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Photoreceptor cells have a remarkable capacity to adapt the sensitivity and speed of their responses to ever changing conditions of ambient illumination. Recent studies have revealed that a major contributor to this adaptation is the phenomenon of light-driven translocation of key signaling proteins into and out of the photoreceptor outer segment, the cellular compartment where phototransduction takes place. So far, only two such proteins, transducin and arrestin, have been established to be involved in this mechanism. To investigate the extent of this phenomenon we examined additional photoreceptor proteins that might undergo light-driven translocation, focusing on three Ca(2+)-binding proteins, recoverin and guanylate cyclase activating proteins 1 (GCAP1) and GCAP2. The changes in the subcellular distribution of each protein were assessed quantitatively using a recently developed technique combining serial tangential sectioning of mouse retinas with Western blot analysis of the proteins in the individual sections. Our major finding is that light causes a significant reduction of recoverin in rod outer segments, accompanied by its redistribution toward rod synaptic terminals. In both cases the majority of recoverin was found in rod inner segments, with approximately 12% present in the outer segments in the dark and less than 2% remaining in that compartment in the light. We suggest that recoverin translocation is adaptive because it may reduce the inhibitory constraint that recoverin imposes on rhodopsin kinase, an enzyme responsible for quenching the photo-excited rhodopsin during the photoresponse. To the contrary, no translocation of rhodopsin kinase itself or either GCAP was identified.

Purpose: To determine the underlying molecular genetic basis of a retinal dystrophy identified in a 4-generation family and to examine the phenotype and the degree of intrafamilial variability., Design: Prospective case series., Participants: Six affected individuals from a nonconsanguineous British family., Methods: Detailed ophthalmologic examination, color fundus photography, autofluorescence imaging, and electrophysiologic assessment were performed. Blood samples were taken for DNA extraction, and mutation screening of GUCA1A, the gene encoding guanylate cyclase-activating protein 1 (GCAP1), was undertaken., Results: All affected subjects complained of mild photophobia and reduced central and color vision. Onset was between the third and fifth decade, with subsequent gradual deterioration of visual acuity and color vision. Visual acuity ranged between 6/9 and counting fingers. Color vision was either absent or markedly reduced along all 3 color axes. A range of macular appearances was seen, varying from mild retinal pigment epithelial disturbance to extensive atrophy. Electrophysiologic testing revealed a range of electrophysiologic abnormalities: isolated cone electroretinography abnormalities, reduced cone and rod responses (with cone loss greater than rod), and isolated macular dysfunction. The 4 coding exons of GUCA1A were screened for mutations in affected and unaffected family members. A single transition, A319G, causing a nonconservative missense substitution, Tyr99Cys, segregated uniquely in all affected subjects., Conclusions: The Tyr99Cys GUCA1A mutation has been previously shown to cause autosomal dominant progressive cone dystrophy. This is the first report of this mutation also causing both cone-rod dystrophy and isolated macular dysfunction. The phenotypic variation described here exemplifies the intrafamilial heterogeneity of retinal dysfunction that can be observed in persons harboring the same mutation and chromosomal segment.

The rod outer segment membrane guanylate cyclase type 1 (ROS-GC1), originally identified in the photoreceptor outer segments, is a member of the subfamily of Ca(2+)-modulated membrane guanylate cyclases. In phototransduction, its activity is tightly regulated by its two Ca(2+)-sensor protein parts, GCAP1 and GCAP2. This study maps the GCAP2-modulatory site in ROS-GC1 through the use of multiple techniques involving surface plasmon resonance binding studies with soluble ROS-GC1 constructs, coimmunoprecipitation, functional reconstitution experiments with deletion mutants, and peptide competition assays. The findings show that the sequence motif of the core GCAP2-modulatory site is Y965-N981 of ROS-GC1. The site is distinct from the GCAP1-modulatory site. It, however, partially overlaps with the S100B-regulatory site. This indicates that the Y965-N981 motif tightly controls the Ca(2+)-dependent specificity of ROS-GC1. Identification of the site demonstrates an intriguing topographical feature of ROS-GC1. This is that the GCAP2 module transmits the Ca(2+) signals to the catalytic domain from its C-terminal side and the GCAP1 module from the distant N-terminal side.

Purpose: To elucidate the phenotypic and biochemical characteristics of a novel mutation associated with autosomal dominant cone-rod dystrophy (adCORD)., Methods: Twenty-three family members of a CORD pedigree underwent clinical examinations, including visual acuity tests, standardized full-field ERG, and fundus photography. Genomic DNA was screened for mutations in GCAP1 exons using DNA sequencing and single-strand conformational polymorphism (SSCP) analysis. Function and stability of recombinant GCAP1-L151F were tested as a function of [Ca(2+)], and its structure was probed by molecular dynamics., Results: Affected family members experienced dyschromatopsia, hemeralopia, and reduced visual acuity by the second to third decade of life. Electrophysiology revealed a nonrecordable photopic response with later attenuation of the scotopic response. Affected family members harbored a C-->T transition in exon 4 of the GCAP1 gene, resulting in an L151F missense mutation affecting the EF hand motif 4 (EF4). This change was absent in 11 unaffected family members and in 100 unrelated normal subjects. GCAP1-L151F stimulation of photoreceptor guanylate cyclase was not completely inhibited at high physiological [Ca(2+)], consistent with a lowered affinity for Ca(2+)-binding to EF4., Conclusions: A novel L151F mutation in the EF4 hand domain of GCAP1 is associated with adCORD. The clinical phenotype is characterized by early cone dysfunction and a progressive loss of rod function. The biochemical phenotype is best described as persistent stimulation of photoreceptor guanylate cyclase, representing a gain of function of mutant GCAP1. Although a conservative substitution, molecular dynamics suggests a significant change in Ca(2+)-binding to EF4 and EF2 and changes in the shape of L151F-GCAP1.

Background: We investigated mutations in the gene coding for guanylate-cyclase activating protein 2 (GCAP2), also known as GUCA1B gene, in Japanese patients with retinitis pigmentosa (RP) and tried to identify phenotypic characteristics associated with mutations in the gene., Subjects and Methods: Genomic DNA samples from 63 unrelated patients with autosomal dominant retinitis pigmentosa (ADRP) and 33 patients with autosomal recessive retinitis pigmentosa (ARRP) were screened by single-strand conformational polymorphism analysis followed by direct sequencing. Clinical features associated with a mutation were demonstrated by visual acuity, visual field testing, fundus photography, and electroretinography., Results: A novel transitional mutation converting GGA to AGA at codon 157 (G157R) was identified. This mutation has been found in three index patients from three independent families. Phenotypic examination of seven members of the three families revealed that this mutation was associated with RP with or without macular involvement in five members, macular degeneration in one member, and asymptomatic normal phenotype in one member. In addition, previously unknown polymorphic changes including V29V, Y57Y, T87I, and L180L were identified., Conclusions: A racial difference exists in the spectrum of mutations and/or polymorphisms in the GCAP 2 gene between British and Japanese populations. Our findings suggest that the mutation in the GCAP 2 gene can cause one form of autosomal dominant retinal dystrophy, with variable phenotypic expression and incomplete penetrance.

