Retinal degeneration-3 protein promotes photoreceptor survival by suppressing activation of guanylyl cyclase rather than accelerating GMP recycling.

Retinal degeneration-3 protein (RD3) deficiency causes photoreceptor dysfunction and rapid degeneration in the rd3 mouse strain and in human Leber's congenital amaurosis, a congenital retinal dystrophy that results in early vision loss. However, the mechanisms responsible for photoreceptor death remain unclear. Here, we tested two hypothesized biochemical events that may underlie photoreceptor death: (i) the failure to prevent aberrant activation of retinal guanylyl cyclase (RetGC) by calcium-sensor proteins (GCAPs) versus (ii) the reduction of GMP phosphorylation rate, preventing its recycling to GDP/GTP. We found that GMP converts to GDP/GTP in the photoreceptor fraction of the retina ∼24-fold faster in WT mice and ∼400-fold faster in rd3 mice than GTP conversion to cGMP by RetGC. Adding purified RD3 to the retinal extracts inhibited RetGC 4-fold but did not affect GMP phosphorylation in wildtype or rd3 retinas. RD3-deficient photoreceptors rapidly degenerated in rd3 mice that were reared in constant darkness to prevent light-activated GTP consumption via RetGC and phosphodiesterase 6. In contrast, rd3 degeneration was alleviated by deletion of GCAPs. After 2.5 months, only ∼40% of photoreceptors remained in rd3/rd3 retinas. Deletion of GCAP1 or GCAP2 alone preserved 68% and 57% of photoreceptors, respectively, whereas deletion of GCAP1 and GCAP2 together preserved 86%. Taken together, our in vitro and in vivo results support the hypothesis that RD3 prevents photoreceptor death primarily by suppressing activation of RetGC by both GCAP1 and GCAP2 but do not support the hypothesis that RD3 plays a significant role in GMP recycling.

Retinal degeneration-3 protein attenuates photoreceptor degeneration in transgenic mice expressing dominant mutation of human retinal guanylyl cyclase.

Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase-activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone-rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca2+GCAPs but also elevates this isozyme's resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S+). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S+ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca2+ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone-rod dystrophy 6 pathology.

GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone-rod dystrophy but not for stationary night blindness.

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone-rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer-6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.

Retinal guanylyl cyclase activation by calcium sensor proteins mediates photoreceptor degeneration in an rd3 mouse model of congenital human blindness.

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. RetGC activity completely failed to accelerate in rd3/rd3GCAPs-/- hybrid photoreceptors, whose photoresponses remained drastically suppressed compared with the WT. However, ∼70% of the hybrid rd3/rd3GCAPs-/- photoreceptors survived past 6 months, in stark contrast to <5% in the nonhybrid rd3/rd3 retinas. GFP-tagged human RD3 inhibited GCAP-dependent activation of RetGC in vitro similarly to the untagged RD3. When transgenically expressed in rd3/rd3 mouse retinas under control of the rhodopsin promoter, the RD3GFP construct increased RetGC levels to near normal levels, restored dark-adapted photoresponses, and rescued rods from degeneration. The fluorescence of RD3GFP in rd3/rd3RD3GFP+ retinas was mostly restricted to the rod photoreceptor inner segments, whereas GCAP1 immunofluorescence was concentrated predominantly in the outer segment. However, RD3GFP became distributed to the outer segments when bred into a GCAPs-/- genetic background. These results support the hypothesis that an essential biological function of RD3 is competition with GCAPs that inhibits premature cyclase activation in the inner segment. Our findings also indicate that the fast rate of degeneration in RD3-deficient photoreceptors results from the lack of this inhibition.

A G86R mutation in the calcium-sensor protein GCAP1 alters regulation of retinal guanylyl cyclase and causes dominant cone-rod degeneration.

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. The G86R-GCAP1 variant activated RetGC at low Ca2+ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca2+ concentrations characteristic of dark-adapted photoreceptors. Ca2+-dependent increase in Trp94 fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca2+ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca2+, and the dose dependence of the G86R GCAP1-RetGC1 complex inhibition by retinal degeneration 3 (RD3) protein was shifted toward higher than normal concentrations. Our results indicate that the flexibility of the hinge region between EF-hands 2 and 3 is required for placing GCAP1-regulated Ca2+ sensitivity of the cyclase within the physiological range of intracellular Ca2+ at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly86, leading to abnormally high affinity for the target enzyme and reduced Ca2+ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca2+ influx in photoreceptors in the dark.

