LISE S. ELIOT*, YADIN DUDAIt, ERIC R. KANDEL*, AND THOMAS W. ABRAMS*f. *Howard Hughes Medical Institute, Center for Neurobiology and Behavior, ...
Proc. Nati. Acad. Sci. USA Vol. 86, pp. 9564-9568, December 1989 Neurobiology
Ca2+/calmodulin sensitivity may be common to all forms of neural adenylate cyclase (classical conditioning/cyclic AMP/associative lering)
LISE S. ELIOT*, YADIN DUDAIt, ERIC R. KANDEL*, AND THOMAS W.
ABRAMS*f
*Howard Hughes Medical Institute, Center for Neurobiology and Behavior, Columbia University, College of Physicians and Surgeons, 722 West 168th Street, New York, NY 10032; tDepartment of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel; and tDepartment of Biology and The Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, PA 19104-6018
Contributed by Eric R. Kandel, July 28, 1989
unbound fractions show comparable activation by Ca2l in the presence of CaM. Moreover, despite this separation into bound and unbound CaM-Sepharose fractions, Ca2+ stimulation of the bound fraction was never enriched over the level of stimulation in the solubilized preparation; such enrichment would be expected if the original population contained a mixture of Ca2+-sensitive and -insensitive cyclases. It is possible to isolate a largely Ca2+-insensitive population of cyclase in the unbound fraction from a CaM-Sepharose column, but only when this column is preceded by other chromatographic methods. Our results suggest that this Ca2+insensitive population results from the degradation or loss of Ca2+ sensitivity during prior chromatography, rather than from the more efficient separation of Ca2+-sensitive and Ca2+-insensitive forms on CaM-Sepharose. Together, these results indicate that the vast majority of cyclase in the CNS can be activated by Ca2+/CaM and furthermore suggest that much or all of the Ca2+-insensitive cyclase that has been described in the literature may be a product of preparative degradation.
ABSTRACT The Ca2_/calmodu~in (CaM)-activated adenylate cyclase has been implicated as playing an important associative role in classical conditioning in both Aplysia and Drosophila. Studies of the cyclase in mammalian cerebral cortex have suggested that Ca2+/CaM sensitivity is confined to a subpopulation of total cyclase activity. We investigated the properties of cyclase from Aplysia, rat, and bovine central nervous system membranes by using CaM-Sepharose chromatography. Although only a minority of total cyclase activity bound to the CaM column, both bound and unbound fractions of cyclase from all three species showed comparable stimulation by Ca21 in the presence of CaM. When solubilized bovine membranes were first depleted of most of their endogenous CaM by prior chromatography, binding to the CaM column was substantially increased and Ca2+ stimulation of the unbound fraction was somewhat reduced. However, this reduction in Ca21 sensitivity resulted from the loss of Ca2+ sensitivity during prior chromatography, rather than from the more efficient separation of Ca2+-sensitive and -insensitive forms. This finding, together with the fact that we never observed any enrichment for Ca2+ sensitivity in the bound fraction over the level in the starting preparation, suggests that the vast majority of the cyclase present in solubilized central nervous system membranes is Ca2+/CaM-sensitive.