Purpose: To describe the clinical features and genetic analysis of a family with an autosomal dominant cone dystrophy (adCD)., Methods: Selected members of a family with an autosomal dominant cone dystrophy underwent ophthalmic evaluation. Blood samples were obtained, genomic DNA was isolated, and genomic fragments were amplified by PCR. Linkage to locus D6S1017 was established. DHPLC mutational analysis and direct sequencing were used to identify a mutation in GUCA1A, the gene encoding the guanylate cyclase activating protein 1 (GCAP1)., Results: Of 24 individuals who are at risk of the disease in a five generation family, 11 members were affected. Clinical presentations included photophobia, color vision defects, central acuity loss, and legal blindness with advanced age. The disease phenotype was observed in the second and third decades of life and segregated in an autosomal dominant fashion. An electroretinogram performed on one proband revealed profoundly subnormal and prolonged photopic and flicker responses, but preserved scotopic ERGs, consistent with a cone dystrophy. Mutational analysis and direct sequencing revealed a C451T transition in GUCA1A, corresponding to a novel L151F mutation in GCAP1. Like the E155G mutation, this mutation occurs in the EF4 hand domain, a region of GCAP1 critical in conferring calcium sensitivity to the protein. The leucine at this position is highly conserved among vertebrate guanylate cyclase activating proteins., Conclusions: A novel L151F missense mutation in the EF4 high affinity Ca2+ binding site of GCAP1 is linked to adCD in a large pedigree. The cone dystrophy in this family shares clinical and electrophysiologic characteristics with other previously described adCD caused by mutations in GUCA1A.

Guanylyl cyclase-activating proteins (GCAPs) are calcium sensor proteins of the EF-hand superfamily that inhibit retinal photoreceptor membrane guanylyl cyclase (retGC) in the dark when they bind Ca(2+) but activate retGC when Ca(2+) dissociates from GCAPs in response to light stimulus. We addressed the difference in exposure of GCAP-2 structure to protein kinase and a protease as indicators of conformational change caused by binding and release of Ca(2+). We have found that unlike its homolog, GCAP-1, the C terminus of GCAP-2 undergoes phosphorylation by cyclic nucleotide-dependent protein kinases (CNDPK) present in the retinal extract and rapid dephosphorylation by the protein phosphatase PP2C present in the retina. Inactivation of the CNDPK phosphorylation site in GCAP-2 by substitutions S201G or S201D, as well as phosphorylation or thiophosphorylation of Ser(201), had little effect on the ability of GCAP-2 to regulate retGC in reconstituted membranes in vitro. At the same time, Ca(2+) strongly inhibited phosphorylation of the wild-type GCAP-2 by retinal CNDPK but did not affect phosphorylation of a constitutively active Ca(2+)-insensitive GCAP-2 mutant. Partial digestion of purified GCAP-2 with Glu-C protease revealed at least two sites that become exposed or constrained in a Ca(2+)-sensitive manner. The Ca(2+)-dependent conformational changes in GCAP-2 affect the areas around Glu(62) residue in the entering helix of EF-hand 2, the areas proximal to the exiting helix of EF-hand 3, and Glu(136)-Glu (138) between EF-hand 3 and EF-hand 4. These changes also cause the release of the C-terminal Ser(201) from the constraint caused by the Ca(2+)-bound conformation.

We explored the possibility that, in the regulation of an effector enzyme by a Ca(2+)-sensor protein, the actual Ca(2+) sensitivity of the effector enzyme can be determined not only by the affinity of the Ca(2+)-sensor protein for Ca(2+) but also by the relative affinities of its Ca(2+)-bound versus Ca(2+)-free form for the effector enzyme. As a model, we used Ca(2+)-sensitive activation of photoreceptor guanylyl cyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg(838)Ser in RetGC-1 found in human patients with cone-rod dystrophy is known to shift the Ca(2+) sensitivity of RetGC-1 regulation by GCAP-1 to a higher Ca(2+) range. We find that at physiological concentrations of Mg(2+) this mutation increases the free Ca(2+) concentration required for half-maximal inhibition of the cyclase from 0.27 to 0.61 microM. Similar to rod outer segment cyclase, Ca(2+) sensitivity of recombinant RetGC-1 is strongly affected by Mg(2+), but the shift in Ca(2+) sensitivity for the R838S mutant relative to the wild type is Mg(2+)-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 for both Ca(2+)-bound and Ca(2+)-free GCAP-1 and found that the net shift in Ca(2+) sensitivity of the R838S RetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclase for the Ca(2+)-free versus Ca(2+)-loaded GCAP-1. Our findings confirm that the dynamic range for RetGC regulation by Ca(2+)/GCAP is determined by both the affinity of GCAP for Ca(2+) and relative affinities of the effector enzyme for the Ca(2+)-free versus Ca(2+)-loaded GCAP.

Purpose: To identify pathogenic mutations in the guanylate cyclase-activating protein 1 (GCAP1) and GCAP2 genes and to characterize the biochemical effect of mutation on guanylate cyclase (GC) stimulation., Methods: The GCAP1 and GCAP2 genes were screened by direct sequencing for mutations in 216 patients and 421 patients, respectively, with various hereditary retinal diseases. A mutation in GCAP1 segregating with autosomal dominant cone degeneration was further evaluated biochemically by employing recombinant proteins, immunoblotting, Ca2+-dependent stimulation of GC, fluorescence emission spectra, and limited proteolysis in the absence and presence of Ca2+., Results: A novel GCAP1 mutation, I143NT (substitution of Ile at codon 143 by Asn and Thr), affecting the EF4 Ca2+-binding loop, was identified in a heterozygote father and son with autosomal dominant cone degeneration. Both patients had much greater loss of cone function versus rod function; previous histopathologic evaluation of the father's eyes at autopsy (age 75 years) showed no foveal cones but a few, scattered cones remaining in the peripheral retina. Biochemical analysis showed that the GCAP1-I143NT mutant adopted a conformation susceptible to proteolysis, and the mutant inhibited GC only partially at high Ca2+ concentrations. Individual patients with atypical or recessive retinitis pigmentosa (RP) had additional heterozygous GCAP1-T114I and GCAP2 gene changes (V85M and F150C) of unknown pathogenicity., Conclusions: A novel GCAP1 mutation, I143NT, caused a form of autosomal dominant cone degeneration that destroys foveal cones by mid-life but spares some cones in the peripheral retina up to 75 years. Properties of the GCAP1-I143NT mutant protein suggested that it is incompletely inactivated by high Ca2+ concentrations as should occur with dark adaptation. The continued activity of the mutant GCAP1 likely results in higher-than-normal scotopic cGMP levels which may, in turn, account for the progressive loss of cones.

