NMR Structure of Retinal Guanylate Cyclase Activating Protein 5 (GCAP5) with R22A Mutation That Abolishes Dimerization and Enhances Cyclase Activation.

Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with ∼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.

The Transition of Photoreceptor Guanylate Cyclase Type 1 to the Active State.

Membrane-bound guanylate cyclases (GCs), which synthesize the second messenger guanosine-3', 5'-cyclic monophosphate, differ in their activation modes to reach the active state. Hormone peptides bind to the extracellular domain in hormone-receptor-type GCs and trigger a conformational change in the intracellular, cytoplasmic part of the enzyme. Sensory GCs that are present in rod and cone photoreceptor cells have intracellular binding sites for regulatory Ca2+-sensor proteins, named guanylate-cyclase-activating proteins. A rotation model of activation involving an α-helix rotation was described as a common activation motif among hormone-receptor GCs. We tested whether the photoreceptor GC-E underwent an α-helix rotation when reaching the active state. We experimentally simulated such a transitory switch by integrating alanine residues close to the transmembrane region, and compared the effects of alanine integration with the point mutation V902L in GC-E. The V902L mutation is found in patients suffering from retinal cone-rod dystrophies, and leads to a constitutively active state of GC-E. We analyzed the enzymatic catalytic parameters of wild-type and mutant GC-E. Our data showed no involvement of an α-helix rotation when reaching the active state, indicating a difference in hormone receptor GCs. To characterize the protein conformations that represent the transition to the active state, we investigated the protein dynamics by using a computational approach based on all-atom molecular dynamics simulations. We detected a swinging movement of the dimerization domain in the V902L mutant as the critical conformational switch in the cyclase going from the low to high activity state.

NMR and EPR-DEER Structure of a Dimeric Guanylate Cyclase Activator Protein-5 from Zebrafish Photoreceptors.

Retinal guanylate cyclases (RetGCs) are regulated by a family of guanylate cyclase-activating proteins (called GCAP1-7). GCAPs form dimers that bind to Ca2+ and confer Ca2+ sensitive activation of RetGC during visual phototransduction. The GCAP5 homologue from zebrafish contains two nonconserved cysteine residues (Cys15 and Cys17) that bind to ferrous ion, which stabilizes GCAP5 dimerization and diminishes its ability to activate RetGC. Here, we present NMR and EPR-DEER structural analysis of a GCAP5 dimer in the Mg2+-bound, Ca2+-free, Fe2+-free activator state. The NMR-derived structure of GCAP5 is similar to the crystal structure of Ca2+-bound GCAP1 (root-mean-square deviation of 2.4 Å), except that the N-terminal helix of GCAP5 is extended by two residues, which allows the sulfhydryl groups of Cys15 and Cys17 to become more solvent exposed in GCAP5 to facilitate Fe2+ binding. Nitroxide spin-label probes were covalently attached to particular cysteine residues engineered in GCAP5: C15, C17, T26C, C28, N56C, C69, C105, N139C, E152C, and S159C. The intermolecular distance of each spin-label probe in dimeric GCAP5 (measured by EPR-DEER) defined restraints for calculating the dimer structure by molecular docking. The GCAP5 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H18, Y21, M25, F72, V76, and W93, as well as an intermolecular salt bridge between R22 and D71. The structural model of the GCAP5 dimer was validated by mutations (H18E/Y21E, H18A/Y21A, R22D, R22A, M25E, D71R, F72E, and V76E) at the dimer interface that disrupt dimerization of GCAP5 and affect the activation of RetGC. We propose that GCAP5 dimerization may play a role in the Fe2+-dependent regulation of cyclase activity in zebrafish photoreceptors.

A novel missense variant in the EML1 gene associated with bilateral ribbon-like subcortical heterotopia leads to ciliary defects.

