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A. Lopez-Colome, A. Parrales; Molecular Analysis of a Consensus Sequence for Casein Kinase 2 (CK2) in the NR1 Subunit of Retina NMDA Receptor. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3167. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Glutamate is the major excitatory neurotransmitter in the vertebrate retina and exerts its effects through the activation of distinct receptor subtypes located on postsynaptic neurons. The calcium-permeable NMDAR subtype plays a central role in retinal transmission, and has been implicated in differentiation, plasticity and excitotoxicity in this tissue. We have identified a novel alternatively spliced cassette (C3) in the C-terminal region of the channel-forming NR1 subunit of the NMDAR in chick retina, coding for a consensus sequence for CK2 phosphorylation (EETSEH). Importantly, CK2 has been implicated in cell cycle and circadian rhythms, in which the retina plays a key role. The purpose of this study was to develop a model system for the in vitro analysis of the NR1subunit C3 cassette phosphorylation by CK2 in the retina.
We generated fusion proteins of NR1 C-terminal containing or lacking the C3 cassette, with Xpress epitope. These constructs were overexpressed in HEK cells, and CK2 phosphorylation was analyzed for both proteins. Finally, co-immunoprecipitation of CK2 with the fusion proteins was probed.
Using this model, we demonstrated that CK2 and C3 cassette of NR1 subunit do not interact, in spite of the presence of the consensus sequence.
We conclude that such failure to interact is due to the presence of a basic residue (His) within the C3 cassette, and of a stop codon following CK2 consensus sequence. These results suggest that proteins bearing a consensus sequence for CK2 phosphorylation require the presence of acidic residues at the C-terminal of the target sequence in order to achieve functional activation.
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