. This i n f e r e n c e has b e e n substantia t e d by the d e m o n s t r a t i o n that a 7 prote~n e x p r e s s e d in Xenopus oocytes can function as a nicotinic l i g a n d - g a t e d ion c h a n n e l sensitive to f u n c t i o n a l b l o c k a d e by Bgt 4. W h e r e a s t h e subunit c o m p o s i t i o n s o f n A C h R f o u n d in v e r t e b r a t e muscle o r o f m u s c l e - t y p e n A C h R from electric tissue o f electric fish have b e e n k n o w n for m o r e t h a n a d e c a d e 2'2°, t h e r e is less c e r t a i n t y a b o u t the c o m p o s i t i o n o f nBgtS. Toxin b i n d i n g m a c r o m o l e c u l e s i s o l a t e d from several tissue sources by various investig a t o r s have b e e n r e p o r t e d to c o m p r i s e b e t w e e n o n e
Correspondence: A.M. Joy, Neuro-Oncology Research, Division of Neurology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013, USA. * Present address: Neuro-oncology Research, Division of Neurology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013, USA. ** Present address: Iatric Corp., 2330 South Industrial Park Drive, Tempe, AZ 85282, USA.
96
nBgtS derived from cells of different clonal lines. Preliminary reports of parts of this work have appeared 25.
and four subunits with apparent molecular sizes ( M app) ranging from 45 to 72 kDa 3'5'8'24'28. This lack of consensus could reflect, among other things, susceptibility to proteolysis of nBgtS, just as there is for nAChR, or use of tissues containing heterogeneous populations of nBgtS. As a complement to affinity purification techniques, and in order to minimize the likelihood that expression of multiple nBgtS subtypes could complicate data interpretation, photolabile, radiolabeled Bgt derivatives were used in affinity labeling protocols to characterize
1000000
,
,
,
MATERIALS AND METHODS
Preparation and characterization of derivatized toxin Preparation and characterization of 1~I-labeled Bgt (I-Bgt) was as described n, except that the final ion exchange chromatography to separate native from mono- and di-iodinated toxin was not performed. The bifunctional photoaffinity reagent, N-5-azido-2-nitrobenzoylaminoacetimidate-HC1 (ANB-AI), was a generous gift of Dr. William Allison of the University of California at San Diego. Photoaffinity derivatized toxin was prepared as described 17. Briefly,
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50
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Fig. 1. Saturation curves for A-IoBgt binding to membranes. Crude membrane fractions were prepared from cell lines as described in the text. Membranes from Torpedo elecfroplax (A), TE671 cells (B), IMR-32 cells (C), or SH-SYSY cells (D) were incubated with the specified concentration of A-I-Bgt for 2 h in the dark. Unbound toxin was removed via two cycles of centrifug~ition at 40,000x g for 10 rain in the dark, and bound toxin was quantitated by counting the pellet on a gamma counter. Non-specific binding was determined in the presence of a 100-fold excess of native, underivatized toxin. Data displayed are representative of a minimum of 3 separate experiments.
97 radioiodinated toxin was incubated overnight at 25°C with excess ANB-AI ( l m g / m l ) in 100 mM Na2CO 3, pH 9. This and all following procedures were carried out in the dark. Unreacted ANB-AI was removed by eluting the reaction mixture from a Sephadex G-10 column with 0.1 mM Na2PO4, pH 7.4. Derivatized and radiolabeled toxin (A-I-Bgt)was characterized as described 11. The derivatized toxin was stored in the dark at - 2 0 ° C and used within 5 days.
