RD3, the protein associated with Leber congenital amaurosis type 12, is required for guanylate cyclase trafficking in photoreceptor cells

Abstract

Guanylate cyclases, GC1 and GC2, are localized in the light-sensitive outer segment compartment of photoreceptor cells, where they play a crucial role in phototransduction by catalyzing the synthesis of cGMP, the second messenger of phototransduction, and regulating intracellular Ca 2+ levels in combination with the cGMP-gated channel. Mutations in GC1 are known to cause Leber congenital amaurosis type 1 (LCA1), a childhood disease associated with severe vision loss. Although the enzymatic and regulatory properties of guanylate cyclases have been studied extensively, the molecular determinants responsible for their trafficking in photoreceptors remain unknown. Here we show that RD3, a protein of unknown function encoded by a gene associated with photoreceptor degeneration in humans with Leber congenital amaurosis type 12 (LCA12), the rd3 mouse, and rcd2 collie, colocalizes and interacts with GC1 and GC2 in rod and cone photoreceptor cells of normal mice. GC1 and GC2 are undetectable in photoreceptors of the rd3 mouse deficient in RD3 by immunofluorescence microscopy. Cell expression studies show that RD3 mediates the export of GC1 from the endoplasmic reticulum to endosomal vesicles, and that the C terminus of GC1 is required for RD3 binding. Our results indicate that photoreceptor degeneration in the rd3 mouse, rcd2 dog, and LCA12 patients is caused by impaired RD3-mediated guanylate cyclase expression and trafficking. The resulting deficiency in cGMP synthesis and the constitutive closure of cGMP-gated channels might cause a reduction in intracellular Ca 2+ to a level below that required for long-term photoreceptor cell survival. membrane trafficking | photoreceptor degeneration | vision | calcium homeostasis | retinal disease mechanisms

Impaired Association of Retinal Degeneration-3 with Guanylate Cyclase-1 and Guanylate Cyclase-activating Protein-1 Leads to Leber Congenital Amaurosis-1

“…The GCAP-6B12 monoclonal antibody was generated in our laboratory from mice immunized with an affinity-purified GST fusion protein containing the C-terminal 45 amino acids of the mouse GCAP1 that had no homology with GCAP2. Polyclonal antibodies for RD3 and GC1 were generated against the last 16-amino acid C-terminal peptide of the mouse GC1 and RD3; details are presented in Table 1 (15).…”

“…A GC1 trafficking model proposes a vesicular mechanism (25) that aids other integral and peripheral membrane proteins in moving from the ER to the POS (4). We previously showed that GC1 traffics to the POS by interacting with RD3 (15). We also showed that RD3 negatively regulates GC1, probably during protein trafficking (26).…”

“…Loss of GC1 function is the most common feature of LCA1, and it is thought to be the cause of photoreceptor death in LCA1 patients (1, 9 -14). LCA12 is another severe form of retinal dystrophy caused by mutations in the gene encoding retinal degeneration 3 (RD3) protein (15). We showed earlier that RD3 is crucial for the stability and expression of GC1 and that the interactions between RD3 and GC1 are essential for GC1 trafficking to the photoreceptor outer segment (POS) (15).…”

The R838S Mutation in Retinal Guanylyl Cyclase 1 (RetGC1) Alters Calcium Sensitivity of cGMP Synthesis in the Retina and Causes Blindness in Transgenic Mice

“…Two RetGC isozymes expressed in photoreceptors, RetGC1 and RetGC2 (6 -8), bind calcium-sensor proteins, GCAPs (7)(8)(9)(10)(11)(12), which decelerate the cyclase activity at high intracellular calcium concentrations in the dark and accelerate it in the light, when the influx of calcium through the cGMP-gated channels is shut off (13)(14)(15)(16)(17)(18)(19). RetGC also binds retinal degeneration 3 (RD3) protein (20 -21), which prevents cyclase activation by GCAPs (22)(23) and promotes accumulation of RetGC1 in the outer segments (21,24,25). RetGC1 (human gene GUCY2D) accounts for most of the cGMP synthesis in mammalian photoreceptors (26,27), therefore mutations in RetGC1 that disable the cyclase activity (28 -33) cause recessive blindness at birth, Leber congenital amaurosis type 1 (LCA1) (33), mostly a non-degenerative or partially degenerative (31,32) loss-of-function hereditary retinal disease.…”

Positive selection rather than relaxation of functional constraint drives the evolution of vision during chicken domestication

“…ZNF469 encodes a zinc-finger protein and mutations in this gene are associated with brittle cornea syndrome and keratoconus, a common inherited ocular disorder resulting in progressive corneal thinning [30,31]. RD3 on the other hand encodes retinal degeneration 3 protein, and mutations in this gene cause Leber congenital amaurosis type 12, a disease that results in photoreceptor degeneration [32].…”