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

The Biochemical Journal
Hiroshi TokumitsuHiroyuki Sakagami

Abstract

Intracellular Ca2+-dependent cellular responses are often mediated by the ubiquitous protein CaM (calmodulin), which, upon binding Ca2+, 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 abunda...Continue Reading

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Citations

Feb 18, 2012·The Journal of Biological Chemistry·Hiroaki YaguchiShigetsugu Hatakeyama
Feb 8, 2013·Biochimica Et Biophysica Acta·Ulf Strauss, Anja U Bräuer
Mar 26, 2013·Liver International : Official Journal of the International Association for the Study of the Liver·Joji TaniRyoji Kobayashi
Sep 17, 2013·Journal of Neuroscience Research·Tomio HashimotoMitsuhiko Yamada
Dec 17, 2015·EMBO Molecular Medicine·Bernardo Stutz, Tamas L Horvath
Dec 31, 2020·Molecular Biology of the Cell·Shalini MenonStephanie L Gupton
Aug 3, 2017·Biochemical and Biophysical Research Communications·Kyohei SakaneHiroshi Tokumitsu
May 22, 2021·Journal of Clinical Laboratory Analysis·Huanzheng LiShaohua Tang

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