GUCY2D Cone-Rod Dystrophy-6 Is a "Phototransduction Disease" Triggered by Abnormal Calcium Feedback on Retinal Membrane Guanylyl Cyclase 1.

The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone-rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAPs). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their nontransgenic siblings by 3-4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5-2.2-fold higher than in nontransgenic controls. Similarly, the dim flash response amplitude in R838S+ rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark-current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca2+ phototransduction feedback and abnormally high free-Ca2+ concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice in GCAP1,2+/+ or GCAP1,2+/- backgrounds, was prevented in GCAP1,2-/- mice lacking Ca2+ feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a "phototransduction disease," which means it is associated with increased free-cGMP and Ca2+ levels in photoreceptors.SIGNIFICANCE STATEMENT In a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1, GUCY2D), a mutation associated with early progressing congenital blindness, cone-rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase mediated by guanylyl cyclase activating proteins (GCAPs), which are calcium-sensor proteins. The abnormal calcium sensitivity of the cyclase increases cGMP-gated dark current in the rod outer segments, reshapes rod photoresponses, and triggers photoreceptor death. This work is the first to demonstrate a direct physiological effect of GUCY2D CORD6-linked mutation on photoreceptor physiology in vivo It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.

Functional Study and Mapping Sites for Interaction with the Target Enzyme in Retinal Degeneration 3 (RD3) Protein.

Retinal degeneration 3 (RD3) protein, essential for normal expression of retinal membrane guanylyl cyclase (RetGC) in photoreceptor cells, blocks RetGC catalytic activity and stimulation by guanylyl cyclase-activating proteins (GCAPs). In a mouse retina, RD3 inhibited both RetGC1 and RetGC2 isozymes. Photoreceptors in the rd3/rd3 mouse retinas lacking functional RD3 degenerated more severely than in the retinas lacking both RetGC isozymes, consistent with a hypothesis that the inhibitory activity of RD3 has a functional role in photoreceptors. To map the potential target-binding site(s) on RD3, short evolutionary conserved regions of its primary structure were scrambled and the mutations were tested for the RD3 ability to inhibit RetGC1 and co-localize with the cyclase in co-transfected cells. Substitutions in 4 out of 22 tested regions, (87)KIHP(90), (93)CGPAI(97), (99)RFRQ(102), and (119)RSVL(122), reduced the RD3 apparent affinity for the cyclase 180-700-fold. Changes of amino acid sequences outside the Lys(87)-Leu(122) central portion of the molecule either failed to prevent RD3 binding to the cyclase or had a much smaller effect. Mutations in the (93)CGPAI(97) portion of a predicted central α-helix most drastically suppressed the inhibitory activity of RD3 and disrupted RD3 co-localization with RetGC1 in HEK293 cells. Different side chains replacing Cys(93) profoundly reduced RD3 affinity for the cyclase, irrespective of their relative helix propensities. We conclude that the main RetGC-binding interface on RD3 required for the negative regulation of the cyclase localizes to the Lys(87)-Leu(122) region.

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

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. The R838S RetGC1 expression in rod outer segments reduced inhibition of cGMP production in the transgenic mouse retinas at the free calcium concentrations typical for dark-adapted rods. The transgenic mice demonstrated early-onset and rapidly progressed with age decline in visual responses from the targeted rods, in contrast to the longer lasting preservation of function in the non-targeted cones. The decline in rod function in the retina resulted from a progressive degeneration of rods between 1 and 6 months of age, with the severity and pace of the degeneration consistent with the extent to which the Ca2+ sensitivity of the retinal cGMP production was affected. Our study presents a new experimental model for exploring cellular mechanisms of the CORD6-related photoreceptor death. This mouse model provides the first direct biochemical and physiological in vivo evidence for the Arg838 substitutions in RetGC1 being the culprit behind the pathogenesis of the CORD6 congenital blindness.

Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State.

GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca(2+)-sensitive activation of retinal guanylyl cyclase 1 (RetGC1). We present NMR resonance assignments, residual dipolar coupling data, functional analysis, and a structural model of GCAP1 mutant (GCAP1(V77E)) in the Ca(2+)-free/Mg(2+)-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca(2+)-dependent differences for residues 170-174. An NMR-derived model of GCAP1(V77E) contains Mg(2+) bound at EF2 and looks similar to Ca(2+) saturated GCAP1 (root mean square deviations = 2.0 Å). Ca(2+)-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164-174 called the Ca(2+) switch helix). Ca(2+)-induced shortening of the Ca(2+) switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca(2+) switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca(2+) binding affinity and abolished RetGC1 activation. We conclude that Ca(2+)-dependent conformational changes in the Ca(2+) switch helix are important for activating RetGC1 and provide further support for a Ca(2+)-myristoyl tug mechanism.

Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface.

The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met(823) was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg(822). The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met(823) or Arg(822) was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg(822) and Met(823).

Evaluating the role of retinal membrane guanylyl cyclase 1 (RetGC1) domains in binding guanylyl cyclase-activating proteins (GCAPs).

Retinal membrane guanylyl cyclase 1 (RetGC1) regulated by guanylyl cyclase-activating proteins (GCAPs) controls photoreceptor recovery and when mutated causes blinding disorders. We evaluated the principal models of how GCAP1 and GCAP2 bind RetGC1: through a shared docking interface versus independent binding sites formed by distant portions of the cyclase intracellular domain. At near-saturating concentrations, GCAP1 and GCAP2 activated RetGC1 from HEK293 cells and RetGC2(-/-)GCAPs1,2(-/-) mouse retinas in a non-additive fashion. The M26R GCAP1, which binds but does not activate RetGC1, suppressed activation of recombinant and native RetGC1 by competing with both GCAP1 and GCAP2. Untagged GCAP1 displaced both GCAP1-GFP and GCAP2-GFP from the complex with RetGC1 in HEK293 cells. The intracellular segment of a natriuretic peptide receptor A guanylyl cyclase failed to bind GCAPs, but replacing its kinase homology and dimerization domains with those from RetGC1 restored GCAP1 and GCAP2 binding by the hybrid cyclase and its GCAP-dependent regulation. Deletion of the Tyr(1016)-Ser(1103) fragment in RetGC1 did not block GCAP2 binding to the cyclase. In contrast, substitutions in the kinase homology domain, W708R and I734T, linked to Leber congenital amaurosis prevented binding of both GCAP1-GFP and GCAP2-GFP. Our results demonstrate that GCAPs cannot regulate RetGC1 using independent primary binding sites. Instead, GCAP1 and GCAP2 bind with the cyclase molecule in a mutually exclusive manner using a common or overlapping binding site(s) in the Arg(488)-Arg(851) portion of RetGC1, and mutations in that region causing Leber congenital amaurosis blindness disrupt activation of the cyclase by both GCAP1 and GCAP2.

Functional study of two biochemically unusual mutations in GUCY2D Leber congenital amaurosis expressed via adenoassociated virus vector in mouse retinas.

