title: Identification and characterization of PRG-1 as a neuronal calmodulin-binding protein.
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 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.
authors.affiliation: Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan. tokumit@med.kagawa-u.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tokumitsu
authors.forename: Naoya
authors.initials: N
authors.lastname: Hatano
authors.forename: Mitsumasa
authors.initials: M
authors.lastname: Tsuchiya
authors.forename: Saki
authors.initials: S
authors.lastname: Yurimoto
authors.forename: Tomohito
authors.initials: T
authors.lastname: Fujimoto
authors.forename: Naoki
authors.initials: N
authors.lastname: Ohara
authors.forename: Ryoji
authors.initials: R
authors.lastname: Kobayashi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sakagami
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011509:Proteoglycans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D011509:Proteoglycans; C476330:Plppr4 protein, rat; D010744:Phosphoric Monoester Hydrolases