- Retinal degeneration-3 (RD3) protein protects photoreceptors from degeneration by preventing retinal guanylyl cyclase (RetGC) activation via calcium-sensing guanylyl cyclase–activating proteins (GCAP), and RD3 truncation causes severe congenital blindness in humans and other animals. The three-dimensional structure of RD3 has recently been established, but the molecular mechanisms of its inhibitory binding to RetGC remain unclear. Here, we report the results of probing 133 surface-exposed residues in RD3 by single substitutions and deletions to identify side chains that are critical for the inhibitory binding of RD3 to RetGC.
- Deficiency of RD3 (retinal degeneration 3) protein causes recessive blindness and photoreceptor degeneration in humans and in the rd3 mouse strain, but the disease mechanism is unclear. Here, we present evidence that RD3 protects photoreceptors from degeneration by competing with guanylyl cyclase-activating proteins (GCAPs), which are calcium sensor proteins for retinal membrane guanylyl cyclase (RetGC). RetGC activity in rd3/rd3 retinas was drastically reduced but stimulated by the endogenous GCAPs at low Ca2+ concentrations.
- The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca2+ concentrations decline, and decelerates the cyclase in the dark, when Ca2+ concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 (GUCA1A) gene in a family with a dominant retinopathy. The G86R substitution in a “hinge” region connecting EF-hand domains 2 and 3 in GCAP1 strongly interfered with its Ca2+-dependent activator-to-inhibitor conformational transition.
- Retinal degeneration 3 (RD3) protein promotes accumulation of retinal membrane guanylyl cyclase (RetGC) in the photoreceptor outer segment and suppresses RetGC activation by guanylyl cyclase–activating proteins (GCAPs). Mutations truncating RD3 cause severe congenital blindness by preventing the inhibitory binding of RD3 to the cyclase. The high propensity of RD3 to aggregate in solution has prevented structural analysis. Here, we produced a highly soluble variant of human RD3 (residues 18–160) that is monomeric and can still bind and negatively regulate RetGC.
- Substitutions of Arg838 in the dimerization domain of a human retinal membrane guanylyl cyclase 1 (RetGC1) linked to autosomal dominant cone-rod degeneration type 6 (CORD6) change RetGC1 regulation in vitro by Ca2+. In addition, we find that R838S substitution makes RetGC1 less sensitive to inhibition by retinal degeneration-3 protein (RD3). We selectively expressed human R838S RetGC1 in mouse rods and documented the decline in rod vision and rod survival. To verify that changes in rods were specifically caused by the CORD6 mutation, we used for comparison cones, which in the same mice did not express R838S RetGC1 from the transgenic construct.