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RetGC-1 and RetGC-2 are photoreceptor membrane guanylyl cyclases that are regulated by the Ca2+-binding protein, GCAP-2. We used a protease protection assay to localize regions of the intracellular domains of RetGCs important for the interaction with GCAP-2 and found that GCAP-2 reduces the access of trypsin to a site in the kinase homology domain (KHD) of RetGC-1. The protective effect of GCAP-2 is independent of Ca2+. We also found that RetGC-2 and GCAP-2 interact cooperatively with high affinity, but RetGC-1 and GCAP-2 interact noncooperatively with low affinity. By analyzing RetGC-1/RetGC-2 chimeras we demonstrated that the affinity and cooperativity of the interaction with GCAP-2 is dictated by the structure of the KHD. These findings suggest that GCAP-2 interacts constituitively with the KHDs of RetGC-1 and RetGC-2 and that cGMP synthesis is controlled by Ca2+-dependent conformational changes in the RetGC/GCAP complex.

Photoreceptor membrane guanylate cyclases (RetGC) are regulated by calcium-binding proteins, GCAP-1 and GCAP-2. At Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, guanylate cyclase-activating protein (GCAPs) activate RetGC, and at free Ca2+ concentrations above 500 nM, characteristic of dark-adapted photoreceptors, GCAPs inhibit RetGC. A mutation, Y99C, in human GCAP-1 was recently found to be linked to autosomal dominant cone dystrophy in a British family (Payne, A. M., Downes, S. M., Bessant, D. A. R., Taylor, R., Holder, G. E., Warren, M. J., Bird, A. C., and Bhattachraya, S. S. (1998) Hum. Mol. Genet. 7, 273-277). We produced recombinant Y99C GCAP-1 mutant and tested its ability to activate RetGC in vitro at various free Ca2+ concentrations. The Y99C mutation does not decrease the ability of GCAP-1 to activate RetGC. However, RetGC stimulated by the Y99C GCAP-1 remains active even at Ca2+ concentration above 1 microM. Hence, the cyclase becomes constitutively active within the whole physiologically relevant range of free Ca2+ concentrations. We have also found that the Y99C GCAP-1 can activate RetGC even in the presence of Ca2+-loaded nonmutant GCAPs. This is consistent with the fact that cone degeneration was dominant in human patients who carried such mutation (Payne, A. M., Downes, S. M., Bessant, D. A. R. , Taylor, R., Holder, G. E., Warren, M. J., Bird, A. C., and Bhattachraya, S. S. (1998) Hum. Mol. Genet. 7, 273-277). A similar mutation, Y104C, in GCAP-2 results in a different phenotype. This mutation apparently does not affect Ca2+ sensitivity of GCAP-2. Instead, the Y104C GCAP-2 stimulates RetGC less efficiently than the wild-type GCAP-2. Our data indicate that cone degeneration associated with the Y99C mutation in GCAP-1 can be a result of constitutive activation of cGMP synthesis.

GCAP1 stimulates photoreceptor guanylate cyclase (GC) in bleached vertebrate photoreceptors when [Ca2+]free decreases but is inactivated when cytoplasmic [Ca2+]free increase after dark adaptation. A Y99C mutation in GCAP1 has recently been found to be associated with autosomal dominant cone dystrophy. We show that the GCAP1(Y99C) mutant and native GCAP1 are highly effective in stimulation of photoreceptor GC1. The Ca2+ sensitivity of the mutant GCAP1, however, is markedly altered, causing reduced but persistent stimulation of GC1 under physiological dark conditions. These results are consistent with a model in which enhanced GC activity in dark-adapted cones leads to elevated levels of cytoplasmic cGMP. Alterations in physiological cGMP levels are also associated with other retinal degenerations, including Leber's congenital amaurosis.