Heterotopia is a brain malformation caused by a failed migration of cortical neurons during development. Clinical symptoms of heterotopia vary in severity of intellectual disability and may be associated with epileptic disorders. Abnormal neuronal migration is known to be associated with mutations in the doublecortin gene (DCX), the platelet-activating factor acetylhydrolase gene (PAFAH1B1), or tubulin alpha-1A gene (TUBA1A). Recently, a new gene encoding echinoderm microtubule-associated protein-like 1 (EML1) was reported to cause a particular form of subcortical heterotopia, the ribbon-like subcortical heterotopia (RSH). EML1 mutations are inherited in an autosomal recessive manner. Only six unrelated EML1-associated heterotopia-affected families were reported so far. The EML1 protein is a member of the microtubule-associated proteins family, playing an important role in microtubule assembly and stabilization as well as in mitotic spindle formation in interphase. Herein, we present a novel homozygous missense variant in EML1 (NM_004434.2: c.692G>A, NP_004425.2: p.Gly231Asp) identified in a male RSH-affected patient. Our clinical and molecular findings confirm the genotype-phenotype associations of EML1 mutations and RSH. Analyses of patient-derived fibroblasts showed the significantly reduced length of primary cilia. In addition, our results presented, that the mutated EML1 protein did not change binding capacities with tubulin. The data described herein will expand the mutation spectrum of the EML1 gene and provide further insight into molecular and cellular bases of the pathogenic mechanisms underlying RSH.

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.

Structural Characterization of Ferrous Ion Binding to Retinal Guanylate Cyclase Activator Protein 5 from Zebrafish Photoreceptors.

Sensory guanylate cyclases (zGCs) in zebrafish photoreceptors are regulated by a family of guanylate cyclase activator proteins (called GCAP1-7). GCAP5 contains two nonconserved cysteine residues (Cys15 and Cys17) that could in principle bind to biologically active transition state metal ions (Zn2+ and Fe2+). Here, we present nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) binding analyses that demonstrate the binding of one Fe2+ ion to two GCAP5 molecules (in a 1:2 complex) with a dissociation constant in the nanomolar range. At least one other Fe2+ binds to GCAP5 with micromolar affinity that likely represents electrostatic Fe2+ binding to the EF-hand loops. The GCAP5 double mutant (C15A/C17A) lacks nanomolar binding to Fe2+, suggesting that Fe2+ at this site is ligated directly by thiolate groups of Cys15 and Cys17. Size exclusion chromatography analysis indicates that GCAP5 forms a dimer in the Fe2+-free and Fe2+-bound states. NMR structural analysis and molecular docking studies suggest that a single Fe2+ ion is chelated by thiol side chains from Cys15 and Cys17 in the GCAP5 dimer, forming an [Fe(SCys)4] complex like that observed previously in two-iron superoxide reductases. Binding of Fe2+ to GCAP5 weakens its ability to activate photoreceptor human GC-E by decreasing GC activity >10-fold. Our results indicate a strong Fe2+-induced inhibition of GC by GCAP5 and suggest that GCAP5 may serve as a redox sensor in visual phototransduction.

Two retinal dystrophy-associated missense mutations in GUCA1A with distinct molecular properties result in a similar aberrant regulation of the retinal guanylate cyclase.

Two recently identified missense mutations (p. L84F and p. I107T) in GUCA1A, the gene coding for guanylate cyclase (GC)-activating protein 1 (GCAP1), lead to a phenotype ascribable to cone, cone-rod and macular dystrophies. Here, we present a thorough biochemical and biophysical characterization of the mutant proteins and their distinct molecular features. I107T-GCAP1 has nearly wild-type-like protein secondary and tertiary structures, and binds Ca(2+) with a >10-fold lower affinity than the wild-type. On the contrary, L84F-GCAP1 displays altered tertiary structure in both GC-activating and inhibiting states, and a wild type-like apparent affinity for Ca(2+). The latter mutant also shows a significantly high affinity for Mg(2+), which might be important for stabilizing the GC-activating state and inducing a cooperative mechanism for the binding of Ca(2+), so far not been observed in other GCAP1 variants. Moreover, the thermal stability of L84F-GCAP1 is particularly high in the Ca(2+)-bound, GC-inhibiting state. Molecular dynamics simulations suggest that such enhanced stability arises from a deeper burial of the myristoyl moiety within the EF1-EF2 domain. The simulations also support an allosteric mechanism connecting the myristoyl moiety to the highest-affinity Ca(2+) binding site EF3. In spite of their remarkably distinct molecular features, both mutants cause constitutive activation of the target GC at physiological Ca(2+). We conclude that the similar aberrant regulation of the target enzyme results from a similar perturbation of the GCAP1-GC interaction, which may eventually cause dysregulation of both Ca(2+) and cyclic GMP homeostasis and result in retinal degeneration.