Background: Inhaled nitric oxide (NO) is a potent and selective pulmonary vasodilator, which induces cGMP synthesis by activating soluble guanylate cyclase (sGC) in ventilated lung regions. Carbon monoxide (CO) has also been proposed to influence smooth muscle tone via activation of sGC. We examined whether direct stimulation of sGC by BAY 41-2272 would produce pulmonary vasodilation and augment the pulmonary responses to inhaled NO or CO., Methods and Results: In awake, instrumented lambs, the thromboxane analogue U-46619 was intravenously administered to increase mean pulmonary arterial pressure to 35 mm Hg. Intravenous infusion of BAY 41-2272 (0.03, 0.1, and 0.3 mg x kg(-1) x h(-1)) reduced mean pulmonary arterial pressure and pulmonary vascular resistance and increased transpulmonary cGMP release in a dose-dependent manner. Larger doses of BAY 41-2272 also produced systemic vasodilation and elevated the cardiac index. N(omega)-nitro-l-arginine methyl ester abolished the systemic but not the pulmonary vasodilator effects of BAY 41-2272. Furthermore, infusing BAY 41-2272 at 0.1 mg x kg(-1) x h(-1) potentiated and prolonged the pulmonary vasodilation induced by inhaled NO (2, 10, and 20 ppm). In contrast, inhaled CO (50, 250, and 500 ppm) had no effect on U-46619-induced pulmonary vasoconstriction before or during administration of BAY 41-2272., Conclusions: In lambs with acute pulmonary hypertension, BAY 41-2272 is a potent pulmonary vasodilator that augments and prolongs the pulmonary vasodilator response to inhaled NO. Direct pharmacological stimulation of sGC, either alone or in combination with inhaled NO, may provide a novel approach for the treatment of pulmonary hypertension.

The guanylate cyclase-activating proteins (GCAPs), Ca2+-binding proteins of the calmodulin gene superfamily, function as regulators of photoreceptor guanylate cyclases. In contrast to calmodulin, which is active in the Ca2+-bound form, GCAPs stimulate GCs in the [Ca2+]-free form and inhibit GCs upon Ca2+ binding. In vertebrate retinas, at least two GCAP1 and two GCs are present, a third GCAP3 is expressed in humans and fish, and at least five additional GCAP4-8 genes have been identified or are predicted in zebrafish and pufferfish. Missense mutations in GCAP1 (Y99C, I143NT, E155G, and P50L) have been associated with autosomal dominant cone dystrophy. Absence of GCAP1/2 in mice delays recovery of the photoresponse, a phenotype consistent with delay in cGMP synthesis. In the absence of GCAP2, GCAP1 supports the generation of wild-type flash responses in both rod and cone cells. Recent progress revealed an unexpected complexity of the GC-GCAP system, pointing, out a number of unsolved questions., (Copyright 2004 Elsevier Inc.)

Purpose: To examine the spatial and temporal characteristics of cone cell survival and the expression of guanylate cyclase-activating proteins (GCAPs) in the guanylate cyclase (GC)-1 knockout (KO) mouse retina., Methods: Immunohistochemical analyses with peanut agglutinin and an antibody specific for cone transducin were used to examine cone cell survival in the GC1 KO retina at 4, 5, 9, 16, and 24 weeks of age. Immunohistochemical and Northern and Western blot analyses were used to examine the expression of GCAP1 and GCAP2 in 4- to 5-week-old mice., Results: The number of cone cells appeared normal throughout the superior and inferior retinal regions in 4- and 5-week-old GC1 KO mice but gradually decreased by 6 months. Cone cell loss was exacerbated in the inferior retinal region, with only 2% to 8% remaining by 6 months of age; however, 40% to 70% of the cone cells survived in the superior region at this age. GCAP1 and GCAP2 protein levels were downregulated in GC1 KO retinas at 4 weeks of age and GCAP1 immunostaining was absent from the photoreceptor outer segments., Conclusions: The results of this study show that the rate of cone cell loss in the GC1 KO mouse is comparable to that previously described in the GUCY1*B chicken and in humans with Leber congenital amaurosis (LCA)-1. The GCAP expression data, when combined with those of previous electrophysiological studies of the GC1 KO mouse retina, provide evidence that GC1-GCAP1 interactions are essential for cone cell function in mice and that GC2 and GCAP2 activities contribute to the rod cell response in the absence of GC1.

Guanylate cyclase-activating proteins (GCAPs) are neuronal calcium sensors that activate membrane bound guanylate cyclases (EC 4.6.1.2.) of vertebrate photoreceptor cells when cytoplasmic Ca2+ decreases during illumination. GCAPs contain four EF-hand Ca2+-binding motifs, but the first EF-hand is nonfunctional. It was concluded that for GCAP-2, the loss of Ca2+-binding ability of EF-hand 1 resulted in a region that is crucial for targeting guanylate cyclase [Ermilov, A.N., Olshevskaya, E.V. & Dizhoor, A.M. (2001) J. Biol. Chem.276, 48143-48148]. In this study we tested the consequences of mutations in EF-hand 1 of GCAP-1 with respect to Ca2+ binding, Ca2+-induced conformational changes and target activation. When the nonfunctional first EF-hand in GCAP-1 is replaced by a functional EF-hand the chimeric mutant CaM-GCAP-1 bound four Ca2+ and showed similar Ca2+-dependent changes in tryptophan fluorescence as the wild-type. CaM-GCAP-1 neither activated nor interacted with guanylate cyclase. Size exclusion chromatography revealed that the mutant tended to form inactive dimers instead of active monomers like the wild-type. Critical amino acids in EF-hand 1 of GCAP-1 are cysteine at position 29 and proline at position 30, as changing these to glycine was sufficient to cause loss of target activation without a loss of Ca2+-induced conformational changes. The latter mutation also promoted dimerization of the protein. Our results show that EF-hand 1 in wild-type GCAP-1 is critical for providing the correct conformation for target activation., (Copyright 2004 FEBS)

The guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins of the calmodulin (CaM) gene superfamily that function in the regulation of photoreceptor guanylate cyclases (GCs). In the mammalian retina, two GCAPs (GCAP 1-2) and two transmembrane GCs have been identified as part of a complex regulatory system responsive to fluctuating levels of free Ca(2+). A third GCAP, GCAP3, is expressed in human and zebrafish (Danio rerio) retinas, and a guanylate cyclase-inhibitory protein (GCIP) has been shown to be present in frog cones. To explore the diversity of GCAPs in more detail, we searched the pufferfish (Fugu rubripes) and zebrafish (Danio rerio) genomes for GCAP-related gene sequences (fuGCAPs and zGCAPs, respectively) and found that at least five additional GCAPs (GCAP4-8) are predicted to be present in these species. We identified genomic contigs encoding fuGCAPl-8, fuGCIP, zGCAPl-5, zGCAP7 and zGCIP. We describe cloning, expression and localization of three novel GCAPs present in the zebrafish retina (zGCAP4, zGCAP5, and zGCAP7). The results show that recombinant zGCAP4 stimulated bovine rod outer segment GC in a Ca(2+)-dependent manner. RT-PCR with zGCAP specific primers showed specific expression of zGCAPs and zGCIP in the retina, while zGCAPl mRNA is also present in the brain. In situ hybridization with anti-sense zGCAP4, zGCAP5 and zGCAP7 RNA showed exclusive expression in zebrafish cone photoreceptors. The presence of at least eight GCAP genes suggests an unexpected diversity within this subfamily of Ca(2+)-binding proteins in the teleost retina, and suggests additional functions for GCAPs apart from stimulation of GC. Based on genome searches and EST analyses, the mouse and human genomes do not harbor GCAP4-8 or GCIP genes.