Guanylyl cyclase-activating peptide II (GCAP-II), an endogenous ligand of particulate guanylyl cyclase C (GC-C), is processed from the precursor protein and circulates in human blood. GCAP-II consists of 24 amino acid residues and contains two disulfide bridges. The correct disulfide paring of GCAP-II is an absolute requirement for its biological activity. This study shows that the folding of the peptide from the reduced form yields a peptide with the native disulfide paring as a minor product and with non-native ones as major products, regardless of the presence or absence of reduced and oxidized glutathione. The results suggest that GCAP-II does not possess sufficient information to permit the adoption of the native conformation and to effectively form the correct disulfide pairing and, as a result, that GCAP-II is correctly folded by assistance of a factor(s) such as an intra- or intermolecular chaperone. We studied whether a peptide in the pro-leader sequence of the precursor protein (proGCAP-II) contains sufficient information to facilitate the folding of GCAP-II. For this purpose, we prepared proGCAP-II in Escherichia coli by a recombinant technique and examined the disulfide-coupled folding of proGCAP-II from the reduced form. proGCAP-II was quantitatively recovered with the correctly folded structure from the reduced form both in the presence and in the absence of reduced and oxidized glutathione. The protein contains only disulfide linkages at the same positions as the mature form of proGCAP-II, GCAP-II, and the biologically active isomer of GCAP-II in the molecule. These results provide evidence that the propeptide of proGCAP-II is a critical factor in the formation of the correct disulfide paring in the folding of the protein.

Purpose: To explore the distribution of guanylyl cylase-activating proteins 1 and 2 (GCAP1 and GCAP2) in the mammalian retina., Methods: Cryostat and vibratome vertical sections and wholemount retinas from mouse, rat, cat, bovine, monkey, and human eyes were prepared for immunocytochemistry and viewing by light and confocal microscopy., Results: In all mammalian retinas investigated, intense GCAP1 immunoreactivity (GCAP1-IR) was seen in cone photoreceptor inner and outer segments, cell bodies, and synaptic regions. Intensity of the GCAP1-IR was strong in inner segments of rods in all species but weaker in outer segments-particularly so in primates and cats. GCAP2 immunoreactivity (GCAP2-IR) was weak in bovine, mouse, and rat cones but was intense in human and monkey cones. In all species except primates, GCAP2 staining was intense in rod inner and outer segments. In primates GCAP2-IR was intense in the rod inner segment but faint in the rod outer segment. A striking difference from the GCAP1 pattern of immunoreactivity was seen with GCAP2 antibodies as far as the inner retina was concerned. GCAP2-IR was evident in certain populations of bipolar, amacrine, and ganglion cells in all species., Conclusions: GCAP1 and GCAP2, which are involved in Ca2+-dependent stimulation and inhibition of photoreceptor guanylyl cyclase, can be detected in mammalian photoreceptor inner and outer segments, consistent with their physiological function. The occurrence of both GCAPs in the synaptic region of the photoreceptors indicates participation of these proteins in pathways other than regulation of phototransduction. The occurrence of GCAP2 in inner retinal neurons is indicative of second-messenger chemical transduction, possibly in metabotropic glutamate, gamma-aminobutyric acid (GABA) receptor, and nitric oxide-activated neural circuits.

The second messengers cAMP and inositol-1,4,5-triphosphate have been implicated in olfaction in various species. The odorant-induced cGMP response was investigated using cilia preparations and olfactory primary cultures. Odorants cause a delayed and sustained elevation of cGMP. A component of this cGMP response is attributable to the activation of one of two kinetically distinct cilial receptor guanylyl cyclases by calcium and a guanylyl cyclase-activating protein (GCAP). cGMP thus formed serves to augment the cAMP signal in a cGMP-dependent protein kinase (PKG) manner by direct activation of adenylate cyclase. cAMP, in turn, activates cAMP-dependent protein kinase (PKA) to negatively regulate guanylyl cyclase, limiting the cGMP signal. These data demonstrate the existence of a regulatory loop in which cGMP can augment a cAMP signal, and in turn cAMP negatively regulates cGMP production via PKA. Thus, a small, localized, odorant-induced cAMP response may be amplified to modulate downstream transduction enzymes or transcriptional events.

