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

Intracellular Ca(2+)-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 PRO-1 (plasticity-related gene-1) as a novel CaM target. A CaM-overlay and an immunoprecipitation assay revealed that PRO-1 is capable of binding the Ca(2+)/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 Ca(2+)-dependent manner, with a relatively high affinity (a K(d) value for Ca(2+)/CaM of 8 nM). Various PRO-1 mutants indicated that the Ca(2+)/CaM-binding region of PRG-1 is located between residues Ser(554) and Gin(588), and that Trp(559) and Ile(578) potentially anchor PRO-1 to CaM. This is supported by pronounced changes in the fluorescence emission spectrum of Trp(559) in the PRO-1 peptide (residues 554-588) upon binding to Ca(2+)/CaM, showing the stoichiometrical binding of the PRO-1 peptide with Ca(2+)/CaM. Immunoblot analyses revealed that the PRO-1 protein is abundant in brain, but is weakly expressed in the testes. Immunohistochemical analysis revealed that PRO-1 is highly expressed in forebrain structures and in the cerebellar cortex. Furthermore, PRO-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 PRO-1 may be involved in postsynaptic functions regulated by intracellular Ca(2+)-signalling.