Abstract
Purpose: :
To investigate the molecular and cellular mechanisms of the interaction between Arr1 and NSF in maintaining the normal synaptic function in the mouse photoreceptors.
Methods: :
The interaction of Arr1 and NSF was first identified using co-immunoprecipitation (IP) assays, followed by mass spectrometry (MS) analysis, and verified by immunohistochemical methods. The transcription levels of NSF were analyzed by quantitative RT-PCR. The region in NSF that interacts with Arr1 was defined by constructed NSF-truncated domain segments (amino acids residues 1-744, 1-205, 206-744, 1-477 and 1-206^478-744) and analyzed using gel overlay assay.
Results: :
Co-IP assays of mouse retinal homogenates using Arr1-specific antibody (D9F2) identified NSF by MS analysis and supports their potential interaction. Moreover, protein-protein interactions between Arr1 and NSF were greater in the retina isolated in dark conditions. Immunohistochemical analysis reveals NSF and Arr1 are co-localized in the photoreceptor synapse of the dark-adapted retinas with more intense staining compared to the light-adapted retina. NSF mRNA expression level is significantly increased in dark-adapted compared with light-adapted retinas, while NSF transcription level is decreased in the Arr1-/- retinas compared with control retinas in dark conditions. Gel overlay assay of recombinant Arr1 protein with serial NSF truncated proteins showed that Arr1 binds to the junction of N-terminal (N) and first ATPase (D1) domain of NSF.
Keywords: photoreceptors • synapse • retina