METHODS Tissue Preparation. For each preparation of Aplysia neural cyclase, abdominal and ring ganglia were removed from 20 Aplysia californica (100-200 g) and homogenized on ice in 10 ml of buffer A [20 mM Hepes, pH 7.6/5 mM MgCl2/5 mM EGTA/1 mM dithiothreitol (DTT) containing phenylmethanesulfonyl fluoride (50 ,ug/ml) and protease inhibitors (benzamidine, 1 ,uM; leupeptin, 10 jig/ml; aprotinin, 10 jig/ml)]. The homogenate was filtered through gauze and then centrifuged at 30,000 x g for 30 min. All centrifugations, as well as all chromatography, were done at 4°C. The pellet was washed by resuspension in 10 ml of buffer A and recentrifugation. This membrane pellet ("='10 mg of protein) was solubilized on ice for 30 min in 6 ml of buffer B (25 mM Hepes, pH 7.6/1% Lubrol PX/1 mM EDTA/1 mM DTT containing phenylmethanesulfonyl fluoride and protease inhibitors). Unsolubilized material was pelleted by centrifugation at 100,000 x g for 1 hr. The supernatant (.0.5 mg of protein per ml) was made 5 mM in MgC12 and then frozen in liquid N2. For each preparation of rat brain cyclase, the cerebral cortex of a single Sprague-Dawley rat was homogenized in 120 ml of buffer A and centrifuged as above. The first 30,000 x g pellet was resuspended and an aliquot (5 mg of protein) was washed three times in buffer A (15 ml per wash). Solubilization was as above. For the preparation of bovine cerebral cyclase, the cortices offour cow brains were homogenized in a blender at 4°C with 2.5 liters of buffer A and centrifuged as above. One-tenth of this pellet was further washed three times in buffer A (400 ml
Studies of the neural mechanisms underlying classical conditioning in both Aplysia and Drosophila have led to the proposal that the Ca +/calmodulin (CaM)-sensitive adenylate cyclase may serve as one site of associative interaction between inputs from the conditioned and unconditioned stimuli (1-6). Abrams et al. (7) recently presented evidence that both Ca2", the proposed molecular mediator of the conditioned stimulus, and modulatory transmitter, the mediator of the unconditioned stimulus, can activate the same population of CaM-binding adenylate cyclase molecules. In the present study, we ask the question: how much of the total cyclase population in the central nervous system (CNS) is capable of being activated by Ca2l? Earlier work had indicated that Ca2l sensitivity is confined to a fraction of the total adenylate cyclase activity in brain (8, 9). The strongest evidence for heterogeneity of cyclase came from the study of Westcott et al. (9), who found that only about 20% of the total adenylate cyclase activity in solubilized bovine brain membranes bound to a CaM-Sepharose column and that only this small bound fraction was capable of being activated by Ca2 . We have similarly observed that only a small fraction of the total cyclase activity in Aplysia CNS, as well as in rat and bovine CNS, binds to a CaM-Sepharose column in the presence of Ca2+. But in contrast to the findings of Westcott et al. (9), we find for all three species that both the bound and the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: CaM, calmodulin; CNS, central nervous system; DTT, dithiothreitol; GTP[yS], guanosine 5'-[y-thio]triphosphate.
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Neurobiology: Eliot et al. per wash) and solubilized immediately in 400 ml of buffer B. The solubilization mixture was stirred for 30 min at 40C and then centrifuged as above. The supernatant (2.2 mg of protein per ml) was frozen in liquid N2. For experiments requiring extensive EGTA washing, a small piece (0.3-0.4 g) of the first membrane pellet, which had been frozen in liquid N2, was thawed in buffer A and washed three to six times in buffer A (30 ml per wash). The final pellet (-5 mg of protein) was solubilized in 5 ml of buffer B and centrifuged at 100,000 x g for 1 hr. CaM-AffiMnity Chromatography. Solubilized neural tissue or concentrated column eluate (2-6 ml) was brought to 5 mM MgCl2 and 2 mM CaCl2 and then continuously recirculated for 3 hr over 1 ml of CaM-Sepharose (Pharmacia) that had been prewashed in buffer C (50 mM Hepes, pH 7.6/0.1% Lubrol PX/1 mM EDTA/5 mM MgCl2/1 mM DTT containing protease inhibitors, 0.2 mg of human serum albumin per ml, and 2 mM CaCl2). The resin was washed at 0.25 ml/min with 10-15 ml of buffer C, and then the bound proteins were eluted with buffer C containing 5 mM EGTA and lacking CaCl2. Before assay of Ca2' stimulation, the