Journal of Molecular Neuroscience

, Volume 5, Issue 3, pp 133–148

Rapid purification, site-directed mutagenesis, and initial characterization of recombinant RC3/neurogranin

  • Dan D. Gerendasy
  • Steven R. Herron
  • Kenneth K. Wong
  • Joseph B. Watson
  • J. Gregor Sutcliffe
Article

DOI: 10.1007/BF02736729

Cite this article as:
Gerendasy, D.D., Herron, S.R., Wong, K.K. et al. J Mol Neurosci (1994) 5: 133. doi:10.1007/BF02736729

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.

Index Entries

RC3 neurogranin GAP-43 neuromodulin calmodulin protein kinase C 

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Copyright information

© Humana Press Inc 1995

Authors and Affiliations

  • Dan D. Gerendasy
    • 1
  • Steven R. Herron
    • 1
  • Kenneth K. Wong
    • 1
  • Joseph B. Watson
    • 2
  • J. Gregor Sutcliffe
    • 1
  1. 1.Department of Molecular BiologyThe Scripps Research InstituteLa Jolla
  2. 2.Department of Psychiatry and Biobehavioral SciencesMental Retardation Center and Brain Research Institute, UCLA School of MedicineLos Angeles

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