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.

Guanylyl cyclase-activating proteins (GCAP) are EF-hand Ca(2+)-binding proteins that activate photoreceptor guanylyl cyclase (RetGC) in the absence of Ca(2+) and inhibit RetGC in a Ca(2+)-sensitive manner. The reported data for the RetGC inhibition by Ca(2+)/GCAPs in vitro are in disagreement with the free Ca(2+) levels found in mammalian photoreceptors (Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., and Fain, G. L. (2002) J. Physiol. (Lond.) 542, 843-854). We have found that binding of Mg(2+) dramatically affects both Ca(2+)-dependent conformational changes in GCAP-1 and Ca(2+) sensitivity of RetGC regulation by GCAP-1 and GCAP-2. Lowering free Mg(2+) concentrations ([Mg](f)) from 5.0 mm to 0.5 mm decreases the free Ca(2+) concentration required for half-maximal inhibition of RetGC ([Ca]((1/2))) by recombinant GCAP-1 and GCAP-2 from 1.3 and 0.2 microm to 0.16 and 0.03 microm, respectively. A similar effect of Mg(2+) on Ca(2+) sensitivity of RetGC by endogenous GCAPs was observed in mouse retina. Analysis of the [Ca]((1/2)) changes as a function of [Mg](f) in mouse retina shows that the [Ca]((1/2)) becomes consistent with the range of 23-250 nm free Ca(2+) found in mouse photoreceptors only if the [Mg](f) in the photoreceptors is near 1 mm. Our data demonstrate that GCAPs are Ca(2+)/Mg(2+) sensor proteins. While Ca(2+) binding is essential for cyclase activation and inhibition, Mg(2+) binding to GCAPs is critical for setting the actual dynamic range of RetGC regulation by GCAPs at physiological levels of free Ca(2+).

The regulation of cGMP levels is central to the normal process of phototransduction in both cone and rod photoreceptor cells. Two of the proteins involved in this process are the enzyme, retinal guanylate cyclase (retGC), and its activating protein (GCAP) through which activity is regulated via changes in cellular Ca2+ levels. Dominant cone-rod dystrophies arising from changes in retGC1 are essentially restricted to mutations in codon 838 and result in the replacement of a conserved arginine residue with either cysteine, histidine or serine. In all three cases, the effect of the substitution on the in vitro cyclase activity is a loss of Ca2+ sensitivity arising from an increased stability of the coiled-coil domain of the protein dimer and retention of cyclase activity. In contrast, mutations in the Ca2+-coordinating EF hands of GCAP1 result in dominant cone dystrophy; the consequences of these mutations is a reduced ability of the mutant protein to regulate retGC activity in response to changes in Ca2+ levels. Functionally therefore, the retGC2 and GCAP2 mutations are similar in reducing the feedback inhibition of Ca2+ on cyclase activity and thereby on cGMP levels in the photoreceptors.

It has been believed that retinal guanylyl cyclase (retGC), a key enzyme in the cGMP recovery to the dark state, is solely activated by guanylyl cyclase-activating proteins (GCAPs) in a Ca2+-sensitive manner. However, a question has arisen as to whether the observed GCAP stimulation of retGC is sufficient to account for the cGMP recovery because the stimulated activity measured in vitro is less than the light/GTP-activated cGMP phosphodiesterase activity. Here we report that the retGC activation by GCAPs is larger than previously reported and that a preincubation with adenine nucleotide is essential for the large activation. Under certain conditions, ATP is two times more effective than adenylyl imidodiphosphate (AMP-PNP), a hydrolysis-resistant ATP analog; however, this study mainly used AMP-PNP to focus on the role of adenine nucleotide binding to retGC. When photoreceptor outer segment homogenates are preincubated with AMP-PNP (EC50 = 0.65 +/- 0.20 mM), GCAP2 enhanced the retGC activity 10-13 times over the control rate. Without AMP-PNP, GCAP2 stimulated the control activity only 3-4-fold as in previous reports. The large activation is due to a GCAP2-dependent increase in Vmax without an alteration of retGC affinity for GCAP2 (EC50 = 47.9 +/- 2.7 nM). GCAP1 stimulated retGC activity in a similar fashion but with lower affinity (EC50 = 308 nM). In the AMP-PNP preincubation, low Ca2+ concentrations are not required, and retGC exists as a monomeric form. This large activation is accomplished through enhanced action of GCAPs as shown by Ca2+ inhibition of the activity (IC50 = 178 nM). We propose that retGC is activated by a two-step mechanism: a conformational change by ATP binding to its kinase homology domain under high Ca2+ concentrations that allows large enhancement of GCAP activation under low Ca2+ concentrations.

Mediated by guanylate cyclase-activating proteins (GCAPs), cytoplasmic Ca2+ levels regulate the activity of photoreceptor guanylate cyclase (GC) and the synthesis of cGMP, the internal transmitter of phototransduction. When GCAP1 is expressed in transgenic mice on a GCAP null background, it restores the wild-type flash responses in rod photoreceptors. In this communication, we explored the role of GCAP1 in cone photoreceptors by using electroretinograms (ERGs). Under cone isolation conditions, ERGs recorded from mice lacking both GCAP1 and GCAP2 had normal amplitudes of the saturated a-wave and b-wave. However, recordings from these mice demonstrated a widened b-wave and increased sensitivity of both M- and UV-cone systems. Paired-flash ERGs revealed a delayed recovery of both the cone driven b-wave and a-wave and suggest that the delay originated from the photoreceptors. To test whether GCAP1 could restore normal cone response recovery, mice that expressed only transgenic GCAP1 in the absence of wild-type GCAP expression were tested. Immunohistochemical analysis demonstrated that cones of these mice expressed high levels of GCAP1. Paired-flash ERGs showed that the recovery of the cone-driven a-wave was restored to normal, whereas recovery of the cone-driven b-wave was slightly faster than that observed in wild-type mice. These studies reveal that, similar to rods, deletion of GCAP1 and GCAP2 delays the recovery of light responses in cones and GCAP1 restores the recovery of cone responses in the absence of GCAP2.

Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca(2+)-binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca(2+). Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca(2+)-dependent binding of recoverin to Phenyl-Sepharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca(2+)](free) (10 microM) than at low [Ca(2+)](free) (10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca(2+)](free) in recoverin and at low [Ca(2+)](free) in GCAP2. Such different changes of hydrophobicity evoked by Ca(2+) appear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.

Guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins with a fatty acid (mainly myristic acid) that is covalently attached at the N terminus. Myristoylated forms of GCAP were produced in E. coli by coexpression of yeast N-myristoyl-transferase. Proteins with nearly 100% degree of myristoylation were obtained after chromatography on a reversed phase column. Although proteins were denatured by this step, they could be successfully refolded. Nonmyristoylated GCAPs activated bovine photoreceptor guanylate cyclase 1 less efficiently than the myristoylated forms. Maximal activity of guanylate cyclase at low Ca(2+)-concentration decreased about twofold, when GCAPs lacked myristoylation. In addition, the x-fold activation of cyclase was lower with nonmyristoylated GCAPs. Myristoylation of GCAP-2 had no influence on the apparent affinity for photoreceptor guanylate cyclase 1, but GCAP-1 has an about sevenfold higher affinity for cyclase, when it is myristoylated. We conclude that myristoylation of GCAP-1 and GCAP-2 is important for fine tuning of guanylate cyclase activity.

