Abstract
RC3/Neurogranin is a postnatal-onset, forebrain-specific, thyroid hormone-regulated, protein kinase C (PKC) substrate that binds calmodulin (CaM) and accumulates in dendritic spines. We bacterially expressed and purified RC3 and, for comparison, GAP-43/neuromodulin to near homogeneity using relatively simple procedures. We then raised antisera against recombinant RC3 that does not crossreact with GAP-43 and is suitable for immunohistochemical analysis of brain slices. We also constructed over 30 RC3 sequence variants by PCR-mediated, site-directed mutagenesis, and purified four of these to near homogeneity. The elution profiles displayed by RC3 and sequence variants during purification on CaM-Sepharose columns suggest that two different affinity forms of the RC3-CaM complex coexist when Ca2+ is absent and that GAP-43-CaM interactions are far more sensitive to salt than those that occur between recombinant RC3 and CaM. Variant proteins in which serine 36 was changed failed to serve as a substrate for PKC, implicating this as the target residue.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alexander K. A., Watkins B. T., Doyle G. S., Walsh K. A., and Storm D. R. (1988) Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein.J. Biol. Chem. 263, 7544–7549.
Andreasen T. J., Leutje C. W., Heideman W., and Storm D. R. (1983) Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes.Biochemistry 22, 4615–4618.
Apel E. D., Byford M. F., Au D., Walsh K. A., and Storm D. R. (1990) Identification of the protein kinase C phosphorylation site in neuromodulin.Biochemistry 29, 2330–2335.
Baudier J., Bronner C., Kligman D., and Cole R. D. (1989) Protein kinase C substrates from bovine brain. Purification and characterization of neuromodulin, a neuron-specific calmodulin-binding protein.J. Biol. Chem. 264, 1824–1828.
Baudier J., Deloulme J. C., Van Dorsselaer A., Black D., and Matthes H. W. (1991) Purification and characterization of a brain-specific protein kinase C substrate, neurogranin.J. Biol. Chem. 266, 229–237.
Benowitz L. I. and Perrone-Bizzozero N. I. (1991a) The expression of GAP-43 in relation to neuronal growth and plasticity: when, where, how, and why?Prog. Brain Res. 89, 69–87.
Benowitz L. I. and Perrone-Bizzozero N. I. (1991b) The relationship of GAP-43 to the development and plasticity of synaptic connections.Ann. NY Acad. Sci. 627, 58–74.
Chapman E. R., Au D., Alexander K. A., Nicolson T. A., and Storm D. R. (1991) Characterization of the calmodulin binding domain of neuromodulin.J. Biol. Chem. 266, 207–213.
Chen S.-J., Klann E., and Sweatt J. D. (1993) Proceedings of the 23rd Annual Meeting of the Society for Neuroscience, Washington, DC, Abstract #703.5.
Cimler B. M., Giebelhaus D. H., Wakim B. T., Storm D. R., and Moon R. T. (1987) Characterization of murine cDNAs encoding P-57, a neural-specific calmodulin-binding protein.J. Biol. Chem. 262, 12,158–12,163.
Cimler B. M., Andreasen T. J., Andreasen K. I., and Storm D. R. (1985) P-57 is a neural specific calmodulin-binding protein.J. Biol. Chem. 260, 10,784–10,788.
Coggins P. J. and Zwiers H. (1991) B-50 (GAP-43): biochemistry and functional neurochemistry of a neuron-specific phosphoprotein.J. Neurochem. 53, 1895–1901.
De Graan P. N., Oestreicher A. B., Schotman P., and Schrama L. H. (1991) Protein kinase C substrate B-50 (GAP-43) and neurotransmitter release.Prog. Brain Res. 89, 187–207.
Dekker L. V., De Graan P. N., and Gispen W. H. (1991) Transmitter release: target of regulation by protein kinase C?Prog. Brain Res. 89, 209–233.
Deloulme J. C., Sensenbrenner M., and Baudier J. (1991) A rapid purification method for neurogranin, a brain specific calmodulin-binding protein kinase C substrate.FEBS Lett. 282, 183–188.