PURPOSE: To test, in living photoreceptors, two mutations, S248W and R1091x, in the GUCY2D gene linked to Leber congenital amaurosis 1 (LCA1) that fail to inactivate the catalytic activity of a heterologously expressed retinal membrane guanylyl cyclase 1 (RetGC1). METHODS: GUC2YD cDNA constructs coding for wild-type human (hWT), R1091x, and S248W GUCY2D under the control of the human rhodopsin kinase promoter were expressed in Gucy2e-/-Gucy2f-/- knockout (GCdKO) mouse retinas, which lack endogenous RetGC activity. The constructs were delivered via subretinally injected adenoassociated virus (AAV) vector in one eye, leaving the opposite eye as the non-injected negative control. After testing with electroretinography (ERG), the retinas extracted from the AAV-treated and control eyes were used in guanylyl cyclase activity assays, immunoblotting, and anti-RetGC1 immunofluorescence staining. RESULTS: Cyclase activity in retinas treated with either hWT or R1091x GUCY2D transgenes was similar but was undetectable in the S248W GUCY2D-treated retinas, which starkly contrasts their relative activities when heterologously expressed in human embryonic kidney (HEK293) cells. Rod and cone ERGs, absent in GCdKO, appeared in the hWT and R1091x GUCY2D-injected eyes, while the S248W mutant failed to restore scotopic ERG response and enabled only rudimentary photopic ERG response. The hWT and R1091x GUCY2D immunofluorescence was robust in the rod and cone outer segments, whereas the S248W was detectable only in the sparse cone outer segments and sporadic photoreceptor cell bodies. Robust RetGC1 expression was detected with immunoblotting in the hWT and R1091x-treated retinas but was marginal at best in the S248W GUCY2D retinas, despite the confirmed presence of the S248W GUCY2D transcripts. CONCLUSIONS: The phenotype of S248W GUCY2D in living retinas did not correlate with the previously described normal biochemical activity of this mutant when heterologously expressed in non-photoreceptor cell culture. This result suggests that the S248W mutation contributes to LCA1 by hampering the expression, processing, and/or cellular transport of GUCY2D, rather than its enzymatic properties. In contrast, the effective restoration of rod and cone function by R1091x GUCY2D is paradoxical and does not explain the severe loss of vision typical for LCA1 associated with that mutant allele.

Identification of target binding site in photoreceptor guanylyl cyclase-activating protein 1 (GCAP1).

Retinal guanylyl cyclase (RetGC)-activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. We mapped GCAP1 residues comprising the RetGC1 binding site by mutagenizing the entire surface of GCAP1 and testing the ability of each mutant to bind RetGC1 in a cell-based assay and to activate it in vitro. Mutations that most strongly affected the activation of RetGC1 localized to a distinct patch formed by the surface of non-metal-binding EF-hand 1, the loop and the exiting helix of EF-hand 2, and the entering helix of EF-hand 3. Mutations in the binding patch completely blocked activation of the cyclase without affecting Ca(2+) binding stoichiometry of GCAP1 or its tertiary fold. Exposed residues in the C-terminal portion of GCAP1, including EF-hand 4 and the helix connecting it with the N-terminal lobe of GCAP1, are not critical for activation of the cyclase. GCAP1 mutants that failed to activate RetGC1 in vitro were GFP-tagged and co-expressed in HEK293 cells with mOrange-tagged RetGC1 to test their direct binding in cyto. Most of the GCAP1 mutations introduced into the "binding patch" prevented co-localization with RetGC1, except for Met-26, Lys-85, and Trp-94. With these residues mutated, GCAP1 completely failed to stimulate cyclase activity but still bound RetGC1 and competed with the wild type GCAP1. Thus, RetGC1 activation by GCAP1 involves establishing a tight complex through the binding patch with an additional activation step involving Met-26, Lys-85, and Trp-94.

Determining consequences of retinal membrane guanylyl cyclase (RetGC1) deficiency in human Leber congenital amaurosis en route to therapy: residual cone-photoreceptor vision correlates with biochemical properties of the mutants.

The GUCY2D gene encodes retinal membrane guanylyl cyclase (RetGC1), a key component of the phototransduction machinery in photoreceptors. Mutations in GUCY2D cause Leber congenital amaurosis type 1 (LCA1), an autosomal recessive human retinal blinding disease. The effects of RetGC1 deficiency on human rod and cone photoreceptor structure and function are currently unknown. To move LCA1 closer to clinical trials, we characterized a cohort of patients (ages 6 months-37 years) with GUCY2D mutations. In vivo analyses of retinal architecture indicated intact rod photoreceptors in all patients but abnormalities in foveal cones. By functional phenotype, there were patients with and those without detectable cone vision. Rod vision could be retained and did not correlate with the extent of cone vision or age. In patients without cone vision, rod vision functioned unsaturated under bright ambient illumination. In vitro analyses of the mutant alleles showed that in addition to the major truncation of the essential catalytic domain in RetGC1, some missense mutations in LCA1 patients result in a severe loss of function by inactivating its catalytic activity and/or ability to interact with the activator proteins, GCAPs. The differences in rod sensitivities among patients were not explained by the biochemical properties of the mutants. However, the RetGC1 mutant alleles with remaining biochemical activity in vitro were associated with retained cone vision in vivo. We postulate a relationship between the level of RetGC1 activity and the degree of cone vision abnormality, and argue for cone function being the efficacy outcome in clinical trials of gene augmentation therapy in LCA1.