Preparation of membranes from cell lines All cell lines (TE671, PC12, SH-SY5Y, and IMR-32 cells were obtained from Drs. G. Crawford, D. Schubert, J. Biedler, and the American Type Culture Collection, respectively) were grown for 5 - 7 days until confluent, under conditions described previously 12. The TE671 clone was originally thought to derive from a human medulIoblastoma 14, but may be a subclone of the RD human rhabdomyosarcoma cell line 26. The PC12 cell line is derived from a rat pheochromocytoma 6 of presumed neural crest origin. Also of presumed neural crest origin are the SH-SY5Y 21 and IMR-3227 human neuroblastoma cell lines. To prepare crude membrane pellets, cells were harvested in Ringers solution supplemented with 5 mM EDTA, 1 mM EGTA, 0.02% NAN3, 1 /xg/ml pepstatin A, 0.5 /zg/ml leupeptin, 2 mM iodoacetamide, 0.5 mM phenylmethylsulfonyl fluoride, 10 p,M aprotinin, 25/~g/ml soybean trypsin inhibitor and 1 mM benzamidine (solution A) and centrifuged at 40,000× g for 10 min to collect cells and broken processes. To prepare membranez, cells were lysed in hypotonic medium by incubating for 1 h at 4°C in 5 mM Tris pH 7.4 supplemented with the protease inhibitors found in solution A. Cells were then homogenized using a polytron homogenizer at setting 90 for 35 s, and cell debris and the nuclear fraction were removed by centrifugation at 18,000x g for 10 min at 4°C. Subsequent 10 min centrifugation of the supernatant at 40,000× g and 4°C was used to collect the crude plasma membrane fraction. The pellet was resuspended in Ringers solution supplemented with protease inhibitors as listed above and used immediately for photoaffinity labeling.
from Torpedo electroplax or TE671, SH-SY5Y or IMR-32 cells was specific and saturable (Fig. 1) and yielded K a values comparable to those for underivatized I-Bgt. The ratio of total to non-specific binding of A-I-Bgt to PC12 cell or rat brain membranes was very small making it difficult to detect specific binding. Analysis of the kinetics of toxin dissociation showed that a proportion, approximately 20%, of A-I-Bgt was irreversibly bound to Torpedo membranes following irradiation (data not shown). Affinity labeling profiles (representative profiles are shown in Fig. 2) for SDS-PAGE-resolved components from Torpedo preparations indicated that five polypeptides with M app of 49, 54, 58, 70, and 74 kDa were consistently and prominently reactive with A-I-Bgt, whereas an additional band of about 44 kDa was labeled with a varying degree of intensity from experiment to experiment. Assuming that one toxin molecule (8 kDa) was incorporated per labeled band, the corrected M app of polypeptides that were most reactive with A-I-Bgt are 41, 46, 50, 62, and 66 kDa. Labeling of each band was apparently specific, since incubation in the presence of excess unlabeled toxin strongly in-
Preparation of Torpedo and rat brain membranes Torpedo californica electroplax and crude rat brain membranes
>-
n-
were prepared as described 11 except that all buffers contained protease inhibitors (solution A). Some preparations of brain membranes solubilized in 1% Triton X-100 were subjected to affinity purification on agarose columns derivatized with the principal toxin from Naja naja siamensis venom essentially as described 16'~5.
Photoaffinity labeling All steps were carried out in the dark until photolysis of A-I-Bgt in the reaction mixture. The optimal ratio of A-I-Bgt to receptor to achieve the best ratio of specific to non-specific binding was determined prior to photoaffinity labeling by conducting saturation assays 11. Subsequently, membranes were incubated with the determined optimal concentration of A-I-Bgt for 2 h at 25°C. Unbound toxin was removed by three cycles of dilution in Ringers solution and centrifugation at 40,000 x g for 10 rain at 4°C. The toxin-membrane complex was photolyzed by exposure of membranes at 4°C to a sunlamp for 15 min, with shaking. Any remaining unbound toxin was removed by an additional centrifugation for 10 min at 40,000 x g. In parallel, other membrane aliquots were labeled in the presence of a 100-fold excess of native toxin to determine the specificity of the labeling reaction. Labeled membranes were solubilized in sample buffer and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis ( S D S - P A G E ) on a 7.5-15% gradient gel by the method of Laemmli 9. Autoradiographs were obtained by exposing the dried gel to Cronex 4 X-ray film for 2-7 days, and m app of labeled bands were calibrated with reference to standard proteins of known mass visualized on the stained gel. RESULTS
Typically, A-I-Bgt preparations had a specific activity of 30 cpm/fmol. Binding of A-I-Bgt to membranes
97kDa--,
66kDa--,
43kDa--,
EXCESSBGT-
+
-
+
-
+
-
+
Fig. 2. S D S - P A G E analysis of membranes photoaffinity labeled with A-I-Bgt. Crude membrane preparations from the specified cell lines or tissue were incubated in the dark with A-I-Bgt for 2 h, and unbound toxin was removed via three cycles of centrifugation at 40,000× g at 5°C for 10 min. Chilled membranes were then irradiated for 15 min, followed by another 40,000× g centrifugation to remove remaining unbound toxin. Labeled membranes were then solubilized in sample buffer, separated on a 7.5-15% gradient gel, and visualized by autoradiography for 3-5 days. Non-specific binding was determined by incubating samples with a 100-fold excess of native, underivatized toxin. The photoaffinity labeling pattern is representative of the results obtained from a minimum of 3 separate experiments.
