Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: receptors • excitatory neurotransmitters • protein structure/function