April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Insights Into the Molecular Basis of Leber Congenital Amaurosis (LCA12) Associated With Mutations in RD3
Author Affiliations & Notes
  • S. Azadi
    Dept of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
  • L. L. Molday
    Dept of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
  • R. S. Molday
    Dept of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
  • Footnotes
    Commercial Relationships  S. Azadi, None; L.L. Molday, None; R.S. Molday, None.
  • Footnotes
    Support  NIH Grant EY 02422; CIHR Grant (MT 5822); MVRF Grant
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 6451. doi:
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    • Get Citation

      S. Azadi, L. L. Molday, R. S. Molday; Insights Into the Molecular Basis of Leber Congenital Amaurosis (LCA12) Associated With Mutations in RD3. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6451.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : RD3 is an evolutionarily conserved 195 aa protein of unknown subcellular localization and function. Mutations in RD3 are responsible for a form of Leber Congenital Amaurosis (LCA12) and early onset retinal degeneration in the rd3 mouse and rcd2 dog. The goal of this study was to determine the localization of RD3, identify interacting proteins, and define the molecular basis for retinal degeneration due to a deficiency in RD3.

Methods: : Anti-RD3 antibodies were made and used to localize RD3 in the retina of 21 day old wild-type (WT) and rd3 mice by confocal microscopy. Co-immunoprecipitation, tandem mass spectrometry (MS/MS), and western blotting were used to identify proteins that interact with RD3. The effect of RD3 deficiency on protein expression and trafficking to rod and cone outer segments (OS) was evaluated by confocal microscopy and western blotting.

Results: : In agreement with earlier proteomic studies, RD3 was found in rod and cone OS of WT mice. It was not detected in photoreceptors of rd3 mice. Guanylate cyclases, GC1 and GC2, co-precipitated with RD3 on an anti-RD3 affinity matrix as analyzed by MS/MS and western blotting. The direct interaction of RD3 with GC1 was confirmed by co-precipitating GC1 with RD3 from HEK293 cells co-expressing these proteins. Confocal microscopy showed that GC1 and GC2 were undetectable in rods and cones of rd3 mice and the absence of GC1 was confirmed by western blotting. GCAP1 and GCAP2 were also mislocalized in the rd3 mice. In contrast, the CNG channel, ABCA4, PDE, and rhodopsin localized normally to the OS of rd3 mice.

Conclusions: : RD3 interacts and co-localizes with GC1 and GC2 in rod and cone outer segments. It plays a crucial role in the stabilization and trafficking of GCs and associated GCAPs in photoreceptor cells. Retinal degeneration in LCA12 patients and the rd3 mouse is most likely due to the loss in the GC expression and targeting as a result of RD3 deficiency. The phenotype resulting from RD3 deficiency is consistent with the phenotype of LCA1 patients with mutations in GC1 and GC1/GC2 double knockout mice.

Keywords: retinal degenerations: hereditary • retinal degenerations: cell biology • proteins encoded by disease genes 
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