98 hibited formation of covalent toxin-polypeptide complexes (Fig. 2). Membranes prepared from the TE671 cell line consistently displayed incorporation of label into complexes with M ~pp of 47, 50, 55, and 63 kDa and variable degrees of labeling of a band at 42 kDa (Fig. 2). Virtually complete inhibition of labeling occurred for reactions run in the presence of excess native toxin (representative example shown in Fig. 2). Assuming that there is one covalently-attached toxin per polypeptide, corrected m app are 39, 42, 47, and 55 kDa for the prominently labeled bands. Labeling patterns for membranes prepared from IMR-32 or SH-SY5Y cell lines were very similar to those for TE671 cells, although excess native toxin was less effective in inhibiting formation of complexes between polypeptides and A-I-Bgt (Fig. 2). Corrected M ~°° for prominently toxin-reactive components of these neural crest-derived cell lines were about 40, 43, 48, and 57 kDa; there also was a variable degree of labeling of bands with corrected M ~pp of 33, 53 and 60 kDa. A similar labeling pattern was observed for PC12 cell preparations, but specific labeling was difficult to detect. Similar affinity labeling patterns showing four prominently and specifically labeled bands also were obtained for crude rat brain membrane preparations as well as for detergent-solubilized rat brain preparations that had been subjected to affinity purification on Naja naja toxin-agarose columns 25. DISCUSSION Prominent and apparently specific photoaffinity labeling with A-I-Bgt has been found to occur to five components from Torpedo electroplax and four components from TE671 cells, each of which express muscletype nAChR, and to four components from neuron-like cells of the IMR-32 and SH-SY5Y clonal lines, which express nBgtS. The major conclusions of this study are that at least four of the photoaffinity labeled components in each preparation most likely represent subunits of nAChR or nBgtS, and that the similarities in labeling patterns suggest that the subunit composition of mammalian nBgtS is like that for mammalian muscle-type nAChR (as found in TE671 cells). Three technical issues warrant comment. One is that whereas photoaffinity labeling of polypeptide components from SH-SY5Y and IMR-32 cells or from PC-12 or rat brain membrane preparations (data not shown) could be progressively inhibited by increasing the native toxin preincubation period or by increasing the concentration of competing native toxin in the reaction mixture, complete abolition of affinity labeling
was not achieved. This outcome contrasts with the full block by native toxin of affinity labeling of polypeptides from TE671 cell and Torpedo preparations, and occurred even in samples where there was a high ratio of specific to non-specific binding of A-I-Bgt to membrane polypeptides immediately prior to photolysisaided cross-linking. We have no fully satisfactory explanation for these observations. It is possible that a proportion of 'non-specific' A-I-Bgt binding (i.e., binding of A-I-Bgt in the presence of excess native toxin) actually represents binding to 'specific' sites, perhaps due to the irreversible nature of A-I-Bgt binding a n d / o r due to rebinding of A-I-Bgt during sunlamp illumination. This explanation is corroborated by the incidental observation that affinity labeling patterns are perfectly preserved as rat brain preparations are subjected to affinity purification on toxin columns just prior to affinity labeling 25, but that there is little-to-no improvement in the ratio of specific to non-specific labeling as evinced by autoradiographic analysis. A second technical issue is that, despite extensive precautions to limit proteolysis, variability in levels of labeling of lower M app species occurred for all preparations. We suggest that polypeptides with corrected M app of 36 kDa (Torpedo; a similar-sized polypeptide previously has been noted as a major degradation product of muscle-type nAChR subunits), 34 kDa (TE671 cells), and 33 kDa (other cells) represent degradation products of other, larger, toxin-reactive polypeptides. The observation that there was much more consistent labeling of other bands argues against artifacts due to massive proteolytic degradation. A third comment is that labeled polypeptides with M ~pp greater than 70 kDa were observed, however there was no consistent labeling of these bands. It is not possible to establish