Purpose: To identify gene arrangement, chromosomal localization, and expression pattern of mouse guanylate cyclase activating proteins GCAP1 and GCAP2, retina-specific Ca2+-binding proteins, and photoreceptor guanylate cyclase activators., Methods: The GCAP1 and GCAP2 genes were cloned from genomic libraries and sequenced. The chromosomal localization of the GCAP array was determined using fluorescent in situ hybridization. The expression of GCAP1 and GCAP2 in mouse retinal tissue was determined by immunocytochemistry., Results: In this study, the mouse GCAP1 and GCAP2 gene array, its chromosomal localization, RNA transcripts, and immunolocalization of the gene products were fully characterized. The GCAP tail-to-tail array is located at the D band of chromosome 17. Each gene is transcribed into a single transcript of 0.8 kb (GCAP1) and 2 kb (GCAP2). Immunocytochemistry showed that both GCAP genes are expressed in retinal photoreceptor cells, but GCAP2 was nearly undetectable in cones. GCAP2 was also found in amacrine and ganglion cells of the inner retina. Light-adapted and dark-adapted retinas showed no significant difference in the distribution of the most intense GCAP2 staining within the outer segment and outer plexiform layers., Conclusions: Identical GCAP gene structures and the existence of the tail-to-tail gene array in mouse and human suggest an ancient gene duplication-inversion event preceding mammalian diversification. Identification of both GCAPs in synaptic regions, and of GCAP2 in the inner retina suggest roles of these Ca-binding proteins in addition to regulation of phototransduction.

The ROS-GC subfamily of membrane guanylate cyclases is at present represented by two members: ROS-GC1 and ROS-GC2. A unique functional feature of this subfamily is that it is intracellularly modulated in low Ca2+ concentration by calmodulin-like Ca(2+)-binding proteins termed GCAPs, 1 and 2, and the modulation is consistent with its linkage to phototransduction. The present study shows that: (1) GCAP2 is a specific modulator of ROS-GC2; (2) through systematic remodeling of ROS-GC modules, the study also shows that the modulated domain resides within the amino acid segment 736-1020. This domain is distinct form the corresponding GCAP1-modulated ROS-GC1 domain. Thus, GCAP1 and GCAP2 act through different ROS-GCs and through two different cyclase domains.

Two guanylate-cyclase-activating proteins (GCAP) encoded by a tail-to-tail gene array have been characterized in the mammalian retina. Using frog retina as a model, we obtained evidence for the presence of a photoreceptor Ca2+-binding protein closely related to GCAP. This protein (206 amino acids) does not stimulate guanylate cyclase (GC) in low [Ca2+], but inhibits GC in high [Ca2+], and is therefore termed guanylate-cyclase-inhibitory protein (GCIP). Sequence analysis indicates that GCIP and GCAP1 and GCAP2 have diverged substantially, but conserved domains present in all vertebrate GCAP are present in GCIP. Moreover, partial characterization of the GCIP gene showed that the positions of two introns in the GCIP gene are identical to positions of corresponding introns of the mammalian GCAP gene array. As to the major differences between GCIP and GCAP, the fourth EF hand Ca2+-binding motif of GCIP is disabled for Ca2+ binding, and GCIP does not stimulate GC. Monoclonal and polyclonal antibodies raised against recombinant GCIP identified high levels of GCIP in the inner segments, somata and synaptic terminals of frog cone photoreceptors. The results suggest that GCIP is a Ca2+-binding protein of the GCAP/recoverin subfamily. Its localization in frog cones closely resembles that of GC in mammalian cones. GCIP inhibits GC at high free [Ca2+], competing with GCAP1 and GCAP2 for GC regulatory sites.

The retinas of the retinal degeneration (rd) chicken are fully developed and possess normal morphology at hatching but fail to respond to light stimulation. Analyses of retinal cGMP, the internal messenger of phototransduction, show that the amount of cGMP in predegenerate, fully developed rd/rd photoreceptors is 5-10 times less than that seen in normal photoreceptor cells. We show that the low levels of cGMP in rd chicken retina are a consequence of a null mutation in the photoreceptor guanylate cyclase (GC1) gene. Thus, the rd chicken is a model for human Leber's congenital amaurosis. Absence of GC1 in rd retina prevents phototransduction and affects survival of rods and cones but does not interfere with normal photoreceptor development.