In visual transduction, guanylate cyclase-activating proteins (GCAPs) activate the membrane-bound guanylate cyclase 1 (ROS-GC1) to synthesize cGMP under conditions of low cytoplasmic [Ca2+]free. GCAPs are neuronal Ca2+-binding proteins with three functional EF-hands and a consensus site for N-terminal myristoylation. GCAP-1 and GCAP-2 regulated ROS-GC1 activities differently. The myristoyl group in GCAP-1 had a strong influence on the Ca2+-dependent regulation of ROS-GC1 (shift in IC50). In contrast, myristoylation of GCAP-2 did not change the cyclase activation profile (no shift in IC50). Thus, the myristoyl group controlled the Ca2+-sensitivity of GCAP-1, but not that of GCAP-2. The myristoyl group restricted the accessibility of one cysteine in GCAP-1 and GCAP-2 observed by measuring the time-dependent thiol reactivity of cysteines. This shielding effect was not relieved when Ca2+ was buffered by EGTA. We applied surface plasmon resonance (SPR) spectroscopy to monitor the Ca2+-dependent binding of myristoylated and nonmyristoylated GCAP-1 and GCAP-2 to immobilized phospholipid membranes. None of the GCAPs exhibited a Ca2+-myristoyl switch as observed for recoverin. Thus, the myristoyl group controls the Ca2+-sensitivity of GCAP-1 (not that of GCAP-2) by an allosteric mechanism, but this control step does not involve a myristoyl switch.

Single photon responses were compared in wild-type and transgenic retinal rods with and without guanylate cyclase activating protein (GCAP) to disrupt Ca(2+)-dependent feedback regulation of guanylate cyclase (see Burns et al. in this issue of Neuron). The results provided new insights into the molecular mechanisms underlying phototransduction.

In retinal rods, Ca(2+) exerts negative feedback control on cGMP synthesis by guanylate cyclase (GC). This feedback loop was disrupted in mouse rods lacking guanylate cyclase activating proteins GCAP1 and GCAP2 (GCAPs(-/-)). Comparison of the behavior of wild-type and GCAPs(-/-) rods allowed us to investigate the role of the feedback loop in normal rod function. We have found that regulation of GC is apparently the only Ca(2+) feedback loop operating during the single photon response. Analysis of the rods' light responses and cellular dark noise suggests that GC normally responds to light-driven changes in [Ca(2+)] rapidly and highly cooperatively. Rapid feedback to GC speeds the rod's temporal responsiveness and improves its signal-to-noise ratio by minimizing fluctuations in cGMP.

cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system.

Purpose: To determine the activity, cell specificity, and developmental expression profiles of fragments of the chicken guanylate cyclase activating protein (GCAP)-1 promoter., Methods: The intrinsic activities of five GCAP1 promoter-luciferase constructs were measured in transiently transfected primary chicken embryonic retinal cultures. Lentivirus vectors carrying GCAP1 promoter-nlacZ transgenes were used to examine the cell specificities and temporal expression characteristics of selected promoter fragments in developing retina., Results: Three of the five GCAP1 promoter fragments exhibited significant activity in vitro. The expression characteristics of the promoter fragments in vivo varied as a function of promoter length. Expression of nlacZ driven by the 0.6- and 1.7-kb GCAP1 promoter fragments was first observed on embryonic day (E)12 and was restricted to the inner nuclear layer (INL). By E16, nlacZ staining was also detected in the outer nuclear layer (ONL). Expression of nlacZ driven by the 4.2-kb GCAP1 promoter fragment was not observed until E16 and was restricted to the ONL., Conclusions: The general organization of regulatory cis elements within the GCAP1 promoter is different from other photoreceptor-specific gene promoters. Elements located within 0.3 kb upstream of the transcription start point are capable of producing efficient gene expression; however, additional elements located within 4.0 kb upstream of the transcription start point are necessary to confer on the fragment the cell specificity and developmental expression characteristics of the native GCAP1 promoter. The results of the current study show that the lentiviral vector system is a useful tool for the characterization of the promoters of genes expressed in neural retina.

Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca(2+) and cGMP. Ca(2+) regulates the activity of guanylate cyclase (GC) and the synthesis of cGMP by acting on a GC-activating protein (GCAP). While this action is critical for rapid termination of the light response, the GCAP responsible has not been identified. To test if GCAP1, one of two GCAPs present in mouse rods, supports the generation of normal flash responses, transgenic mice were generated that express only GCAP1 under the control of the endogenous promoter. Paired flash responses revealed a correlation between the degree of recovery of the rod a-wave and expression levels of GCAP1. In single cell recordings, the majority of the rods generated flash responses that were indistinguishable from wild type. These results demonstrate that GCAP1 at near normal levels supports the generation of wild-type flash responses in the absence of GCAP2.

Among single-spanning transmembrane receptors (sTMRs), two guanylyl cyclase receptors, GC1 and GC2, are critically important during phototransduction in vertebrate retinal photoreceptor cells. Ca(2+)-free forms of guanylyl cyclase-activating proteins (GCAPs) stimulate GCs intracellularly by a molecular mechanism that is not fully understood. To gain further insight into the mechanism of activation and specificity among these proteins, for the first time, several soluble and active truncated GCs and fusion proteins between intracellular domains of GCs and full-length GCAPs were generated. The GC activity of myristoylated GCAP--(437-1054)GC displayed typical [Ca(2+)] dependence, and was further enhanced by ATP and inhibited by guanylyl cyclase inhibitor protein (GCIP). The myristoyl group of GCAP1 appeared to be critical for the inhibition of GCs at high [Ca(2+)], even without membranes. In contrast, calmodulin (CaM)--(437-1054)GC1 fusion protein was inactive, but could be stimulated by exogenous GCAP1. In a series of experiments, we showed that the activation of GCs by linked GCAPs involved intra- and intermolecular mechanisms. The catalytically productive GCAP1--(437-1054)GC1 complex can dissociate, allowing binding and stimulation of the GC1 fusion protein by free GCAP1. This suggests that the intramolecular interactions within the fusion protein have low affinity and are mimicking the native system. We present evidence that the mechanism of GC activation by GCAPs involves a dimeric form of GCs, involves direct interaction between GCs and GCAPs, and does not require membrane components. Thus, fusion proteins may provide an important advance for further structural studies of photoreceptor GCs and other sTMRs with and without different forms of regulatory proteins.

Recoverin and the guanylate cyclase activating proteins (GCAPs) are calcium-sensing proteins in retinal rod and cone cells that belong to the EF-hand superfamily and serve as important calcium sensors in vision. Recoverin and the OCAP proteins are myristoylated at their amino-terminus and are targeted to retinal disc membranes by a myristoyl switch. Here, we present the three-dimensional, atomic-resolution structures of recombinant myristoylated recoverin containing 0, 1 and 2 calcium ions (Ca2+) bound and unmyristoylated GCAP-2 with 3 Ca2+ bound as determined by nuclear magnetic resonance. The Ca2+-induced structural changes in these proteins are important for elucidating their membrane-targeting mechanisms and for understanding the molecular mechanism of Ca2+-sensitive regulation of phototransduction.