at this time whether these complexes represent toxin binding to a single or to more than one polypeptide, toxin labeling of higher molecular weight nAChR subunits or nonspecifically labeled proteins. Corrected M '~pp for four of the A-I-Bgt-reactive components from Torpedo membranes (41, 50, 62, and 66 kDa) are in close agreement with consensus values of M app for subunits of affinity-purified Torpedo nAChR (40, 50, 60, and 65 kDa 2'2°) and derived values of M app for photoaffinity-labeled polypeptides obtained in previous toxin cross-linking studies 1'7'17. This congruence of results coupled with the finding that a nicotinic ligand as small as d-tubocurarine can affinity label y and 6 as well as ~ subunits 19 supports the interpretation that these photoaffinity-labeled peptides are Torpedo nAChR subunits. Whereas it is well established that the 41 kDa a subunit of Torpedo nAChR harbors a binding site for acetylcholine and Bgt 2'1°'2°, it
99 seems that the interaction of Bgt with a subunits places toxin in close proximity to all of the other subunits. An alternative explanation is that some or all of the non-a subunits are labeled through intermolecular rather than intramolecular interactions, nAChR in Torpedo electroplax membranes are known to be densely packed, possibly bringing non-a subunits in close proximity to the a subunit of a neighboring nAChR. Bearing in mind the fact that interpretation of affinity labeling patterns is always complicated by the possibilities, for example, that there are differing degrees of post-translational modification (e.g., phosphorylation, glycosylation, myristylation) of candidate nAChR or nBgtS subunits and that alternative splicing of nAChR-encoding m R N A could give rise to translation products of differing size, it is interesting to note that prominent affinity labeling was obtained of a fifth polypeptide with a corrected m app of about 46 kDa from Torpedo membranes. This band was not reported to be evident in earlier studies 1'v'17, but could represent a major degradation product of a higher m app subunit, a 41 kDa subunit complexed with two toxin molecules, or a non-nAChR protein that is in close proximity to the toxin binding site and is labeled by derivatized toxin. It is reasonable to make a simple interpretation that photoaffinity-labeled bands identified in TE671 cell preparations correspond to subunits of mammalian muscle-type nAChR. The corrected size (39 kDa) of the smallest affinity-labeled band is very similar to that for the smallest photoaffinity-labeled band from Torpedo preparations (41 kDa) and for the TE671 cell nAChR a subunit (42 kDa) identified by Luther et al. ~3. However, corrected M app of the three largest out of the four predominate toxin-reactive polypeptides from TE671 cells (42, 47, and 55 kDa) are at least 10% lower than corrected m app for the three largest photoaffinity-labeled bands from Torpedo membranes (50, 62, and 66 kDa; this study) and for corresponding subunits of affinity-purified nAChR from Torpedo electroplax or from TE671 cells 13 (about 53, 55, and 62 kDa). These results may simply reflect laboratory-tolaboratory variation in apparent sizes of equivalent, SDS-PAGE-resolved species. However, it is also possible that affinity labeling could identify non-nAChR proteins that are in close proximity to nAChR (e.g., the photoaffinity-labeled 47 kDa component of TE671 cells may correspond to the similarly-sized, fifth photoaffinity-labeled band of Torpedo membranes) while failing to engage in intermolecular labeling of authentic components of loosely-packed nAChR that might be labeled intermolecularly in more densely-packed,
nAChR-rich membranes. Thus, whereas there is perfect correspondence between photoaffinity labeling and affinity purification studies in identification of 42 and 55 kDa polypeptides from TE671 cell nAChR ( a and y subunits), the current study might have failed to identify TE671 cell nAChR beta and 6 subunits identified by Luther et al. 13. Moreover, other discrepancies in these data might reflect selective proteolytic degradation or influences of any of a number of factors as discussed above. There was consistent incorporation of affinity label into components with corrected M app of about 40, 43, 48, and 57 kDa from membrane preparations of IMR32 and SH-SY5Y cells. The simplest interpretation of this data is that these polypeptides represent subunits of