We report a mutation (Y99C) in guanylate cyclase activator 1A (GUCA1A), the gene for guanylate cyclase activating protein (GCAP1), in a family with autosomal dominant cone dystrophy. Linkage analysis excluded all the known cone and cone-rod dystrophy loci, except the chromosome 6p21.1 region. This is known to contain the RDS gene, which is associated with dominant cone-rod dystrophy. Screening of the RDS gene by heteroduplex analysis and direct sequencing failed to demonstrate sequence changes in the coding region of this gene. The gene for GCAP1, a calcium binding protein which is highly expressed in photoreceptor outer segments, is also located in 6p21.1. It was screened for mutations, and all affected individuals showed a single base pair missense mutation (A-->G) at codon 99 in exon 2 of this gene generating a tyrosine-to-cysteine change in the GCAP1 protein. This change was absent from 206 unrelated normal controls. We propose that this change would at least disrupt the EF3handof GCAP1 thereby preventing calcium binding and consequently interfere with activation. The resulting effect on cGMP production would predictably modify the number of open cGMP gated cation channels, and could explain the ultimate demise of cone photoreceptor cells.

Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca2+-binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca2+-binding proteins from the calmodulin superfamily, the Ca2+-free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca2+-sensitive modulation of GC1 activity. The change from a Ca2+-bound to a Ca2+-free form of GCAP1 increased susceptibility of Ca2+-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca2+-bound form, only the N-terminus (myristoylated Gly2-Lys9) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca2+-free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca2+-dependent inactivation of GCAP1. GCAP1(E75D,E111D,E155D) mutant did not bind Ca2+ and stimulated GC1 in a [Ca2+]-independent manner. GCAP1 and GCAP2, but not S-100beta, a high [Ca2+]free activator of GC1, competed with the triple mutant at high [Ca2+]free, inhibiting GC1 with similar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1.

The ROS-GC is one of the two subfamilies of membrane guanylate cyclases. It distinguishes itself from the other surface receptor subfamily in that its members are not regulated by extracellular peptides; instead, they are modulated by intracellular Ca2+ signals. There are two members of the subfamily, ROS-GC1 and ROS-GC2. An intriguing feature of ROS-GC1 is that it has two Ca2+ switches. One switch inhibits the enzyme at micromolar concentrations of Ca2+, and the other stimulates. The inhibitory switch is linked to phototransduction, and it is likely that the stimulatory switch is linked to retinal synaptic activity. Ca2+ acts indirectly via Ca(2+)-binding proteins, GCAPs and CD-GCAP. GCAPs modulate the inhibitory switching component of the cyclase and CD-GCAP turns on the activation signaling switch. The activating switch of ROS-GC2 has not so far been scrutinized. The present study shows that CD-GCAP is linked to the activation signaling switch of ROS-GC2, but the linkage is about 10-fold weaker than that of the ROS-GC1. Thus, CD-GCAP is a specific ROS-GC1 activator. Furthermore, through a series of expression studies on the mutants involving deletion, building of hybrids, and reconstruction of a heterologous cyclase, the study confirms that the CD-GCAP regulated switch resides within the amino acid segment 736-1053 of the cyclase.

Regulation of phototransduction in photoreceptor cells occurs via several feedback loops. Some of these circuits involve the neuron-specific Ca2+-binding proteins recoverin and guanylyl cyclase-activating protein. Recent findings suggest that these proteins are also involved in retinal diseases. Another Ca2+-regulated process in the retina is the synthesis of nitric oxide, a substance of potential neurotoxicity. This review discusses several Ca2+-mediated processes in the retina.

Calcium-dependent guanylate cyclase activator protein (CD-GCAP) is a low-molecular-weight retinal calcium-binding protein which activates rod outer segment guanylate cyclase (ROS-GC) in a calcium-dependent manner. This investigation was undertaken to determine the protein's structure and identity. Partial amino acid sequencing (72% of the protein), mass spectral analysis, cloning, and immunological studies revealed that CD-GCAP is identical to S100beta, another low-molecular-weight calcium-binding protein whose structure was known. We had shown earlier that the latter protein, which is usually called S100b (S100betabeta or dimer of S100beta), also activates ROS-GC but that the Vmax of activated cyclase was about 50% lower than when stimulated by CD-GCAP. S100b also required about 15 times more calcium (3.2 x 10(-)5 vs 1.5 x 10(-)6 M) for half-maximal stimulation of cyclase. To investigate the possibility that CD-GCAP is a post-translationally modified form of S100b, both proteins were treated with 1 M hydroxylamine which is known to deacylate proteins. After the treatment, CD-GCAP did not activate cyclase while S100b activation remained unaffected suggesting that CD-GCAP could not be a modified form of S100b. Hydroxylamine also broke down CD-GCAP into smaller fragments while leaving S100b intact. It therefore appeared that in spite of identical primary structures, the conformations of the two proteins were different. We then investigated the possibility that the purification procedures of the two proteins, which were quite different, could have contributed to such conformational differences: CD-GCAP purification included a step of heating at 75 degrees C in 5 mM Ca, while S100b purification included zinc affinity chromatography. To test the influence of these treatments on the properties of the proteins, CD-GCAP was subjected to zinc affinity chromatography and purified as S100b (CD-GCAP-->S100b) and S100b was heated in Ca and purified as CD-GCAP (S100b-->CD-GCAP). Cyclase activation, calcium-sensitivity, and hydroxylamine-lability measurements revealed that CD-GCAP-->S100b is identical to S100b and that S100b-->CD-GCAP is identical to CD-GCAP. Taken together the results demonstrate that CD-GCAP and S100b are one and the same protein and that their functional differences are due to different interconvertible conformational states.