Cyclic GMP is essential for the ability of rods and cones to respond to the light stimuli. Light triggers hydrolysis of cGMP and stops the influx of sodium and calcium through the cGMP-gated ion channels. The consequence of this event is 2-fold: first, the decrease in the inward sodium current plays the major role in an abrupt hyperpolarization of the cellular membrane; secondly, the decrease in the Ca2+ influx diminishes the free intracellular Ca2+ concentration. While the former constitutes the essence of the phototransduction pathway in rods and cones, the latter gives rise to a potent feedback mechanism that accelerates photoreceptor recovery and adaptation to background light. One of the most important events by which Ca2+ feedback controls recovery and light adaptation is synthesis of cGMP by guanylyl cyclase. Two isozymes of membrane photoreceptor guanylyl cyclase (retGC) have been identified in rods and cones that are regulated by Ca2+-binding proteins, GCAPs. At low intracellular concentrations of Ca2+ typical for light-adapted rods and cones GCAPs activate RetGC, but concentrations above 500 nM typical for dark-adapted photoreceptors turn them into inhibitors of retGC. A variety of mutations found in GCAP and retGC genes have been linked to several forms of human congenital retinal diseases, such as dominant cone degeneration, cone-rod dystrophy and Leber congenital amaurosis.

Lowered concentration of Ca2+ ions, resulting from illumination of the photoreceptor cell, is the signal for resynthesis of cGMP by retina-specific guanylyl cyclase (retGC). This Ca2+-dependent activation of retGC is mediated by Ca2+-binding proteins named GCAPs (guanylyl cyclase-activating proteins) and contributes to the recovery of photoreceptor cell to the dark state. Three different GCAPs (GCAP1, GCAP2 and GCAP3) are identified in vertebrate retina to date. In this chapter we describe their discovery, methods of purification, properties, and possible modes of action.

In photoreceptor cells cGMP is the second messenger that transduces light into an electrical response. Regulation of cGMP synthesis by Ca2+ is one of the key mechanisms by which Ca2+ exerts negative feedback to the phototransduction cascade in the process of light adaptation. This Ca2+ feedback to retinal guanylyl cyclases (Ret-GCs) is conferred by the guanylate cyclase-activating proteins (GCAPs). Mutations in GCAP1 that disrupt the Ca2+ regulation of Ret-GCs in vitro have been associated with severe human vision disorders. This chapter focuses on recent data obtained from biochemical and electrophysiological studies of GCAP1/GCAP2 knockout mice and other GCAP transgenic mice, addressing: 1. the quantitative aspects of the Ca2+-feedback to Ret-GCs in regulating the light sensitivity and adaptation in intact rods; 2. functional differences between GCAP1 and GCAP2 in intact rod photoreceptors; and 3. whether GCAP mutants with impaired Ca2+ binding lead to retinal disease in vivo by constitutive activation of Ret-GCs and elevation of intracellular cGMP, as predicted from in vitro studies.

Calmodulin-like neuronal Ca2+-binding proteins (NCBPs) are expressed primarily in neurons and contain a combination of four functional and nonfunctional EF-hand Ca2+-binding motifs. The guanylate cyclase-activating proteins 1-3 (GCAP1-3), the best characterized subgroup of NCBPs, function in the regulation of transmembrane guanylate cyclases 1-2 (GC1-2). The pairing of GCAPs and GCs in vivo depends on cell expression. Therefore, we investigated the expression of these genes in retina using in situ hybridization and immunocytochemistry. Our results demonstrate that GCAP1, GCAP2, GC1 and GC2 are expressed in human rod and cone photoreceptors, while GCAP3 is expressed exclusively in cones. As a consequence of extensive modification, the GCAP3 gene is not expressed in mouse retina. However, this lack of evolutionary conservation appears to be restricted to only some species as we cloned all three GCAPs from teleost (zebrafish) retina and localized them to rod cells, short single cones (GCAP1-2), and all subtypes of cones (GCAP3). Furthermore, sequence comparisons and evolutionary trace analysis coupled with functional testing of the different GCAPs allowed us to identify the key conserved residues that are critical for GCAP structure and function, and to define class-specific residues for the NCBP subfamilies.

Guanylyl cyclase activating proteins (GCAPs) are Ca2+-binding proteins of the EF-hand superfamily, through which the intracellular calcium regulates cGMP synthesis in vertebrate photoreceptors. GCAPs play an essential role in the calcium feedback mechanism that controls recovery and light adaptation of rods and cones. Moreover, mutations in at least one of the GCAPs have already been linked to two forms of congenital human retinal diseases. The GCAPs represent a separate small subfamily among the EF-hand proteins that are structurally similar to recoverin, but demonstrate a number of unique regulatory properties. When in the Ca2+-free conformation (as in light-adapted photoreceptors), GCAPs stimulate photoreceptor membrane guanylyl cyclase (retGC), but when the intracellular free Ca2+ concentrations ([Ca2+]free) rise (as in dark-adapted photoreceptors), GCAPs turn into retGC inhibitors. In GCAPs, site-directed mutagenesis has been successfully used to identify a number of structural elements that contribute to their specific function as guanylyl cyclase regulators. These elements include EF-hand Ca2+-binding loops and various other regions in the GCAP primary structure involved in multiple protein-protein interactions within the retGC/GCAP complex.

The Ca(2+)-dependent activation of retina-specific guanylyl cyclase (retGC) is mediated by guanylyl cyclase-activating proteins (GCAPs). Here we report for the first time detection of a 19 kDa protein (p19) with GCAP properties in extracts of rat retina and pineal gland. Both extracts stimulate synthesis of cGMP in rod outer segment (ROS) membranes at low (30 nM) but not at high (1 microM) concentrations of Ca(2+). At low Ca(2+), immunoaffinity purified p19 activates guanylyl cyclase(s) in bovine ROS and rat retinal membranes. Moreover, p19 is recognized by antibodies against bovine GCAP1 and, similarly to other GCAPs, exhibits a Ca(2+)-dependent electrophoretic mobility shift.

Guanylate cyclase-activating proteins (GCAPs) control the activity of membrane bound guanylate cyclases in vertebrate photoreceptor cells. They form a permanent complex with guanylate cyclase 1 (ROS-GC1) at low and high Ca2+-concentrations. Five different target regions of GCAP-1 have been identified in ROS-GC1 at rather distant sites. These findings could indicate a multipoint attachment site for GCAP-1 or, alternatively, the presence of transient binding sites with short contact to GCAP-1. In addition some data are consistent with the operation of one or more transducer units, that represent regulatory regions without being direct binding sites. A permanent ROS-GC1/GCAP-1 complex is physiologically significant, since it allows a very short response time of cyclase activity when the intracellular Ca2+-concentration changes. Thereby, activation of cyclase participates in speeding up the recovery of the photoresponse after illumination and restores the circulating dark current.

