Identification and characterization of PRG-1 as a neuronal calmodulin-binding protein.
Biochem J
; 431(1): 81-91, 2010 Oct 01.
Article
em En
| MEDLINE
| ID: mdl-20653564
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteoglicanas
/
Proteínas de Ligação a Calmodulina
/
Calmodulina
/
Monoéster Fosfórico Hidrolases
/
Neurônios
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Biochem J
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Japão