In photoreceptor outer segments, particulate guanylyl cyclase (RetGC) is stimulated at low intracellular Ca2+ concentrations by guanylyl cyclase activating protein (GCAP), a Ca2+-sensitive activator, to resynthesize light-depleted cGMP. In washed outer segment membranes, we find that GCAP-stimulable RetGC is rapidly inactivated at physiological temperatures (30-37 degrees C). Under the same conditions, RetGC remains competent for stimulation by S-100 protein preparations or Mn2+/Triton X-100, indicating that the cyclase catalytic domain remains functional. GCAPs and adenine nucleotides protect against inactivation. Protection by GCAPs is independent of Ca2+ concentration, suggesting that there is a Ca2+-independent interaction between GCAP and RetGC. Protection by ATP (EC50 = 150 microM) is not due to phosphorylation, since the nonhydrolyzable analogue adenylyl imidodiphosphate (AMP-PNP) protects equally well. In addition to their roles in protection, ATP and AMP-PNP also slowly stimulate cyclase activity. In parallel with the functional change in RetGC at physiological temperatures, we also observe a structural change. A 62-kDa intracellular fragment of RetGC-1 becomes more sensitive to cleavage by trypsin after preincubation at 30 degrees C unless ATP, AMP-PNP, or GCAP is present. Adenine nucleotides and GCAPs thus protect RetGC structurally, as well as functionally.

Guanylyl cyclase-activating protein 2 (GCAP-2) is a recoverin-like calcium-binding protein that regulates photoreceptor guanylyl cyclase (RetGC) (Dizhoor, A. M., and Hurley, J. B. (1996) J. Biol. Chem. 271, 19346-19350). It was reported that myristoylation of a related protein, GCAP-1, was critical for its affinity for RetGC (Frins, S., Bonigk, W., Muller, F., Kellner, R., and Koch, K.-W. (1996) J. Biol. Chem. 271, 8022-8027). We demonstrate that the N terminus of GCAP-2, like those of other members of the recoverin family of Ca2+-binding proteins, is fatty acylated. However, unlike other proteins of this family, more GCAP-2 is present in the membrane fraction at low Ca2+ than at high Ca2+ concentrations. We investigated the role of the N-terminal fatty acyl residue in the ability of GCAP-2 to regulate RetGCs. Myristoylated or nonacylated GCAP-2 forms were expressed in Escherichia coli. Wild-type GCAP-2 and the Gly2 --> Ala2 GCAP-2 mutant, which is unable to undergo N-terminal myristoylation, were also expressed in mammalian HEK293 cells. We found that compartmentalization of GCAP-2 in photoreceptor outer segment membranes is Ca2+- and ionic strength-sensitive, but it does not require the presence of the fatty acyl group and does not necessarily directly reflect GCAP-2 interaction with RetGC. The lack of myristoylation does not significantly affect the ability of GCAP-2 to stimulate RetGC. Nor does it affect the ability of the Ca2+-loaded form of GCAP-2 to compete with the GCAP-2 mutant that constitutively activates RetGC. We conclude that while Ca2+ binding plays a major regulatory role in GCAP-2 function, it does not operate through a calcium-myristoyl switch similar to the one found in recoverin.

Guanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca2+, a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC in bovine rod outer segment homogenates. These results suggest that although GCAP1 is involved in the Ca2+-sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.