flow-through and eluate fractions were each brought to 5 mM Ca2' and 5 mM EGTA; Ca2' and EGTA were then reduced to 20o of total cyclase activity in the CNS, because any larger population would cause the Ca2+ stimulation of fraction I to be detectably lower than that of fraction II when EGTA washing is the only pretreatment. Our conclusions differ from those of Rosenberg and Storm (19). Those authors prepared an antiserum against a preparation of guanosine 5'-[f3-y-imido]triphosphate-preactivated bovine brain adenylate cyclase that had been purified through a number of steps, including CaM-Sepharose. This antiserum effectively precipitated adenylate cyclase activity only from the bound, and not from the flow-through, fraction of a CaM-Sepharose column. It also precipitated only 60% of the total cyclase activity in bovine brain membranes. The authors concluded that bovine brain contains two distinct isozymes of adenylate cyclase, with the Ca2+-sensitive form presumably representing 60% of the total activity. However, Rosenberg and Storm did not demonstrate that the precipitated cyclase activity showed Ca2+ stimulation while the activity in the supernatant did not. Thus, it is not clear that this antiserum
Proc. Natl. Acad. Sci. USA 86 (1989)
truly segregates Ca2l-sensitive and -insensitive forms of cyclase. Given the lability of the Ca2`/CaM sensitivity of cyclase, the convincing identification of a Ca2+-insensitive form probably requires the cloning, expression, and characterization of all adenylate cyclase species in the CNS. Recently, one species of adenylate cyclase that binds to CaM has been cloned from bovine brain (20). The enzyme expressed from this clone was stimulated by Ca2+/CaM. A second, highly homologous cDNA was subsequently isolated, but it is not yet known whether the cyclase encoded by this clone is Ca2+-sensitive (R. Reed, personal communication). Dual Regulation of Adenylate Cyclase and Associative Learning. Abrams et al. (7) recently demonstrated that the CaM-binding adenylate cyclase can be dually activated by Ca2+ and the stimulatory GTP-binding protein, Gs. The finding that the vast majority of cyclase in Aplysia and mammalian CNS is Ca2+-sensitive similarly suggests that the same catalytic units that are stimulated by receptor via Gs will also be stimulated by Ca2+/CaM. Indeed, these results indicate that Ca2+ regulation may be a general feature of adenylate cyclase activation in the CNS. Thus, the enzyme could function generally in nervous systems to temporally associate transmitter input with neuronal activity or any of a variety of other stimuli that increase intracellular Ca2 . We thank K. Karl for technical assistance. We thank Y. Yovell, R. Hawkins, D. Sweatt, M. Klein, and H. Bayley for comments on an earlier draft of the manuscript. 1. Abrams, T. W. & Kandel, E. R. (1988) Trends Neurosci. 11, 128-135. 2. Abrams, T. W. (1985) Cell. Mol. Neurobiol. 5, 23-145. 3. Kandel, E. R., Abrams, T., Bernier, L., Carew, T. J., Hawkins, R. D. & Schwartz, J. H. (1983) Cold Spring Harbor Symp. Quant. Biol. 48, 821-830. 4. Ocorr, K. A., Walters, E. T. & Byrne, J. H. (1985) Proc. Natl. Acad. Sci. USA 82, 2548-2552. 5. Dudai, Y. & Zvi, S. (1984) Neurosci. Lett. 47, 119-124. 6. Livingstone, M. S., Sziber, P. P. & Quinn, W. G. (1984) Cell 37, 205-215. 7. Abrams, T. W., Karl, K. & Kandel, E. R. (1989) J. Neurosci., in press. 8. Brostrom, C. O., Brostrom, M. A., Wolff, D. J. (1977) J. Biol. Chem. 252, 5677-5685. 9. Westcott, K. R., LaPorte, D. C. & Storm, D. R. (1979) Proc. Natl. Acad. Sci. USA 76, 204-208. 10. Salomon, Y. (1979) Adv. Cycleic Nucleotide Res. 10, 35-55. 11. Yovell, Y., Dudai, Y. & Abrams, T. W. (1986) Soc. Neurosci. Abstr. 12, 400. 12. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 13. Wolff, D. J., Poirier, P. G., Brostrom, C. 0. & Brostrom, M. A. (1977) J. Biol. Chem. 252, 4108-4117. 14. Gilman, A. G. (1984) Cell 36, 577-579. 15. Klee, C. B. & Newton, D. L. (1985) in Control and Manipulation and Calcium Movement, ed. J. R. Parratt (Raven, New York), pp. 131-146. 16. Malnoe, A. & Cox, J. A. (1985) J. Neurochem. 45, 1163-1171. 17. Toscano, W. A., Westcott, K. R., LaPorte, D. C. & Storm, D. R. (1979) Proc. Natl. Acad. Sci. USA 76, 5582-5586. 18. MacNeil, S., Lakey, T. & Tomlinson, S. (1985) Cell Calcium 6, 213-226. 19. Rosenberg, G. B. & Storm, D. R. (1987) J. Biol. Chem. 262, 7623-7628. 20. Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R. & Gilman, A. G. (1989) Science 244, 1558-1564.
