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S.-P. Huang, B. M. Brown, C. M. Craft; Visual Arrestin 1 is Essential for Photoreceptor Synaptic Regulation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4134.
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© ARVO (1962-2015); The Authors (2016-present)
Arrestin1 (Arr1) and N-ethylmaleimide sensitive factor (NSF) interact in vitro and in vivo. We evaluated potential molecular and cellular mechanisms in maintaining normal synaptic function in mouse photoreceptors lacking Arr1.
Protein-protein interaction of Arr1 and NSF was verified by immunoblots, immunohistochemical (IHC) methods, and NSF-Arr1 domains were defined using GST pulldown assays. Quantitative RT-PCR measured transcriptional levels of NSF and other synaptic-enriched encoded genes, in vitro binding assays determined the role of ATP in Arr1-NSF interaction, colorimetric phosphate quantitation measured Arr1’s modulation of NSF ATPase activity, co-precipitation assay determined the disassembly activity of NSF, and in vivo FM1-43 staining examined the exocytosis rate in the photoreceptor synapse.
GST pulldown assay of Arr1 with NSF truncated proteins showed that Arr1 binds to NSF N-terminal (N) and first ATPase (D1) domain junction. Arr1 increases NSF ATPase activity and NSF disassembly activities in a dose-dependent manner. The interactions between Arr1 and NSF are greater in the retina isolated in dark conditions and IHC analysis reveals more intense dual-localization staining in the photoreceptor synapse of dark-adapted (DA) wild type (WT) retinas compared to light-adapted (LA) WT retinas. NSF mRNA expression level is significantly increased in DA compared with LA retinas, while NSF transcription level is decreased in the Arr1-/- retinas compared with WT retinas in dark conditions. Synaptic uptake of FM1-43 revealed a decrease of synaptic activity in Arr1-/- retina.
Our results document that NSF acts as a functional partner for Arr1 in the photoreceptor synapse and their interactions are greater in dark conditions. Arr1 enhances the NSF ATPase activity and increases the NSF disassembly activities, which are crucial for normal NSF functions in sustaining a higher rate of exocytosis and the compensatory endocytosis to retrieve and recycle vesicle membrane and proteins in the photoreceptor synapses. Deletion of Arr1 also leads to a reduced level of mRNA expression of NSF, vGLUT1, EAAT5 and VAMP2 and a potential to markedly depress the exocytosis rate in the photoreceptor synapse. This study demonstrates a critical and essential functional role of Arr1 in the photoreceptor synapse and provides novel insights into the potential molecular mechanisms of inherited retinal diseases, such as Oguchi disease & ARR1-associated retinitis pigmentosa.
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