In rod and cone photoreceptor cells, activation of particulate guanylate cyclase (retGC1) is mediated by a Ca2+-binding protein termed GCAP1, that detects changes in [Ca2+]free. In this study, we show that N-acylated GCAP1 restored Ca2+ sensitivity of native and recombinant photoreceptor retGC1. ATP increased the affinity of retGC1 for GCAP1 and accelerated catalysis. Using peptides derived from the GCAP1 sequence, we found that at least three regions, encompassing the N-terminus, the EF-1 motif, and the EF-3 motif, were likely involved in the interaction with retGC1. Mutation of 2Gly to Ala (GCAP1-G2A), which abolished myristoylation and a 25 amino acid truncation at the N-terminus (delta25-GCAP1) reduced retGC1-stimulating activity dramatically, while deletion of 10 amino acids (delta10-GCAP1) reduced the specific activity by only approximately 60% and modified the Ca2+ sensitivity. At 10(-6) M [Ca2+]free, in conditions that inactivated native GCAP1, retGC1 showed significant activity in the presence of delta10-GCAP1. Native and all three mutant forms of GCAP1 had similar affinities for Ca2+ as demonstrated by gel filtration and the changes in tryptophan fluorescence. All mutants bound to ROS membranes in a Ca2+-independent manner, except delta25-GCAP1, which was mostly soluble. These findings suggest that the N-terminal region is important in tethering of GCAP1 to the ROS membranes.

GCAP1 and GCAP2 are related Ca(2+)-binding proteins that activate photoreceptor guanylate cyclase(s). We showed previously that the human GCAP1 gene, consisting of four exons, is located at 6p21.1 (locus designation GUCA). To identify the chromosomal location of the GCAP2 gene, we first cloned its cDNA and determined its intron-exon distribution by PCR analysis. The results show that the introns of the GCAP2 gene are positioned exactly as in the GCAP1 gene and are nearly double in size. Sequence similarity between the two genes, however, is limited to portions of exons 1 and 2. The GCAP1 and GCAP2 genes are transcribed into single mRNA species (1.7 and 2.2 kb, respectively) and are detectable only in the retina by Northern blotting. The GCAP2 gene was found by somatic human-hamster hybrid panel analysis and FISH to reside at GUCA in a region indistinguishable from that of GCAP1. PCR analysis with exon 4-specific primers showed that the genes are in a tail-to-tail array less than 5 kb apart and altogether span less than 20 kb of genomic DNA. The identical gene structures and loci of GCAP1 and GCAP2, and the identical function of the gene products, are consistent with gene duplication event.

Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous form of retinal degeneration. The genes for the beta-subunit of rod phosphodiesterase (PDEB), rhodopsin (RHO), peripherin/RDS (RDS) and the rod outer segment membrane protein 1 (ROM1), as well as loci at 6p and 1q, have previously been reported as the cause of ARRP. In order to determine whether they are responsible for the disease in Spanish pedigrees, linkage and homozygosity studies using markers at these loci were carried out on 47 Spanish ARRP families. SSCP analysis was performed to search for mutations in the genes cosegregating with the disease in particular pedigrees. Three homozygous mutations in the PDEB gene were found, thus accounting for 6% of the cases. No other disease-causing mutation was observed in the other genes analysed, nor was significant evidence found for the involvement of the loci at 6p or 1q. On the basis of these data, it is unlikely that these genes and loci account for a considerable proportion of ARRP cases.

Guanylyl cyclase activator proteins GCAP-1 and GCAP-2 (Dizhoor et al. , 1995, Gorczyca et al., 1995) are members of a recently identified subclass of EF-hand type Ca2+-binding proteins that respond to Ca2+ differently than any other known members of the EF-hand superfamily. GCAPs acquire an activating conformation only in their Ca2+-free form. Free Ca2+ concentrations corresponding to levels in dark-adapted vertebrate photoreceptors inhibit the ability of GCAPs to activate photoreceptor guanylyl cyclases (RetGCs). We studied the effects of mutations that block binding of Ca2+ to the EF-hands of GCAP-2. Unlike other EF-hand proteins, which fail to activate their target when their EF-hands are inactivated by mutations, GCAP-2 with any single EF-hand inactivated remains active and is 3-6 times less sensitive to the inhibitory effect of Ca2+. Inactivation of any two or all three EF-hands produces active forms of GCAP-2 that are insensitive to inhibition by physiological intracellular concentrations of Ca2+. Unexpectedly we also found that activation of RetGCs by a Ca2+-insensitive mutant is inhibited by Ca2+-loaded wild type GCAP-2. We propose the following. 1) GCAP-2 can exist in two extreme functional forms: an apo form that activates RetGCs and a Ca2+-loaded form that blocks activation of RetGCs. 2) All three EF-hands of GCAP-2 contribute to the inhibitory effect of Ca2+. 3) Inactivation of two or three EF-hands is sufficient to shift the "activator-inhibitor" transition outside the physiological range of intracellular free Ca2+.

