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Seifollah Azadi, Rahel Zulliger, Robert Molday, Muna Naash; Impaired formation of RD3-GCAP1-GC1 complex causes Leber congenital Amaurosis type1. Invest. Ophthalmol. Vis. Sci. 2013;54(15):734.
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Leber congenital amaurosis type 1 (LCA1) is a severe retinal dystrophy manifesting from early infancy as poor vision or blindness and is caused by loss-of-function mutations in guanylate cyclase 1 (GC1). We have recently reported that retinal degeneration 3 (RD3) directly interacts with GC1 and is necessary for trafficking of GC to photoreceptor (PR) outer segments (OSs). In the absence of RD3, OS targeting of GC1 and GC activating protein1 (GCAP1) is impaired. We hypothesize that GCAP1 and RD3 complex with GC1 in the endoplasmic reticulum (ER) before they are targeted to the OS. Our goal is to gain more insights into the role of this complex in causing LCA1.
All the known LCA1 mutations in GC1 were reconstructed in an expression vector using mutagenesis. Interactions between GC1 (WT and LCA1 mutants) and RD3 were evaluated by reciprocal immunoprecipitation (R-IP) in HEK293 cells using corresponding antibodies conjugated to columns. A direct binding assay was applied to analyze the binding of RD3 with GCAP1. Expression in COS7 cells was used to verify the interaction between RD3 and GCAP1. The interactions between RD3, GC1 and GCAP1 were studied using cells transfected with full-length RD3, GCAP1 and either WT or mutant GC1.
Our in vitro data suggest that RD3 directly interacts with GCAP1 and GC1, and that this interaction is required for plasma membrane (PM) targeting. This complex is trafficked to the PM in rab11 containing vesicles. Furthermore, mutagenesis studies showed that LCA1 disease-causing mutations impair RD3-GC1 binding and PM targeting. Our in vivo analysis supports the hypothesis that complex assembly of these three proteins is required for proper OS targeting.
These data suggests that GCAP1 and RD3 complex with GC1 in the ER to target GC1 to PR OS and interruption of this assembly leads to LCA1. These findings advance our understandings of the mechanisms underlie LCA1 and create an important foundation for the development of therapeutic strategies to treat this severe form of blindness.
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