Photoreceptor Guanylate Cyclase (GUCY2D) Mutations Cause Retinal Dystrophies by Severe Malfunction of Ca2+-Dependent Cyclic GMP Synthesis.

Over 100 mutations in GUCY2D that encodes the photoreceptor guanylate cyclase GC-E are known to cause two major diseases: autosomal recessive Leber congenital amaurosis (arLCA) or autosomal dominant cone-rod dystrophy (adCRD) with a poorly understood mechanism at the molecular level in most cases. Only few mutations were further characterized for their enzymatic and molecular properties. GC-E activity is under control of neuronal Ca2+-sensor proteins, which is often a possible route to dysfunction. We investigated five recently-identified GC-E mutants that have been reported in patients suffering from arLCA (one large family) and adCRD/maculopathy (four families). Microsatellite analysis revealed that one of the mutations, c.2538G > C (p.K846N), occurred de novo. To better understand the mechanism by which mutations that are located in different GC-E domains develop different phenotypes, we investigated the molecular consequences of these mutations by expressing wildtype and mutant GC-E variants in HEK293 cells. Analyzing their general enzymatic behavior, their regulation by Ca2+ sensor proteins and retinal degeneration protein 3 (RD3) dimerization domain mutants (p.E841K and p.K846N) showed a shift in Ca2+-sensitive regulation by guanylate cyclase-activating proteins (GCAPs). Mutations in the cyclase catalytic domain led to a loss of enzyme function in the mutant p.P873R, but not in p.V902L. Instead, the p.V902L mutation increased the guanylate cyclase activity more than 20-fold showing a high GCAP independent activity and leading to a constitutively active mutant. This is the first mutation to be described affecting the GC-E catalytic core in a complete opposite way.

Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1.

Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca2+-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1-101), or its short scaffolding domain (81-101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca2+-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca2+-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca2+-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca2+-signals thereby maintaining efficient phototransduction recovery and light adaptation.

Control of the Nucleotide Cycle in Photoreceptor Cell Extracts by Retinal Degeneration Protein 3.

Retinal degeneration protein 3 (RD3) is crucial for photoreceptor cell survival and linked to Leber Congenital Amaurosis type 12 (LCA12), a hereditary retinal disease in humans. RD3 inhibits photoreceptor guanylate cyclases GC-E and GC-F and is involved in transport of GCs from the inner to the outer segments. Otherwise, its role in photoreceptor physiology is poorly understood. Here, we describe a new function of RD3. Purified RD3 evoked an increase in guanylate kinase activity, an enzyme that is involved in the nucleotide cycle in photoreceptors. We demonstrate a direct interaction between guanylate kinase and RD3 using back-scattering interferometry and show by immunohistochemistry of mouse retina sections that RD3 and guanylate kinase co-localize in photoreceptor inner segments and to a lesser extent in the outer plexiform layer. Our findings point toward a more complex function of RD3 in photoreceptors. The RD3 - guanylate kinase interaction may also play a role in other cellular systems, while the GC - RD3 interaction is exclusive to photoreceptors.

Genotype-functional-phenotype correlations in photoreceptor guanylate cyclase (GC-E) encoded by GUCY2D.

The GUCY2D gene encodes for the photoreceptor guanylate cyclase GC-E that synthesizes the intracellular messenger of photoreceptor excitation cGMP and is regulated by intracellular Ca2+-sensor proteins named guanylate cyclase-activating proteins (GCAPs). Over 140 disease-causing mutations have been described so far in GUCY2D, 88% of which cause autosomal recessive Leber congenital amaurosis (LCA) while heterozygous missense mutations cause autosomal dominant cone-rod degeneration (adCRD). Mutations in GUCY2D are one of the major causes of all LCA cases and are the major cause of adCRD. A single amino acid, arginine at position 838, is likely to be the most sensitive one in GC-E as four single mutations and two complex mutations were reported to affect R838. The biochemical effect of 45 GC-E variants was studied showing a clear genotype-phenotype correlation: LCA-causing mutations either show reduced ability or complete inability to synthesize cGMP from GTP, while CRD-causing mutations are functional, but shift the Ca2+-sensitivity of the GC-E - GCAP complex. Eight animal models of retinal guanylate cyclase deficiency have been reported including knockout (KO) mouse and chicken models. These two models were used for gene augmentation therapy that yielded promising results. Here we integrate the available information on the genetics, biochemistry and phenotype that is related to GUCY2D mutations. These data clearly show that mutation type (missense versus null) and localization (dimerization domain versus other protein domains) are correlated with the pattern of inheritance, impact on enzymatic function and retinal phenotype. Such clear correlation is unique to GUCY2D while mutations in many other retinal disease genes show variable phenotypes and lack of available biochemical assays.