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L. L. Molday, J. M. Holopainen, C. L. Cheng, G. Johal, J. Coleman, F. M. Dyka, T. Hii, J. Ahn, R. S. Molday; Interaction of X-linked Retinitis Pigmentosa Protein 2 (rp2) With N-ethylmaleimide Sensitive Factor (nsf). Invest. Ophthalmol. Vis. Sci. 2009;50(13):5450.
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RP2, the protein encoded by the gene responsible for a form of X-linked Retinitis Pigmentosa (XLRP), has been previously shown to bind Arl3 and serve as a GTPase activating protein (GAP). The goal of this study was to determine the distribution of RP2 in retina and identify proteins that directly bind to RP2 as an essential step in defining the role of RP2 in photoreceptor cell function and XLRP.
Monoclonal antibody, RP2-6H2, generated against the N-terminal region of RP2, was used to localize RP2 in rodent and human retina by immunofluorescence microscopy. Co-immunoprecipitation, mass spectrometry, and western blotting were used to identify proteins that interact with RP2. GST fusion proteins and SDS gel electrophoresis were used to identify protein binding domains and determine the effect of RP2 disease-causing mutations on protein-protein interactions.
Intense immunofluorescence staining of RP2 was observed in the inner segment and synaptic layers of photoreceptors with punctate labeling near the junction of the inner and outer segments. RPE and ganglion cells and the inner plexiform layer were also intensely stained. When a detergent-solubilized retina extract was immunoprecipitated with the RP2-6H2 antibody, NSF co-immunoprecipitated with RP2 as analyzed by mass spectrometry and western blotting. Co-localization of NSF with RP2 was observed in double labeling studies. GST-fusion proteins against various NSF domains localized the RP2 binding site to the N-terminus of NSF. The E138G and ΔI137 disease-linked RP2 mutations eliminated the binding of RP2 to the N-terminal domain of NSF, whereas the R118H, L188P, and R282W mutations had no effect.
Our studies indicate that RP2 interacts strongly and co-localizes with NSF in photoreceptors. This suggests that RP2 may play an important role in vesicle trafficking and/or membrane fusion in photoreceptor cells in addition to functioning as a GAP protein for Arl3.
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