A range of cone and cone-rod dystrophies (CORD) have been observed in man, caused by mutations in retinal guanylate cyclase 1 (RetGC1) and guanylate cyclase activating protein 1 (GCAP 1). The CORD causing mutations in RetGC1 are located at a mutation "hot spot" within the dimerisation domain, where R838 is the key residue. Three disease causing mutations have been found in human GCAP1, resulting in cone or cone-rod degeneration. All three mutations are dominant in their effect although the mechanism by which the P50L mutation exerts its influence remains unclear although it might act due to a haplo-insufficiency, arising from increased susceptibility to protease activity and increased thermal instability. In contrast, loss of Ca2+ sensitivity appears to be the main cause of the diseased state for the Y99C and E155G mutations. The cone and cone-rod dystrophies that are caused by mutations in RetGC1 or GCAP1 arise from a perturbation of the delicate balance of Ca2+ and cGMP within the photoreceptor cells and it is this disruption that is believed to cause cell death. The diseases caused by mutations in RetGC1 and GCAP1 prominently affect cones, consistent with the higher concentrations of these proteins in cone cells.

Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca(2+)-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca(2+)-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca(2+) for the ability to interact with the target enzyme.

Guanylate cyclase-activating protein 1 (GCAP-1) is a Ca(2+)-sensing protein in vertebrate photoreceptor cells. It activates a membrane-bound guanylate cyclase. Three of four cysteines present in wild-type GCAP-1 were accessible to the thiol-modifying reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) in the presence of Ca(2+). Only Cys106 became exposed to the solvent after Ca(2+)-chelation. Since Cys106 is located in EF-hand 3, we could determine an apparent K(D) of 2.9 microM for Ca(2+) binding to this site with a fast off-rate (t approximately 2 ms). We conclude that the rapid dissociation of Ca(2+) from EF-hand 3 in GCAP-1 triggers activation of guanylate cyclase in rod cells.

Guanylyl cyclase-activating proteins are EF-hand Ca(2+)-binding proteins that belong to the calmodulin superfamily. They are involved in the regulation of photoreceptor membrane-associated guanylyl cyclases that produce cGMP, a second messenger of vertebrate vision. Here, we investigated changes in GCAP1 structure using mutagenesis, chemical modifications, and spectroscopic methods. Two Cys residues of GCAP1 situated in spatially distinct regions of the N-terminal domain (positions 18 and 29) and two Cys residues located within the C-terminal lobe (positions 106 and 125) were employed to detect conformational changes upon Ca(2+) binding. GCAP1 mutants with only a single Cys residue at each of these positions, modified with N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, an environmentally sensitive fluorophore, and with (1-oxy-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate, a spin label reagent, were studied using fluorescence and EPR spectroscopy, respectively. Only minor structural changes around Cys(18), Cys(29), Cys(106), and Cys(125) were observed as a function of Ca(2+) concentration. No Ca(2+)-dependent oligomerization of GCAP1 was observed at physiologically relevant Ca(2+) concentrations, in contrast to the observation reported by others for GCAP2. Based on these results and previous studies, we propose a photoreceptor activation model that assumes changes within the flexible central helix upon Ca(2+) dissociation, causing relative reorientation of two structural domains containing a pair of EF-hand motifs and thus switching its partner, guanylyl cyclase, from an inactive (or low activity) to an active conformation.

Objective: To describe the phenotype in 4 families with dominantly inherited cone-rod dystrophy, 1 with an R838C mutation and 1 with an R838H mutation in the guanylate cyclase 2D (GUCY2D) gene encoding retinal guanylate cyclase-1., Methods: Psychophysical and electrophysiological evaluation and confocal laser scanning ophthalmoscopic imaging was performed on 10 affected members of 4 British families., Results: Although subjects had lifelong poor vision in bright light, a major reduction in visual acuity did not occur in most of them until after their late teens. Fundus abnormalities were confined to the central macula, and increasing central atrophy was noted with age. Increased background autofluorescence was observed surrounding the central atrophic area. Electrophysiological testing revealed a marked loss of cone function with only minimal rod involvement, even in older subjects. Photopic and scotopic static perimetry demonstrated central and peripheral cone-mediated threshold elevations with midperipheral sparing., Conclusion: The phenotype associated with autosomal dominant cone-rod dystrophy with either an R838C or R838H mutation in GUCY2D is distinctive, with predominantly cone system involvement. There is some variation in severity within the 3 families with the R838C mutation., Clinical Relevance: Families with the R838C or R838H mutation have a much milder phenotype than the family previously described that had 2 sequence changes, E837D and R838S, in GUCY2D.

Mutations in the gene for guanylate cyclase-activating protein-1 (GCAP1) (GUCA1A) have been associated with autosomal dominant cone dystrophy (COD3). In the present study, a severe disease phenotype in a large white family was initially shown to map to chromosome 6p21.1, the location of GUCA1A. Subsequent single-stranded conformation polymorphism analysis and direct sequencing revealed an A464G transition, causing an E155G substitution within the EF4 domain of GCAP1. Modeling of the protein structure shows that the mutation eliminates a bidentate amino acid side chain essential for Ca2+ binding. This represents the first disease-associated mutation in GCAP1, or any neuron-specific calcium-binding protein within an EF-hand domain, that directly coordinates Ca2+. The functional consequences of this substitution were investigated in an in vitro assay of retinal guanylate cyclase activation. The mutant protein activates the cyclase at low Ca2+ concentrations but fails to inactivate at high Ca2+ concentrations. The overall effect of this would be the constitutive activation of guanylate cyclase in photoreceptors, even at the high Ca2+ concentrations of the dark-adapted state, which may explain the dominant disease phenotype.

A key challenge in studying protein/protein interactions is to accurately identify contact surfaces, i.e. regions of two proteins that are in direct physical contact. Aside from x-ray crystallography and NMR spectroscopy few methods are available that address this problem. Although x-ray crystallography often provides detailed information about contact surfaces, it is limited to situations when a co-crystal of proteins is available. NMR circumvents this requirement but is limited to small protein complexes. Other methods, for instance protection from proteolysis, are less direct and therefore less informative. Here we describe a new method that identifies candidate contact surfaces in protein complexes. The complexes are first stabilized by cross-linking. They are then digested with a protease, and the cross-linked fragments are analyzed by mass spectrometry. We applied this method, referred to as COSUMAS (contact surfaces by mass spectrometry), to two proteins, retinal guanylyl cyclase 1 (RetGC1) and guanylyl cyclase-activating protein-1 (GCAP-1), that regulate cGMP synthesis in photoreceptors. Two regions in GCAP-1 and three in RetGC1 were identified as possible contact sites. The two regions of RetGC1 that are in the vicinities of Cys(741) and Cys(780) map to a kinase homology domain in RetGC1. Their identities as contact sites were independently evaluated by peptide inhibition analysis. Peptides with sequences from these regions block GCAP-1-mediated regulation of guanylyl cyclase at both high and low Ca2+ concentrations. The two regions of GCAP-1 cross-linked to these peptides were in the vicinities of Cys(17) and Cys(105) of GCAP-1. Peptides with sequences derived from these regions inhibit guanylyl cyclase activity directly. These results support a model in which GCAP-1 binds constitutively to RetGC1 and regulates cyclase activity by structural changes caused by the binding or dissociation of Ca2+.

