Identification and characterization of PRG-1 as a neuronal calmodulin-binding protein.
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.
Tokumitsu
Hiroshi
H
Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan. tokumit@med.kagawa-u.ac.jp
Hatano
Naoya
N
Tsuchiya
Mitsumasa
M
Yurimoto
Saki
S
Fujimoto
Tomohito
T
Ohara
Naoki
N
Kobayashi
Ryoji
R
Sakagami
Hiroyuki
H
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
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D011509:Proteoglycans; C476330:Plppr4 protein, rat; D010744:Phosphoric Monoester Hydrolases