Retinal guanylyl cyclase isozyme 1 is the preferential in vivo target for constitutively active GCAP1 mutants causing congenital degeneration of photoreceptors.

Two calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) activate cGMP synthesis in photoreceptor by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) to expedite recovery, but calcium-insensitive constitutively active GCAP1 mutants cause photoreceptor degeneration in human patients and transgenic mice. Although GCAP1 and GCAP2 can both activate RetGC1 and RetGC2 in vitro, we find that GCAP1 selectively regulates RetGC1 in vivo. Furthermore, elimination of RetGC1 but not RetGC2 isozyme reverses abnormal calcium sensitivity of cGMP synthesis and rescues mouse rods in transgenic mice expressing GCAP1 mutants causing photoreceptor disease. Rods expressing mutant GCAP1 not only survive in the absence of RetGC1 but also remain functional, albeit with reduced electroretinography (ERG) amplitudes typical of RetGC1-/- genotype. The rod ERG recovery from a strong flash, only slightly affected in both RetGC1-/- and RetGC2-/- mice, becomes very slow in RetGC1-/- but not RetGC2-/- mice when GCAP2 is not available to provide Ca²âº feedback to the remaining RetGC isozyme. The intrinsic biochemical properties of RetGC and GCAP determined in vitro do not explain the observed phenomena. Instead, our results argue that there must be a cellular mechanism that limits GCAP1 access to RetGC2 and makes RetGC1 isozyme a preferential target for the disease-causing GCAP1 mutants. A more general conclusion from our findings is that nondiscriminatory interactions between homologous effector enzymes and their regulatory proteins permitted by their intrinsic biochemical properties can be effectively restricted in a living photoreceptor.

Calcium-myristoyl Tug is a new mechanism for intramolecular tuning of calcium sensitivity and target enzyme interaction for guanylyl cyclase-activating protein 1: dynamic connection between N-fatty acyl group and EF-hand controls calcium sensitivity.

Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca(2+) sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca(2+) sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca(2+) binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca(2+)-bound and Mg(2+)-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca(2+) sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca(2+) binding to EF-hand 4 and Ca(2+) sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu(80) and Val(180)) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or "tug") between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca(2+) sensitivity.

Enzymatic properties and regulation of the native isozymes of retinal membrane guanylyl cyclase (RetGC) from mouse photoreceptors.

Mouse photoreceptor function and survival critically depend on Ca(2+)-regulated retinal membrane guanylyl cyclase (RetGC), comprised of two isozymes, RetGC1 and RetGC2. We characterized the content, catalytic constants, and regulation of native RetGC1 and RetGC2 isozymes using mice lacking guanylyl cyclase activating proteins GCAP1 and GCAP2 and deficient for either GUCY2F or GUCY2E genes, respectively. We found that the characteristics of both native RetGC isozymes were considerably different from other reported estimates made for mammalian RetGCs: the content of RetGC1 per mouse rod outer segments (ROS) was at least 3-fold lower, the molar ratio (RetGC2:RetGC1) 6-fold higher, and the catalytic constants of both GCAP-activated isozymes between 12- and 19-fold higher than previously measured in bovine ROS. The native RetGC isozymes had different basal activity and were accelerated 5-28-fold at physiological concentrations of GCAPs. RetGC2 alone was capable of contributing as much as 135-165 µM cGMP s(-1) or almost 23-28% to the maximal cGMP synthesis rate in mouse ROS. At the maximal level of activation by GCAP, this isozyme alone could provide a significantly high rate of cGMP synthesis compared to what is expected for normal recovery of a mouse rod, and this can help explain some of the unresolved paradoxes of rod physiology. GCAP-activated native RetGC1 and RetGC2 were less sensitive to inhibition by Ca(2+) in the presence of GCAP1 (EC(50Ca) ∼132-139 nM) than GCAP2 (EC(50Ca) ∼50-59 nM), thus arguing that Ca(2+) sensor properties of GCAP in a functional RetGC/GCAP complex are defined not by a particular target isozyme but the intrinsic properties of GCAPs themselves.