The retina's photoreceptor cells adjust their sensitivity to allow photons to be transduced over a wide range of light intensities. One mechanism thought to participate in sensitivity adjustments is Ca(2+) regulation of guanylate cyclase (GC) by guanylate cyclase-activating proteins (GCAPs). We evaluated the contribution of GCAPs to sensitivity regulation in rods by disrupting their expression in transgenic mice. The GC activity from GCAPs-/- retinas showed no Ca(2+) dependence, indicating that Ca(2+) regulation of GCs had indeed been abolished. Flash responses from dark-adapted GCAPs-/- rods were larger and slower than responses from wild-type rods. In addition, the incremental flash sensitivity of GCAPs-/- rods failed to be maintained at wild-type levels in bright steady light. GCAP2 expressed in GCAPs-/- rods restored maximal light-induced GC activity but did not restore normal flash response kinetics. We conclude that GCAPs strongly regulate GC activity in mouse rods, decreasing the flash sensitivity in darkness and increasing the incremental flash sensitivity in bright steady light, thereby extending the rod's operating range.

Guanylate cyclase-activating protein 1 (GCAP1) and guanylate cyclase-inhibitory protein (GCIP) are calmodulin-related Ca2+-binding proteins expressed in vertebrate photoreceptor cells. GCAP1 activates photoreceptor guanylate cyclase 1 (GC1) at low free [Ca2+] (<50 nM, in the light), but inhibits it at physiological high [Ca2+] (1 microM, in the dark). GCIP, a Ca2+-binding protein from frog retina, inhibits GC1 at approximately 1 microM [Ca2+], but is unable to stimulate cyclase at low [Ca2+]. In this study, we probed the interaction between GCAP1 and GC1 by producing GCAP1/GCIP chimeras and tested their capability to stimulate GC1. We prepared eight pairs of constructs in which the N-terminal portions of GCIP and GCAP1 were successively replaced by corresponding domains of GCAP1, and GCIP, respectively. The expressed proteins were purified and tested for stimulation of GC1 at 50 nM [Ca2+], and their ability to competitively inhibit GC1 stimulation by a Ca2+-insensitive GCAP1 mutant, GCAP1-tm, at high [Ca2+]. While all GCAP1/GCIP chimeras competitively inhibited GC1 stimulation at high [Ca2+] by GCAP1-tm, several of the GCIP/GCAP1 chimeras had no effect. A chimera consisting of residues 1-20 of GCIP and 21-205 of GCAP1 had no effect on GC1 at low [Ca2+], suggesting that the N-terminal region MGNIMDGKSVEELSSTECHQ, which has no sequence similarity to GCIP, is among the key components necessary for GC1 stimulation. A GCAP1/GCIP chimera consisting of residues 1-43 (including nonfunctional EF1) of GCAP1 and residues 56-206 of GCIP stimulated GC1 at low [Ca2+] and inhibited GC1 at high [Ca2+], suggesting that the essential components required to transform an inhibitory to an activating protein are contained within the N-terminal region of GCAP1 (residues 1-43).

Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived observations are reported that help in explaining why a proline-->leucine mutation at position 50 of human GCAP1 results in cone-rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N-terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.

Objective: To describe the phenotype in 3 families with dominantly inherited cone and cone-rod dystrophy with mutations in guanylate cyclase activator 1A (GUCA1A), the gene-encoding guanylate cyclase activator protein-1 (GCAP-1)., Methods: Phenotypic characterization with psychophysical and electrophysiological evaluation and confocal laser scanning ophthalmoscopy was performed in 2 families with a Tyr99Cys mutation and 1 family with a Pro50Leu mutation. Haplotype analysis was performed in the families with Tyr99Cys mutation., Results: The families with a Y99C mutation were shown to be ancestrally related. Decreased visual acuity and loss of color vision occurred after the age of 20 years, followed by progressive atrophy of the central 5 degrees to 10 degrees. Electrophysiological testing revealed generalized loss of cone function, with preservation of rod function. Abnormal rod and cone sensitivities were confined to the central 5 degrees to 10 degrees. Confocal laser scanning ophthalmoscopy imaging showed abnormalities of autofluorescence in early disease. Subjects with a Pro50Leu mutation demonstrated marked variability in expressivity from minimal abnormalities of macular function to cone-rod dystrophy., Conclusions: The phenotype associated with the Y99C mutation in GUCA1A is distinctive, with little variation in expression. By contrast, that associated with the P50L mutation demonstrates variable expressivity., Clinical Relevance: Phenotype-genotype correlation in these 2 mutations demonstrates 2 different phenotypes.

Examination of the role of Ca(2+)-binding proteins (CaBPs) in mammalian retinal neurons has yielded new insights into the function of these proteins in normal and pathological states. In the last 8 years, studies on guanylate cyclase (GC) regulation by three GC-activating proteins (GCAP1-3) led to several breakthroughs, among them the recent biochemical analysis of GCAP1(Y99) mutants associated with autosomal dominant cone dystrophy. Perturbation of Ca(2+) homeostasis controlled by mutant GCAP1 in photoreceptor cells may result ultimately in degeneration of these cells. Here, detailed analysis of biochemical properties of GCAP1(P50L), which causes a milder form of autosomal dominant cone dystrophy than constitutive active Y99C mutation, showed that the P50L mutation resulted in a decrease of Ca(2+)-binding, without changes in the GC activity profile of the mutant GCAP1. In contrast to this biochemically well-defined regulatory mechanism that involves GCAPs, understanding of other processes in the retina that are regulated by Ca(2+) is at a rudimentary stage. Recently, we have identified five homologous genes encoding CaBPs that are expressed in the mammalian retina. Several members of this subfamily are also present in other tissues. In contrast to GCAPs, the function of this subfamily of calmodulin (CaM)-like CaBPs is poorly understood. CaBPs are closely related to CaM and in biochemical assays CaBPs substitute for CaM in stimulation of CaM-dependent kinase II, and calcineurin, a protein phosphatase. These results suggest that CaM-like CaBPs have evolved into diverse subfamilies that control fundamental processes in cells where they are expressed.

The complex sensation of vision begins with the relatively simple photoisomerization of the visual pigment chromophore 11-cis-retinal to its all-trans configuration. This event initiates a series of biochemical reactions that are collectively referred to as phototransduction, which ultimately lead to a change in the electrochemical signaling of the photoreceptor cell. To operate in a wide range of light intensities, however, the phototransduction pathway must allow for adjustments to background light. These take place through physiological adaptation processes that rely primarily on Ca(2+) ions. While Ca(2+) may modulate some activities directly, it is more often the case that Ca(2+)-binding proteins mediate between transient changes in the concentration of Ca(2+) and the adaptation processes that are associated with phototransduction. Recently, combined genetic, physiological, and biochemical analyses have yielded new insights about the properties and functions of many phototransduction-specific components, including some novel Ca(2+)-binding proteins. Understanding these Ca(2+)-binding proteins will provide a more complete picture of visual transduction, including the mechanisms associated with adaptation, and of related degenerative diseases., (Copyright 2000 John Wiley & Sons, Inc.)