nBgtS. Previously, Whiting and Lindstrom 2s used Bgt affinity chromatography to isolate a macromolecufar complex from rat brain consisting of four subunits with M app of 45, 52, 57 and 65 kDa. Kemp et al. 8 used a similar approach to isolate three subunits, also from rat brain, with M app of 49, 53 and 55 kDa. Gotti et al. 5 used toxin columns to isolate nBgtS from the IMR-32 cell line and reported candidate subunit m app of 52, 60, and 67 kDa, sometimes along with a band of 48 kDa. The total number of presently-described photoaffinity-labeled peptides and some of the deduced sizes of those polypeptides most closely correlate with the results of Whiting and Lindstrom, but all of these results could be rationalized based on many of the arguments advanced above. Recent insight into the make-up of nBgtS has been obtained from a study demonstrating that peptides based on the sequence of the nAChR a5 gene are capable of specifically binding Bgt ~5. Since, PC12, SHSY5Y, and IMR-32 cells express mRNA that hybridizes under high stringency conditions with rat a5 cDNA probes TM (Lukas, Norman and Lucero; unpublished results), these results suggest that an a5 gene product could be a component of nBgtS identified in the current study. Antibodies raised against unique sequences of a 7 and a8 gene products recognize 57 and 60 kDa polypeptides, respectively, on Western blots of nBgtS purified on toxin columns 23. The 57 kDa polypeptide identified in the current study may correspond to a product of the a 7 gene. Alpha7 gene products appear to be capable of forming homooligomers that can function as Bgt-sensitive nicotinic agonist-gated ion channels 4, but the current results suggest that nBgtS have more than 1 subunit and are similar in subunit make-up to muscle-type nAChR. The current results also suggest that nBgtS derived from IMR-32 or SH-SY5Y and possibly the PC12 cell lines are very similar, and that these clonal cell lines
100 may prove useful in the further elucidation of relationships between nAChR subunit genes, proteins, and functional characteristics. Acknowledgments. We thank Mary Jane Cullen, Donna Kucharski and Linda Lucero for maintenance of cell lines, Merouane Bencherif for helpful comments, and David Schubert, Garrett Crawford and June Biedler for cell stocks. This work was supported by capitalization and faculty endowment funds from the Men's and Women's Boards of the Barrow Neurological Foundation and by grants from the National Institutes of Health (NS16821) and the Council for Tobacco Research, USA, Inc.
REFERENCES 1 Chatrenet, B., Tremeau, O., Bontems, F., Goeldner, M.P., Hirth, C.G. and Menez, A., Topography of toxin-acetylcholine receptor complexes by using photoactivatable toxin derivatives, Proc. Natl. Acad. Sci. USA, 87 (1990) 3378-3382. 2 Conti-Tronconi, B.M. and Raftery, M.A., The nicotinic cholinergic receptor: correlation of molecular stucture with functional properties, Annu. Rev. Biochem., 51 (1982) 491-530. 3 Conti-Tronconi, B.M., Dunn, S.M.J., Barnard, E.A., Dolly, J.O., Lai, F.A., Ray, N. and Raftery, M.A., Brain and muscle nicotinic acetylcholine receptors are different but homologous proteins, Proc. Natl. Acad. Sci. USA, 82 (1985) 5208-5212. 4 Couturier, S., Bertrand, D., Matter, J.-M., Hernandez, M.-C., Bertrand, S., Millar, N., Valera, S., Barkas, T. and Ballivet, M., A neuronal nicotinic acetylcholine receptor subunit (o~7) is developmentally regulated and forms a homo-oligomeric channel blocked by a-BTX, Neuron, 5 (1990) 847-856. 5 Gotti, C., Ogando, A.E. and Clementi, F., The a-bungarotoxin receptor purified from a human neuroblastoma cell line: biochemical and immunological characterization, Neuroscience, 32 (1989) 759-767. 6 Greene, L.A. and Tischler, A.S., Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma, Proc. NatL Acad. Sci. USA, 73 (1976) 2424-2428. 7 Hucho, F., Photoaffinity derivatives of a-bungarotoxin and a-Naja naja siamensis toxin, FEBS Lett., 103 (1979) 27-32. 8 Kemp, G., Bentley, L., McNamee, M.G. and Morley, B.J., Purification and characterization of the a-bungarotoxin binding protein from rat brain, Brain Res., 347 (1985) 274-283. 9 Laemmli, U.K., Cleavage of stuctural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. 10 Lukas, R.J. and Bencherif, M., Int. Rev. Neurobiol., in press. 11 Lukas, R.J., Detection of low-affinity a-bungarotoxin binding sites in the rat central nervous system, Biochemistry, 23 (1984) 1152-1160.
