Igor V. Peshenko

The Y99C Mutation in Guanylyl Cyclase-Activating Protein 1 Increases Intracellular Ca2+ and Causes Photoreceptor Degeneration in Transgenic Mice

Guanylyl cyclase-activating proteins (GCAPs) are Ca2+-binding proteins that activate guanylyl cyclase when free Ca2+ concentrations in retinal rods and cones fall after illumination and inhibit the cyclase when free Ca2+ reaches its resting level in the dark. Several forms of retinal dystrophy are caused by mutations in GUCA1A, the gene coding for GCAP1. To investigate the cellular mechanisms affected by the diseased state, we created transgenic mice that express GCAP1 with a Tyr99Cys substitution (Y99C GCAP1) found in human patients with a late-onset retinal dystrophy (Payne et al., 1998). Y99C GCAP1 shifted the Ca2+ sensitivity of the guanylyl cyclase in photoreceptors, keeping it partially active at 250 nm free Ca2+, the normal resting Ca2+ concentration in darkness. The enhanced activity of the cyclase in the dark increased cyclic nucleotide-gated channel activity and elevated the rod outer segment Ca2+ concentration in darkness, measured by using fluo-5F and laser spot microscopy. In different lines of transgenic mice the magnitude of this effect rose with the Y99C GCAP1 expression. Surprisingly, there was little change in the rod photoresponse, indicating that dynamic Ca2+-dependent regulation of cGMP synthesis was preserved. However, the photoreceptors in these mice degenerated, and the rate of the cell loss increased with the level of the transgene expression, unlike in transgenic mice that overexpressed normal GCAP1. These results provide the first direct evidence that a mutation linked to congenital blindness increases Ca2+ in the outer segment, which may trigger the apoptotic process.

Ca2+ and Mg2+ Binding Properties of GCAP-1 EVIDENCE THAT Mg2+-BOUND FORM IS THE PHYSIOLOGICAL ACTIVATOR OF PHOTORECEPTOR GUANYLYL CYCLASE

Guanylyl cyclase-activating protein 1 (GCAP-1) is an EF-hand protein that activates retinal guanylyl cyclase (RetGC) in photoreceptors at low free Ca2+ in the light and inhibits it in the dark when Ca2+ concentrations rise. We present the first direct evidence that Mg2+-bound form of GCAP-1, not its cation-free form, is the true activator of RetGC-1 under physiological conditions. Of four EF-hand structures in GCAP-1, three bound Ca2+ ions and could exchange Ca2+ for Mg2+. At concentrations of free Ca2+ and Mg2+ typical for the light-adapted photoreceptors, all three metal-binding EF-hands were predominantly occupied by Mg2, and the presence of bound Mg2+ in GCAP-1 was essential for its ability to stimulate RetGC-1. In the Mg2+-bound form of GCAP-1 all three Trp residues became more exposed to the polar environment compared with its apo form. The replacement of Mg2+ by Ca2+ in the EF-hands 2 and 3 further exposed Trp-21 to the solution in a non-metal-binding EF-hand domain 1 that interacts with RetGC. Contrary to that, replacement of Mg2+ by Ca2+ in the EF-hand 4 moved Trp-94 in the entering α-helix of the EF-hand 3 back to the non-polar environment. Our results demonstrate that Mg2+ regulates GCAP-1 not only by adjusting its Ca2+ sensitivity to the physiological conditions in photoreceptors but also by creating the conformation required for RetGC stimulation.

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 Ca2+ concentrations in outer segment. Suppression of Ca2+ 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 Ca2+/Mg2+-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 Ca2+ and Mg2+. In the metal binding EF-hands of GCAP1, different point mutations can selectively block binding of Ca2+ or both Ca2+ and Mg2+ altogether. Activation of retGC at low Ca2+ (light adaptation) or its inhibition at high Ca2+ (dark adaptation) follows a cycle of Ca2+/Mg2+ exchange in GCAPs, rather than release of Ca2+ and its binding by apo-GCAPs. The Mg2+ 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. Mg2+ 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 Ca2+. The Mg2+/Ca2+ exchange in GCAP1 and 2 operates within different range of intracellular Ca2+ concentrations and provides a two-step activation of the cyclase during rod recovery.

A role for GCAP2 in regulating the photoresponse. Guanylyl cyclase activation and rod electrophysiology in GUCA1B knock-out mice.

Cyclic GMP serves as the second messenger in visual transduction, linking photon absorption by rhodopsin to the activity of ion channels. Synthesis of cGMP in photoreceptors is supported by a pair of retina-specific guanylyl cyclases, retGC1 and -2. Two neuronal calcium sensors, GCAP1 and GCAP2, confer Ca2+ sensitivity to guanylyl cyclase activity, but the importance and the contribution of each GCAP is controversial. To explore this issue, the gene GUCA1B, coding for GCAP2, was disrupted in mice, and the capacity for knock-out rods to regulate retGC and generate photoresponses was tested. The knock-out did not compromise rod viability or alter outer segment ultrastructure. Levels of retGC1, retGC2, and GCAP-1 expression did not undergo compensatory changes, but the absence of GCAP2 affected guanylyl cyclase activity in two ways; (a) the maximal rate of cGMP synthesis at low [Ca2+] dropped 2-fold and (b) the half-maximal rate of cGMP synthesis was attained at a higher than normal [Ca2+]. The addition of an antibody raised against mouse GCAP2 produced similar effects on the guanylyl cyclase activity in wild type retinas. Flash responses of GCAP2 knock-out rods recovered more slowly than normal. Knock-out rods became more sensitive to flashes and to steps of illumination but tended to saturate at lower intensities, as compared with wild type rods. Therefore, GCAP2 regulation of guanylyl cyclase activity quickens the recovery of flash and step responses and adjusts the operating range of rods to higher intensities of ambient illumination.

