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Yan Cao, Ekaterina Posokhova, Kirill A. Martemyanov; Identification of Nyctalopin Binding Partners. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4127.
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Synaptic G protein signal transduction plays crucial roles in light response in vertebrate retina. ON-bipolar cells utilize metabotropic glutamate receptor 6 (mGluR6) and depolarize in response to light by the inactivation of G protein, Gαo which ultimately opening transient receptor potential cation channel, subfamily M, member 1 (TRPM1). The dysfunction in the signaling pathway in ON-bipolar cells is a primary cause of congenital stationary night blindness (CSNB). To date, three genes have been identified as the mutated gene in CSNB patients. Besides the GRM6 gene encoding postsynaptic mGluR6 and the TRPM1 gene encoding the channel TRPM1, the third gene, NYX encodes the membrane anchored protein nyctalopin. Although mutations in NYX gene were identified in the majority of X-linked recessive families with complete CSNB, the role of nyctalopin in signal transduction is still unknown. Nyctalopin has a leucine rich repeat motif which is predicted to facilitate different protein-protein interactions by providing a protein interaction interface; therefore, we conducted a search for the interaction partners of nyctalopin.
Interactions between nyctalopin and its binding partners were investigated by immunoprecipitation both in intact retinas and in transfected cells. Experiments in vivo utilized specific anti-nyctalopin antibody whereas a tandem affinity purification (TAP) strategy utilizing genetically engineered tags was used to pull-down nyctalopin from transfected HEK293FT cells. Co-precipitating proteins were identified by LC-MS/MS mass-spectrometry, followed by calculating peptide probabilities using PeptideProphet software.
Using unbiased proteomic strategy in intact retinas we identified TRPM1 as protein co-precipitating with nyctalopin confirming previously reported interaction in yeast cells by candidate approach. We further found that in co-transfected cells nyctalopin was able to form complexes with mGluR6. Additionally, we identified many ER chaperone proteins as nyctalopin binding partners.
Nyctalopin interacts with both mGluR6 and TRPM1, key components of the ON-bipolar signaling pathway. Along with the observations that ER chaperone proteins are recruited to nyctalopin these findings suggest that in ON-bipolar cells nyctalopin is critically involved in the biogenesis of the signal transduction complexes involving both mGluR6 and TRPM1 proteins.
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