Gerendasy D. D., Herron S. R., Watson J. B., and Sutcliffe J. G. (1994) Mutational and biophysical studies suggest RC3/neurogranin regulates calmodulin availability.J. Biol. Chem. 269, 22,420–22,426.
Gispen W. H., Nielander H. B., De Graan P. N., Oestreicher A. B., Schrama L. H., and Schotman P. (1991) Role of the growth-associated protein B-50/GAP-43 in neuronal plasticity.Mol. Neurobiol. 5, 61–85.
Iniguez M. A., Rodriguez-Pena A., Ibarrola N., Morreale de Escobar G., and Bernal J. (1992) Adult rat brain is sensitive to thyroid hormone. Regulation of RC3/neurogranin mRNA.J. Clin. Invest. 90, 554–558.
Karns L. R., Ng S.-C., Freeman J. A., and Fishman M. C. (1987) Cloning of complementary DNA for GAP-43 a neuronal growth-related protein.Science 236, 597–600.
Liu Y. C. and Storm D. R. (1990) Regulation of free calmodulin levels by neuromodulin: neuron growth and regeneration.Trends Pharmacol Sci. 11, 107–111.
McLeod M., Stein M., and Beach D. (1987) The product of the mei3+ gene, expressed under control of the mating-type locus induces meiosis and sporulation in fission yeast.EMBO J. 6, 729–736.
Munoz A., Rodriguez-Pena A., Perez-Castillo A., Ferreiro B., Sutcliffe J. G., and Bernal J. (1991) Effects of neonatal hypothyroidism on rat brain gene expression.Mol. Endocrinol. 5, 273–280.
Represa A., Deloulme J. C., Sensenbrenner M., Ben-Ari Y., and Baudier J. (1990) Neurogranin: immunocytochemical localization of a brain-specific protein kinase C substrate.J. Neurosci. 10, 3782–3792.
Robinson P. J. (1991) The role of protein kinase C and its neuronal substrates dephosphin B-50, and MARCKS in neurotransmitter release.Mol. Neurobiol. 5, 87–130.
Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.Science 239, 487–491.
Sanger F., Nicklen S., and Coulsen A. R. (1977) DNA sequencing with chain-terminating inhibitors.Proc. Natl. Acad. Sci. USA 74, 5463–5467.
Strittmatter S. M. and Fishman M. C. (1991) The neuronal growth cone as a specialized transduction system.Bioessays 13, 127–134.
Strittmatter S. M., Vartanian T., and Fishman M. C. (1992) GAP-43 as a plasticity protein in neuronal form and repair.J. Neurobiol. 23, 507–520.
Studier W. F., Rosenberg A. H., Dunn J. J., and Dubendorff J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes.Methods Enzymol. 185, 60–88.
Towbin H., Staehelin T., and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. USA 76, 4350–4354.
Watson J. B., Battenberg E. F., Wong K. K., Bloom F. E., and Sutcliffe J. G. (1990) Subtractive cDNA cloning of RC3, a rodent cortex-enriched mRNA encoding a novel 78 residue protein.J. Neurosci. Res. 26, 397–408.
Watson J. B., Sutcliffe J. G., and Fisher R. S. (1992) Localization of the protein kinase C phosphorylation/calmodulin-binding substrate RC3 in dendritic spines of neostriatal neuronsProc. Natl. Acad. Sci. USA 89, 8581–8585.
Wojtkowiak Z., Briggs R. C., and Hnilica L. S. (1983) A sensitive method for staining proteins transferred to nitrocellulose sheets.Anal. Biochem. 129, 486.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Gerendasy, D.D., Herron, S.R., Wong, K.K. et al. Rapid purification, site-directed mutagenesis, and initial characterization of recombinant RC3/neurogranin. J Mol Neurosci 5, 133–148 (1994). https://doi.org/10.1007/BF02736729
Issue Date:
DOI: https://doi.org/10.1007/BF02736729