Constitutive Excitation by Gly90Asp Rhodopsin Rescues Rods from Degeneration Caused by Elevated Production of cGMP in the Dark

Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca2+ sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca2+ in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca2+ measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca2+ sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca2+ concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca2+ can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival.

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.

Effects of Ca2+, Mg2+, and Myristoylation on Guanylyl Cyclase Activating Protein 1 Structure and Stability†

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca(2+)-dependent activation of retinal guanylyl cylcase (RetGC) during phototransduction in vision. Here we analyze the energetics of Ca(2+) and Mg(2+) binding to the individual EF-hands, characterize metal-induced conformational changes, and evaluate structural effects of myristoylation as studied by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). GCAP1 binds cooperatively to Ca(2+) at EF3 and EF4 (DeltaH(EF3) = -3.5 kcal/mol, and DeltaH(EF4) = -0.9 kcal/mol) with nanomolar affinity (K(EF3) = 80 nM, and K(EF4) = 200 nM), and a third Ca(2+) binds entropically at EF2 (DeltaH(EF2) = 3.1 kcal/mol, and K(EF2) = 0.9 microM). GCAP1 binds functionally to Mg(2+) at EF2 (DeltaH(EF2) = 4.3 kcal/mol, and K(EF2) = 0.7 mM) required for RetGC activation. Ca(2+) and/or Mg(2+) binding to GCAP1 dramatically alters DSC and NMR spectra, indicating metal-induced protein conformational changes in EF2, EF3, and EF4. Myristoylation of GCAP1 does not significantly alter its metal binding energetics or NMR spectra, suggesting that myristoylation does not influence the structure of the metal-binding EF-hands. Myristoylation also has almost no effect on protein folding stability measured by DSC. NMR resonances of myristate attached to GCAP1 are exchange-broadened, upfield-shifted, and insensitive to Ca(2+), consistent with the myristoyl group being sequestered inside the protein as seen in the crystal structure. We conclude that the protein environment near the myristate is not influenced by Mg(2+) or Ca(2+) binding but instead is constitutively dynamic and may play a role in promoting interactions of GCAP1 with the cyclase.

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.

Modulation of Phosphodiesterase6 Turnoff during Background Illumination in Mouse Rod Photoreceptors

In rod photoreceptors of wild-type mice, background light produces an acceleration of the decay of responses to brief flashes, accompanied by a decrease in the rate-limiting time constant for response decay. In rods in which phosphodiesterase γ (PDEγ) lacks one of its sites of phosphorylation (T35A rods), both the waveform of response decay and the rate-limiting time constant are nearly unaffected by backgrounds. These effects are not the result of the removal of the phosphorylation site per se, because rods lacking both of the phosphorylation sites of PDEγ (T22A/T35A rods) adapt to light in a nearly normal manner. Because PDEγ is one of the proteins of the GTPase activating protein (GAP) complex, our experiments argue for a novel mechanism of photoreceptor light adaptation produced by modulation of GAP-dependent hydrolysis of transducin α GTP. In PDEγ T35A rods, a change in the conformation of the PDEγ subunit may hinder or mask this mechanism, which in mammals appears to be primarily responsible for the quickening of the temporal resolution of the rod response in backgrounds. Modulation of PDE turnoff also helps to prevent premature saturation of the rod in bright backgrounds, thus making an important contribution to light adaptation. Our experiments provide evidence for modulation of GAP protein-dependent response turnoff, which may also play a role in controlling signal duration at hormone receptors and synapses in the CNS.

Activation and Inhibition of Photoreceptor Guanylyl Cyclase by Guanylyl Cyclase Activating Protein 1 (GCAP-1) THE FUNCTIONAL ROLE OF Mg2+/Ca2+ EXCHANGE IN EF-HAND DOMAINS

Guanylyl cyclase activating protein 1 (GCAP-1), a Ca2+/Mg2+ sensor protein that accelerates retinal guanylyl cyclase (RetGC) in the light and decelerates it in the dark, is inactive in cation-free form. Binding of Mg2+ in EF-hands 2 and 3 was essential for RetGC activation in the conditions mimicking light adaptation. Mg2+ binding in EF-hand 2 affected the conformation of a neighboring non-metal binding domain, EF-hand-1, and increased GCAP-1 affinity for RetGC nearly 40-fold compared with the metal-free EF-hand 2. Mg2+ binding in EF-hand 3 increased GCAP-1 affinity for RetGC 5-fold and its maximal RetGC stimulation 2-fold. Mg2+ binding in EF-hand 4 affected neither GCAP-1 affinity for RetGC, nor RetGC activation. Inactivation of Ca2+ binding in EF-hand 4 was sufficient to render GCAP-1 a constitutive activator of RetGC, whereas the EF-hand 3 role in Ca2+-dependent deceleration of RetGC was likely to be through the neighboring EF-hand 4. Inactivation of Ca2+ binding in EF-hand 2 affected cooperativity of RetGC inhibition by Ca2+, but did not prevent the inhibition. We conclude that 1) Mg2+ binding in EF-hands 2 and 3, but not EF-hand 4, is essential for the ability of GCAP-1 to activate RetGC in the light; 2) Mg2+ or Ca2+ binding in EF-hand 3 and especially in EF-hand 2 is required for high-affinity interaction with the cyclase and affects the conformation of the neighboring EF-hand 1, a domain required for targeting RetGC; and 3) RetGC inhibition is likely to be primarily caused by Ca2+ binding in EF-hand 4.