Mapping Calcium-Sensitive Regions in the Neuronal Calcium Sensor GCAP2 by Site-Specific Fluorescence Labeling.

Myristoylation of most neuronal calcium sensor proteins, a group of EF-hand calcium-binding proteins mainly expressed in neuronal tissue, can have a strong impact on protein dynamics and functional properties. Intracellular oscillations of the free Ca(2+) concentration can trigger conformational changes in Ca(2+) sensors. The position and possible movements of the myristoyl group in the photoreceptor cell-specific Ca(2+) sensor GCAP2 are not well-defined but appear to be different from those of the highly homologous cognate GCAP1. We designed and applied a new group of diaminoterephthalate-derived fluorescent probes to label GCAP2 at a covalently attached 12-azido-dodecanoic acid (a myristoyl substitute) and at cysteine residues in critical positions. Fluorescence emission of dye-labeled GCAP2 decreased when going from low (10(-9) M) to high [Ca(2+)] (10(-3) M), reaching a half-maximal effect of fluorescence emission at 0.44 ± 0.07 µM. The modified acyl group can therefore monitor changes in the protein conformation during binding and dissociation of Ca(2+) in the physiological range of free [Ca(2+)]. However, fluorescence quenching studies showed that the dye-acyl chain was shielded from the quencher by an adjacent polypeptide region. Further probing three cysteine positions (C35, C111, and C131) by dye labeling revealed that all positions were also sensitive to a change in [Ca(2+)], but only one (C131) was sensitive to a change in [Mg(2+)]. We suggest a scenario during illumination of the photoreceptor cell in which Ca(2+) dissociates first from low and medium affinity binding sites. These steps are sensed by dyes in cysteines at positions 35 and 111. Release of Ca(2+) from high affinity sites is sensed by regions adjacent to the dye-labeled fatty acid and involves the critical conformational change leading to activating guanylate cyclase.

Regulatory function of the C-terminal segment of guanylate cyclase-activating protein 2.

Neuronal responses to Ca2+-signals are provided by EF-hand-type neuronal Ca2+-sensor (NCS) proteins, which have similar core domains containing Ca2+-binding and target-recognizing sites. NCS proteins vary in functional specificity, probably depending on the structure and conformation of their non-conserved C-terminal segments. Here, we investigated the role of the C-terminal segment in guanylate cyclase activating protein-2, GCAP2, an NCS protein controlling the Ca2+-dependent regulation of photoreceptor guanylate cyclases. We obtained two chimeric proteins by exchanging C-terminal segments between GCAP2 and its photoreceptor homolog recoverin, a Ca2+-sensor controlling rhodopsin kinase (RK) activity. The exchange affected neither the structural integrity of GCAP2 and recoverin nor the Ca2+-sensitivity of GCAP2. Intrinsic fluorescence, circular dichroism, biochemical studies and hydrophobic dye probing revealed Ca2+-dependent conformational transition of the C-terminal segment of GCAP2 occurring in the molecular environment of both proteins. In Ca2+-GCAP2, the C-terminal segment was constrained and its replacement provided the protein with approximately two-fold inhibitory activity towards RK, suggesting that the segment contributes to specific target recognition by interfering with RK-binding. Upon Ca2+-release, it became less constrained and more available for phosphorylation by cyclic nucleotide-dependent protein kinase. The transition from the Ca2+-bound to the apo-state exposed hydrophobic sites in GCAP2, and was associated with its activating function without affecting its dimerization. The released C-terminal segment participated further in photoreceptor membrane binding making it sensitive to phosphorylation. Thus, the C-terminal segment in GCAP2 confers target selectivity, facilitates membrane binding and provides sensitivity of the membrane localization of the protein to phosphorylation by signaling kinases.

