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Dibyendu Chakraborty, Youwen Zhang, Shanta Sarfare, Steven J. Pittler, Muna I. Naash; Knockout or overexpression of Glutamic Acid Rich Protein 2 (GARP2) adversely affects rod structure and function. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1625.
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© ARVO (1962-2015); The Authors (2016-present)
The cyclic nucleotide gated (CNG) channel plays an important role in visual transduction in rod and cone photoreceptors. The rod CNG channel consists of three alpha subunits (CNGA1) and one extended beta subunit (CNGB1) which contains a unique proline- and glutamic acid-rich N terminal extension domain (GARP). It had been shown that both free GARP and CNGB1 can bind to the photoreceptor-specific tetraspanin retinal degeneration slow (RDS) protein. RDS is present in the rim region of rod and cone outer segments (OSs) with its homologous binding partner ROM-1, and is required for photoreceptor OS morphogenesis and stabilization. In the present study, we focus on the importance of RDS-CNGB1 and RDS-GARP complexes in OS morphogenesis and stability.
GARP2-Tg transgenic and Cngb1-/- mice were crossed onto wild-type (WT), Rds+/- and Rds-/- genetic backgrounds. Protein expression was verified by Western blot. Morphological and functional assessments of transgenic and knockout retinas were performed by light and electron microscopy and electroretinography (ERG), respectively. Biochemical properties of RDS complexes were evaluated by non-reducing velocity sedimentation.
Significantly diminished scotopic a-wave amplitudes were observed in Cngb1-/- on both the WT and Rds+/- backgrounds in comparison to WT and Rds+/- controls. A-wave maximum amplitudes were also reduced in GARP2-Tg mice (on the WT and Rds+/-) compared to non-transgenic controls although the reduction was not as severe as that seen in the Cngb1-/-. Ultrastructural analysis showed that Cngb1-/- and GARP2-Tg OSs are shorter than those in the WT. Immunofluorescence and velocity sedimentation analyses revealed that neither RDS/ROM-1 localization nor RDS/ROM-1 oligomerization were affected by the absence of Cngb1 or overexpression of GARP2 protein.
Our results suggest that lack of Cngb1 and overexpression of GARP2 both act additively with RDS haploinsufficiency to decrease rod function. However, this additive effect does not appear to be due to any destabilization or inhibition of RDS higher-order complex formation in the absence of Cngb1 or in the presence of excess GARP2. The precise role of RDS-GARP interactions is still under investigation.
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