Identification and characterization of PRG-1 as a neuronal calmodulin-binding protein

Hiroshi Tokumitsu, Naoya Hatano, Mitsumasa Tsuchiya, Saki Yurimoto, Tomohito Fujimoto, Naoki Ohara, Ryoji Kobayashi, Hiroyuki Sakagami

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Intracellular Ca2+-dependent cellular responses are often mediated by the ubiquitous protein CaM (calmodulin), which, upon binding Ca 2+, can interact with and alter the function of numerous proteins. In the present study, using a newly developed functional proteomic screen of rat brain extracts, we identified PRG-1 (plasticity-related gene-1) as a novel CaM target. A CaM-overlay and an immunoprecipitation assay revealed that PRG-1 is capable of binding the Ca2+/CaM complex in vitro and in transfected cells. Surface plasmon resonance and zero-length cross-linking showed that the C-terminal putative cytoplasmic domain (residues 466-766) of PRG-1 binds equimolar amounts of CaM in a Ca2+-dependent manner, with a relatively high affinity (a Kd value for Ca2+/CaM of 8 nM). Various PRG-1 mutants indicated that the Ca2+/CaM-binding region of PRG-1 is located between residues Ser554 and Gln588, and that Trp559 and Ile578 potentially anchor PRG-1 to CaM. This is supported by pronounced changes in the fluorescence emission spectrum of Trp559 in the PRG-1 peptide (residues 554-588) upon binding to Ca2+/CaM, showing the stoichiometrical binding of the PRG-1 peptide with Ca2+/CaM. Immunoblot analyses revealed that the PRG-1 protein is abundant in brain, but is weakly expressed in the testes. Immunohistochemical analysis revealed that PRG-1 is highly expressed in forebrain structures and in the cerebellar cortex. Furthermore, PRG-1 localizes at the postsynaptic compartment of excitatory synapses and dendritic shafts of hippocampal neurons, but is not present in presynaptic nerve terminals. The combined observations suggest that PRG-1 may be involved in postsynaptic functions regulated by intracellular Ca2+-signalling.

Original languageEnglish
Pages (from-to)81-91
Number of pages11
JournalBiochemical Journal
Volume431
Issue number1
DOIs
Publication statusPublished - Oct 1 2010
Externally publishedYes

Keywords

  • Calmodulin (CaM)
  • Neuron
  • Plasticity-related gene-1 (PRG-1)
  • Postsynapse
  • Proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Tokumitsu, H., Hatano, N., Tsuchiya, M., Yurimoto, S., Fujimoto, T., Ohara, N., Kobayashi, R., & Sakagami, H. (2010). Identification and characterization of PRG-1 as a neuronal calmodulin-binding protein. Biochemical Journal, 431(1), 81-91. https://doi.org/10.1042/BJ20100637