Operation profile of zebrafish guanylate cyclase-activating protein 3.

The expression pattern and property profile of the neuronal Ca(2+) sensor guanylate cyclase-activating protein 3 (zGCAP3) was studied by immunochemical approaches, biophysical methods and enzymatic assays. Using affinity purified antibodies immunoreactivity towards zGCAP3 was weakly detected in the outer and strongly in the inner segments of cone cells as well as in the outer plexiform layer, to a lesser degree also in the inner plexiform and ganglion cell layer of the zebrafish retina. This cellular distribution was independent of a dark/light cycle. Some neuronal Ca(2+) sensors are acylated (mainly myristoylated) at the amino-terminus. Probing larval and adult stages of the developing zebrafish retina indicated that zGCAP3 was first expressed in a non-myristoylated form, but was finally present in the adult retina as a myristoylated protein. While zGCAP3 did not undergo a classical Ca(2+) -myristoyl switch as investigated by surface plasmon resonance spectroscopy, myristoylation had two main other consequences: it enhanced the Ca(2+) -sensitivity of the Ca(2+) -induced conformational change and it stabilized the protein conformation. Differences between myristoylated and non-myristoylated zGCAP3 were also observed in modulating the kinetic and catalytic parameters of the GCAP-target, a membrane bound guanylate cyclase. Thus, the stabilizing effect of the myristoyl group is apparently less important in the larval than in the adult fish.

Expression profiles of three novel sensory guanylate cyclases and guanylate cyclase-activating proteins in the zebrafish retina.

Three membrane bound sensory guanylate cyclases are expressed in photoreceptor cells of the developing and adult zebrafish retina. First appearance of mRNA transcripts was detected by in situ hybridization techniques for all guanylate cyclases between 3 and 4 days post fertilization (dpf), but only one isoform (guanylate cyclase 3) appeared to be specifically expressed in cones of the adult retina. Transcripts of three cone specific guanylate cyclase-activating proteins (zGCAP3, zGCAP4 and zGCAP7) were also detected at 3-4 dpf. The expression onset of the guanylate cyclases and these neuronal calcium sensor proteins mainly overlapped. High guanylate cyclase activities in larval eye preparations and the precisely controlled coexpression of guanylate cyclases and zGCAPs coincide with the onset of visual function at 3-4 dpf.

Diversity of sensory guanylate cyclases in teleost fishes.

Teleost fishes like medaka fish (Oryzias latipes), zebrafish (Danio rerio), and pufferfish (Fugu rubripes) contain in their genomes a larger number of guanylate cyclases and guanylate cyclase-activating proteins than mammals. Based on amino acid sequence alignments a group of transmembrane sensory guanylate cyclases can be identified, which are mainly if not exclusively expressed in sensory organs like the retina and olfactory tissue. Retina specific guanylate cyclases and guanylate cyclase-activating proteins in the zebrafish show dynamic changes in their spatial-temporal expression patterns and transcripts of the corresponding genes appear coincidently with the beginning of cone cell maturation at 3 days post-fertilization. Expression patterns of the guanylate cyclase signaling systems during larval development are correlated with the special habitat challenges of zebrafishes in the wild.

Protein-protein interaction of the putative magnetoreceptor cryptochrome 4 expressed in the avian retina.