Bovine photoreceptor guanylate cyclase (ROS-GC) consists of a single transmembrane polypeptide chain with extracellular and intracellular domains. In contrast to non-photoreceptor guanylate cyclases (GCs) which are activated by hormone peptides, ROS-GC is modulated in low Ca2+ by calmodulin-like Ca(2+)-binding proteins termed GCAPs (guanylate cyclase-activating proteins). In this communication we show that, like the native system, ROS-GC expressed in COS cells is activated 4-6-fold by recombinant GCAP1 at 10 nM Ca2+ and that the reconstituted system is inhibited at physiological levels of Ca2+ (1 microM). A mutant ROS-GC in which the extracellular domain was deleted was stimulated by GCAP1 indistinguishable from native ROS-GC indicating that this domain is not involved in Ca2+ modulation. Deletion of the intracellular kinase-like domain diminished the stimulation by GCAP1, indicating that this domain is at least in part involved in Ca2+ modulation. Replacement of the catalytic domain in a non-photoreceptor GC by the catalytic domain of ROS-GC yielded a chimeric GC that was sensitive to ANF/ATP and to a lesser extent to GCAP1. The results establish that GCAP1 acts at an intracellular domain, suggesting a mechanism of photoreceptor GC stimulation fundamentally distinct from hormone peptide stimulation of other cyclase receptors.

Retinal guanylyl cyclase-1 (RetGC-1) is a membrane guanylyl cyclase found in photoreceptor outer segments. It consists of an apparent extracellular domain (ECD) linked by a single transmembrane segment to an intracellular domain (ICD). Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-binding protein that activates RetGC-1 in a Ca2+-sensitive manner. To establish whether GCAP-2 stimulates RetGC-1 through the ECD or ICD, we made deletion mutants lacking either the ECD or both the ECD and transmembrane domains (TMD) of RetGC-1. Recombinant wild type RetGC-1 and both deletion mutants were expressed in HEK 293 cells, and their sensitivities to GCAP-2, Ca2+, and ATP were compared. Our data demonstrate that both deletion mutants are regulated similarly to wild type RetGC-1 with indistinguishable EC50 values for Ca2+ and similar K1/2 values for activation by GCAP-2. This shows that GCAP-2 functions through the ICD of RetGC-1 and that removal of the ECD and TMD do not significantly alter regulation by these factors. Our data also show that ATP potentiates stimulation of guanylyl cyclase activity by GCAP-2 and that neither the ECD nor the TMD of RetGC-1 participate in its regulation by ATP.

We cloned the guanylate cyclase activating proteins, GCAP1 and GCAP2, from chicken retina and examined their expression in normal and predegenerate rdlrd chicken retina. Northern analyses show that the amounts of the single transcripts encoding GCAP1 and GCAP2 are reduced to about 70% of normal levels in rdlrd retina. Western analyses reveal that GCAP2 levels appear normal in this retina, while GCAP1 levels are reduced by more than 90%. The specific downregulation of GCAP1 in rdlrd retina is consistent with a model for this disease in which activation of guanylate cyclase in the photoreceptors is abnormal, resulting in low levels of cGMP and an absence of phototransduction.