Retinal degeneration 3 (RD3) protein inhibits catalytic activity of retinal membrane guanylyl cyclase (RetGC) and its stimulation by activating proteins.

Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca(2+) concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a noncompetitive manner, and it inhibits the GCAP-stimulated RetGC at low intracellular Ca(2+) levels. RD3 opposes the allosteric activation of the cyclase by GCAP but does not significantly change Ca(2+) sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone-rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca(2+) and GCAPs.

Activation of retinal guanylyl cyclase RetGC1 by GCAP1: stoichiometry of binding and effect of new LCA-related mutations.

Retinal membrane guanylyl cyclase (RetGC) and Ca(2+)/Mg(2+) sensor proteins (GCAPs) control the recovery of the photoresponse in vertebrate photoreceptors, through their molecular interactions that remain rather poorly understood and controversial. Here we have determined the main RetGC isozyme (RetGC1):GCAP1 binding stoichiometry at saturation in cyto, using fluorescently labeled RetGC1 and GCAP1 coexpressed in HEK293 cells. In a striking manner, the equimolar binding of RetGC1 with GCAP1 in transfected HEK293 cells typical for wild-type RetGC1 was eliminated by a substitution, D639Y, in the kinase homology domain of RetGC1 found in a patient with a severe form of retinal dystrophy, Leber congenital amaurosis (LCA). A similar effect was observed with another LCA-related mutation, R768W, in the same domain of RetGC1. In contrast to the completely suppressed binding and activation of RetGC1 by Mg(2+)-liganded GCAP1, neither of these two mutations eliminated the GCAP1-independent activity of RetGC stimulated by Mn(2+). These results directly implicate the D639 (and possibly R768)-containing portion of the RetGC1 kinase homology domain in its primary recognition by the Mg(2+)-bound activator form of GCAP1.

Mg2+/Ca2+ cation binding cycle of guanylyl cyclase activating proteins (GCAPs): role in regulation of photoreceptor guanylyl cyclase.

Photon absorption by photoreceptors activates hydrolysis of cGMP, which shuts down cGMP-gated channels and decreases free Ca(2+) concentrations in outer segment. Suppression of Ca(2+) influx through the cGMP channel by light activates retinal guanylyl cyclase through guanylyl cyclase activating proteins (GCAPs) and thus expedites photoreceptors recovery from excitation and restores their light sensitivity. GCAP1 and GCAP2, two ubiquitous among vertebrate species isoforms of GCAPs that activate retGC during rod response to light, are myristoylated Ca(2+)/Mg(2+)-binding proteins of the EF-hand superfamily. They consist of one non-metal binding EF-hand-like domain and three other EF-hands, each capable of binding Ca(2+) and Mg(2+). In the metal binding EF-hands of GCAP1, different point mutations can selectively block binding of Ca(2+) or both Ca(2+) and Mg(2+) altogether. Activation of retGC at low Ca(2+) (light adaptation) or its inhibition at high Ca(2+) (dark adaptation) follows a cycle of Ca(2+)/Mg(2+) exchange in GCAPs, rather than release of Ca(2+) and its binding by apo-GCAPs. The Mg(2+) binding in two of the EF-hands controls docking of GCAP1 with retGC1 in the conditions of light adaptation and is essential for activation of retGC. Mg(2+) binding in a C-terminal EF-hand contributes to neither retGC1 docking with the cyclase nor its subsequent activation in the light, but is specifically required for switching the cyclase off in the conditions of dark adaptation by binding Ca(2+). The Mg(2+)/Ca(2+) exchange in GCAP1 and 2 operates within different range of intracellular Ca(2+) concentrations and provides a two-step activation of the cyclase during rod recovery.