Migratory birds can sense the Earth's magnetic field and use it for orientation over thousands of kilometres. A light-dependent radical-pair mechanism associated with the visual system is currently discussed as the underlying mechanism of the magnetic compass sense. The blue light receptor cryptochrome 4 (Cry4) is considered as the most likely primary sensory protein that detects the geomagnetic field. Since the protein interaction partners of Cry4 are completely unknown at present, here, we aim to identify potential candidate interaction partners of Cry4 in the avian retina. We used the yeast-two-hybrid system to screen avian cDNA libraries for possible interaction partners of Cry4 in the European robin. The UAS-GAL yeast two hybrid system was applied to confirm a group of candidate Cry4 interaction partners. Six proteins were found to be particularly promising candidates for interacting with European robin Cry4. The identified genes code for guanine nucleotide-binding protein G(t) subunit alpha-2 (GNAT2), long-wavelength-sensitive opsin (LWS, also called iodopsin), guanine nucleotide-binding protein subunit gamma 10 (GNG10), potassium voltage-gated channel subfamily V member 2 (KCNV2), retinol binding protein 1 (RBP1) and retinal G protein-coupled receptor (RGR). All genes are known to be expressed in vertebrate retinae of different species. We conclude by discussing putative signalling pathways that could connect cryptochrome 4 to one or more of these 6 candidates.

The cone-specific calcium sensor guanylate cyclase activating protein 4 from the zebrafish retina.

Guanylate cyclase activating proteins (GCAPs) serve as neuronal Ca(2+)-sensor proteins in vertebrate rod and cone photoreceptor cells. Zebrafish express in their retina a variety of six different GCAPs, of which four are specific for cone cells. One isoform, zGCAP4, is mainly expressed in double cones and long single cones. We cloned the zGCAP4 gene, purified non-myristoylated and myristoylated forms of the protein after heterologous expression in Escherichia coli and studied its properties: zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish retina, showing half-maximal activation at 520-570 nM free Ca(2+) concentration. Furthermore, the Ca(2+)-sensitive activation properties of non-myristoylated and myristoylated zGCAP4 were similar, indicating no influence of the myristoyl moiety on Ca(2+)-sensor function. Myristoylated zGCAP4 showed low affinity for membranes and did not exhibit a Ca(2+)-myristoyl switch, a feature typical of some but not all neuronal Ca(2+)-sensor proteins. However, tryptophan fluorescence studies and Ca(2+)-dependent differences in protease accessibility revealed Ca(2+)-induced conformational changes in myristoylated and non-myristoylated zGCAP4, indicating the operation as a Ca(2+) sensor. Thus, expression and biochemical properties of zGCAP4 are in agreement with its function as an efficient Ca(2+)-sensitive regulator of guanylate cyclase activity in cone vision.

Zebrafish Recoverin Isoforms Display Differences in Calcium Switch Mechanisms.

Primary steps in vertebrate vision occur in rod and cone cells of the retina and require precise molecular switches in excitation, recovery, and adaptation. In particular, recovery of the photoresponse and light adaptation processes are under control of neuronal Ca2+ sensor (NCS) proteins. Among them, the Ca2+ sensor recoverin undergoes a pronounced Ca2+-dependent conformational change, a prototypical so-called Ca2+-myristoyl switch, which allows selective targeting of G protein-coupled receptor kinase. Zebrafish (Danio rerio) has gained attention as a model organism in vision research. It expresses four different recoverin isoforms (zRec1a, zRec1b, zRec2a, and zRec2b) that are orthologs to the one known mammalian variant. The expression pattern of the four isoforms cover both rod and cone cells, but the differential distribution in cones points to versatile functions of recoverin in these cell types. Initial functional studies on zebrafish larvae indicate different Ca2+-sensitive working modes for zebrafish recoverins, but experimental evidence is lacking so far. The aims of the present study are (1) to measure specific Ca2+-sensing properties of the different recoverin isoforms, (2) to ask whether switch mechanisms triggered by Ca2+ resemble that one observed with mammalian recoverin, and (3) to investigate a possible impact of an attached myristoyl moiety. For addressing these questions, we employ fluorescence spectroscopy, surface plasmon resonance (SPR), dynamic light scattering, and equilibrium centrifugation. Exposure of hydrophobic amino acids, due to the myristoyl switch, differed among isoforms and depended also on the myristoylation state of the particular recoverin. Ca2+-induced rearrangement of the protein-water shell was for all variants less pronounced than for the bovine ortholog indicating either a modified Ca2+-myristoyl switch or no switch. Our results have implications for a step-by-step response of recoverin isoforms to changing intracellular Ca2+ during illumination.

Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding.

Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that share a high degree of amino acid sequence homology, but differ in Ca(2+)-binding properties, Ca(2+)-sensitive target regulation and spatial-temporal expression profiles. We here study a general problem in cellular Ca(2+)-sensing, namely how similar Ca(2+)-binding proteins achieve functional selectivity to control finely adjusted cellular responses. We investigated two parameters of critical importance for the trigger and switch function of guanylate cyclase-activating proteins: the myristoylation status and the occupation of Ca(2+)-binding sites with Mg(2+). All zGCAPs can be myristoylated in living cells using click chemistry. Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5. We further determined for all zGCAPs at least two binding sites exhibiting high affinities for Ca(2+) with KD values in the submicromolar range, whereas for other zGCAPs (except zGCAP3) the affinity of the third binding site was in the micromolar range. Mg(2+) either occupied the low affinity Ca(2+)-binding site or it shifted the affinities for Ca(2+)-binding. Hydrodynamic properties of zGCAPs are more influenced by Ca(2+) than by Mg(2+), although to a different extent for each zGCAP. Posttranslational modification and competing ion-binding can tailor the properties of similar Ca(2+)-sensors.

Zebrafish guanylate cyclase type 3 signaling in cone photoreceptors.

The zebrafish guanylate cyclase type 3 (zGC3) is specifically expressed in cone cells. A specifc antibody directed against zGC3 revealed expression at the protein level at 3.5 dpf in outer and inner retinal layers, which increased in intensity between 3.5 and 7 dpf. This expression pattern differed from sections of the adult retina showing strong immunostaining in outer segments of double cones and short single cones, less intense immunoreactivity in long single cones, but no staining in the inner retina. Although transcription and protein expression levels of zGC3 are similar to that of the cyclase regulator guanylate cyclase-activating protein 3 (zGCAP3), we surprisingly found that zGCAP3 is present in a 28-fold molar excess over zGC3 in zebrafish retinae. Further, zGCAP3 was an efficient regulator of guanylate cyclases activity in native zebrafish retinal membrane preparations. Therefore, we investigated the physiological function of zGCAP3 by two different behavioral assays. Using the morpholino antisense technique, we knocked down expression of zGCAP3 and recorded the optokinetic and optomotor responses of morphants, control morphants, and wild type fish at 5-6 dpf. No significant differences in behavioral responses among wild type, morphants and control morphants were found, indicating that a loss of zGCAP3 has no consequences in primary visual processing in the larval retina despite its prominent expression pattern. Its physiological function is therefore compensated by other zGCAP isoforms.

Probing the Ca(2+) switch of the neuronal Ca(2+) sensor GCAP2 by time-resolved fluorescence spectroscopy.

We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca(2+)-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a "wobbling-in-a-cone" model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein-dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.

Differential calcium signaling by cone specific guanylate cyclase-activating proteins from the zebrafish retina.

Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs) than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca(2+)-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1-4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca(2+)-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca(2+) around 30 nM (zGCAP1, 2 and 3) or around 400 nM (zGCAP4, 5 and 7). Zebrafish GCAP isoforms showed also differences in their Ca(2+)/Mg(2+)-dependent conformational changes and in the Ca(2+)-dependent monomer-dimer equilibrium. Direct Ca(2+)-binding revealed that all zGCAPs bound at least three Ca(2+). The corresponding apparent affinity constants reflect binding of Ca(2+) with high (≤ 100 nM), medium (0.1-5 µM) and/or low (≥ 5 µM) affinity, but were unique for each zGCAP isoform. Our data indicate a Ca(2+)-sensor system in zebrafish rod and cone cells supporting a Ca(2+)-relay model of differential zGCAP operation in these cells.