12 Lukas, R.J., Characterization of curaremimetic neurotoxin binding sites on membrane fractions derived from the human medulloblastoma clonal line, TE671, J~ Neurochem., 47 (1986) 17681773. 13 Luther, M.A., Schoepfer, R., Whiting, P., Casey, B., Blatt, Y., Montal, M.S., Montal, M. and Lindstrom, J., A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671, J. Neurosci., 9 (1989) 1082-1096. 14 McAllister, R.M., lsaacs, H., Rongey, R., Peer, M., Au, W., Soukup, S.W. and Gardner, M.B., Establishment of a human medulloblastoma cell line, Int. J. Cancer, 20 (1977) 206-212. 15 McLane, K.E., Wu, X. and Conti-Tronconi, B.M., Identification of a brain acetylcholine receptor a subunit able to bind abungarotoxin, .L Biol. Chem., 265 (1990)9816-9824. 16 Morley, B.J. and Kemp, G.E., Characterization of a putative nicotinic acetylcholine receptor in mammalian brain, Brain Res. Ret'., 3 (1981) 81-104. 17 Nathanson, N.M. and Hall, Z.W., In situ labeling of Torpedo and rat muscle acetylcholine receptor by a photoaffinity derivative of a-bungarotoxin, J. Biol. Chem., 255 (1980) 1698-1703. 18 Norman, S., Lucero, L. and Lukas, R.J., Soc. Neurosci. Abstr., 16 (1990) 681. 19 Pedersen, S.E. and Cohen, J.B., d-Tubocurarine binding sites are located at a-3, and a-6 subunit interfaces of the nicotinic acetylcholine receptor, Proc. Natl. Acad. Sci. USA, 87 (1990) 2785-2789. 20 Popot, J.L. and Changeux, J.P., Nicotinic receptor of acetylcholine: structure of an oligomeric integral membrane protein, Physiol. Rev., 64 (1984) 1162-1239. 21 Ross, R.A., Spengler, B.A. and Biedler, J.L., Coordinate morphological and biochemical interconversion of human neuroblastoma cells, JNCI, 71 (1983) 741-749. 22 Schmidt, J., Biochemistry of nicotinic acetylcholine receptors in the vertebrate brain, Int. Retd. Neurobiol., 30 (1988) 1-38. 23 Schoepfer, R., Conroy, W.G., Whiting, P., Gore, M. and Lindstrom, J., Brain a-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily, Neuron, 5 (1990) 35-48. 24 Seto, A., Arimatsu, Y. and Amano, T., Subunit stucture of a-bungarotoxin binding component in mouse brain, J. Neurochem., 37 (1981) 210-216. 25 Siegel, H.N. and Lukas. R.J., Soc. Neurosci. Abstr., 11 (1985) 1042. 26 Stratton, M.R., Darling, J., Pilkington, G.J., Lantos, P.L., Reeves, B.R. and Cooper, C.S., Characterization of the human cell line TE671, Carcinogenesis, 10 (1989) 899-905. 27 Tumilowicz, J.J., Nichols, W.W., Cholon, J.J. and Greene, A.E., Definition of a continuous human cell line derived from neuroblastoma, Cancer Res., 30 (1970) 2110-2118. 28 Whiting, P. and Lindstrom, J., Purification and characterization of a nicotinic acetylcholine receptor from rat brain, Proc. Natl. Acad. Sci. USA, 84 (1987) 595-599.