The membrane-bound guanylyl cyclase in vertebrate photoreceptor cells is one of the key enzymes in visual transduction. It is highly sensitive to the free calcium concentration ([Ca2+]). The activation process is cooperative and mediated by a novel calcium-binding protein named GCAP (guanylyl cyclase-activating protein). We isolated GCAP from bovine rod outer segments, determined amino acid sequences of proteolytically obtained peptides, and cloned its gene. The Ca2+-bound form of native GCAP has an apparent molecular mass of 20.5 kDa and the Ca2+-free form of 25 kDa as determined by SDS-polyacrylamide gel electrophoresis. Recombinant GCAP was functionally expressed in Escherichia coli. Activation of guanylyl cyclase in vertebrate photoreceptor cells by native acylated GCAP was half-maximal at 100 nM free [Ca2+] with a Hill coefficient of 2.5. Activation by recombinant nonacylated GCAP showed a lower degree of cooperativity (n = 2.0), and half-maximal activation was shifted to 261 nM free [Ca2+]. Immunocytochemically we localized GCAP only in rod and cone cells of a bovine retina.

We have amplified, cloned, and sequenced 583 bp GCAP-II/uroguanylin-specific cDNA from human colon cDNA first strand. The cDNA codes for a putative 112 amino-acid precursor protein including the sequence of uroguanylin and GCAP-II. Northern blot hybridization revealed a high level expression of the GCAP-II gene in human colon, but not in the kidney. This expression of GCAP-II indicates a pivotal role in cGMP-mediated functions of the colon.

The systematic isolation of circulating regulatory peptides which generate cGMP as second messenger resulted in the identification of a novel member of the guanylin family. In the present study we describe the purification and amino acid sequence of a new guanylate cyclase C activating peptide (GCAP-II). GCAP-II contains 24 amino acids in the following sequence: FKTLRTIANDDCELCVNVACTGCL. Its molecular mass is 2597.7 Da. The 16 C-terminal amino acids are identical to uroguanylin from human urine. native and synthetic GCAP-II activate GC-C, the specific guanylate cyclase receptor, of cultured human colon carcinoma (T84) cells. GCAP-II stimulates chloride secretion in isolated human intestinal mucosa mediated by intracellular cGMP increase. GCAP-II specific antibodies were used to localize the peptide by immunohistochemistry in entero-endocrine cells of the colonic mucosa.

Guanylyl cyclase activating protein (GCAP1) has been proposed to act as a calcium-dependent regulator of retinal photoreceptor guanylyl cyclase (GC) activity. Using immunocytochemical and biochemical methods, we show here that GCAP1 is present in rod and cone photoreceptor outer segments where phototransduction occurs. Recombinant and native GCAP1 activate recombinant human retGC (outer segment-specific GC) and endogenous GC(s) in rod outer segment (ROS) membranes at low calcium. In addition, we isolate and clone a retinal homolog, termed GCAP2, that shows approximately 50% identity with GCAP1. Like GCAP1, GCAP2 activates photoreceptor GC in a calcium-dependent manner. Both GCAP1 and GCAP2 presumably act on GCs by a similar mechanism; however, GCAP1 specifically localizes to photoreceptor outer segments, while in these experiments GCAP2 was isolated from extracts of retina but not ROS. These results demonstrate that GCAP1 is an activator of ROS GC, while the finding of a second activator, GCAP2, suggests that a similar mechanism of GC regulation may be present in outer segments, other subcellular compartments of the photoreceptor, or other cell types.

Guanylate cyclase-activating protein (GCAP) is a novel Ca(2+)-binding protein that stimulates synthesis of cGMP in photoreceptors. Molecular cloning of human and mouse GCAP cDNA revealed that the known mammalian GCAPs are more than 90% similar, consist of 201-205 amino acids, and contain three identically conserved EF hand Ca2+ binding sites. The sequence homology with recoverin, a related photoreceptor Ca(2+)-binding protein, is less than 35%. In situ hybridization in primate retinas shows that the GCAP gene is expressed exclusively in photoreceptor inner segments. To investigate the GCAP gene structure, we probed 10 eucaryotic genomic DNAs with a bovine GCAP cDNA under stringent conditions. The results demonstrate that the GCAP gene has been well conserved during evolution of vertebrate species and that each gene is most likely present as a single copy. By genomic cloning, polymerase chain reaction, mapping, and direct sequencing, we show that the human GCAP gene spans approximately 6 kilobases of genomic DNA, and consists of four exons (> 250, 146, 94, and 800 base pairs) separated by three introns (4.5 kilobases, 370 base pairs, and 347 base pairs). Using human/hamster hybrid panels and fluorescent in situ hybridization, the GCAP gene was localized to the short arm of chromosome 6 (p21.1).