Calcium-modulated guanylate cyclase transduction machinery in the photoreceptor--bipolar synaptic region

Rod outer segment membrane guanylate cyclase (ROS-GC) transduction system is a central component of the Ca(2+)-sensitive phototransduction machinery. The system is composed of two parts: Ca(2+) sensor guanylate cyclase activating protein (GCAP) and ROS-GC. GCAP senses Ca(2+) impulses and inhibits the cyclase. This operational feature of the cyclase is considered to be unique and exclusive in the phototransduction machinery. A combination of reconstitution, peptide competition, cross-linking, and immunocytochemical studies has been used in this study to show that the GCAP1/ROS-GC1 transduction system also exists in the photoreceptor synaptic (presynaptic) termini. Thus, the presence of this system and its linkage is not unique to the phototransduction machinery. A recent study has demonstrated that the photoreceptor-bipolar synaptic region also contains a Ca(2+)-stimulated ROS-GC1 transduction system [Duda, T., et al. (2002) EMBO J. 21, 2547-2556]. In this case, S100beta senses Ca(2+) and stimulates the cyclase. The inhibitory and stimulatory Ca(2+)-modulated ROS-GC1 sites are distinct. These findings allow the formation of a new topographic model of ROS-GC1 transduction. In this model, the catalytic module of ROS-GC1 at its opposite ends is flanked by GCAP1 and S100beta modules. GCAP1 senses the Ca(2+) impulse and inhibits the catalytic module; S100beta senses the impulse and stimulates the catalytic module. Thus, ROS-GC1 acts as a bimodal Ca(2+) signal transduction switch in the photoreceptor bipolar synapse.     

Differential Ca(2+) sensor guanylate cyclase activating protein modes of photoreceptor rod outer segment membrane guanylate cyclase signaling

Photoreceptor ROS-GC1 (rod outer segment membrane guanylate cyclase) is a vital component of phototransduction. It is a bimodal Ca(2+) signal transduction switch, operating between 20 and ～1000 nM. Modulated by Ca(2+) sensors guanylate cyclase activating proteins 1 and 2 (GCAP1 and GCAP2, respectively), decreasing [Ca(2+)](i) from 200 to 20 nM progressively turns it "on", as does the modulation by the Ca(2+) sensor S100B, increasing [Ca(2+)](i) from 100 to 1000 nM. The GCAP mode plays a vital role in phototransduction in both rods and cones and the S100B mode in the transmission of neural signals to cone ON-bipolar cells. Through a programmed domain deletion, expression, in vivo fluorescence spectroscopy, and in vitro reconstitution experiments, this study demonstrates that the biochemical mechanisms modulated by two GCAPs in Ca(2+) signaling of ROS-GC1 activity are totally different. (1) They involve different structural domains of ROS-GC1. (2) Their signal migratory pathways are opposite: GCAP1 downstream and GCAP2 upstream. (3) Importantly, the isolated catalytic domain, translating the GCAP-modulated Ca(2+) signal into the generation of cyclic GMP, in vivo, exists as a homodimer, the two subunits existing in an antiparallel conformation. Furthermore, the findings demonstrate that the N-terminally placed signaling helix domain is not required for the catalytic domain's dimeric state. The upstream GCAP2-modulated pathway is the first of its kind to be observed for any member of the membrane guanylate cyclase family. It defines a new model of Ca(2+) signal transduction.     

Mutations in the rod outer segment membrane guanylate cyclase in a cone-rod dystrophy cause defects in calcium signaling.

Rod outer segment guanylate cyclase 1 (ROS-GC1) is a member of the subfamily of Ca(2+)-regulated membrane guanylate cyclases; and it is pivotal for vertebrate phototransduction. Two opposing regulatory modes control the activity of ROS-GC1. At nanomolar concentrations of Ca(2+), ROS-GC1 is activated by Ca(2+)-binding proteins named guanylate cyclase activating proteins (GCAPs). However, at micromolar concentrations of Ca(2+), ROS-GC1 is stimulated by S100beta [also named calcium-dependent (CD) GCAP]. This mode is not linked with phototransduction; instead, it is predicted to be involved in retinal synaptic activity. Two point mutations, E786D and R787C, in ROS-GC1 have been connected with cone-rod dystrophy (CORD6), with only one type of point mutation occurring in each family. The present study shows that the E786D mutation has no effect on the basal catalytic activity of ROS-GC1 and on its activation by GCAP1 and S100beta; however, the mutated cyclase becomes more activated by GCAP2. The R787C mutation has three consequences: (1) it causes major damage to the basal cyclase activity, (2) it makes the cyclase 5-fold more sensitive to activation by GCAP1; and 3) converts the cyclase into a form that is less sensitive to activation by GCAP2 and S100beta. Thus, the two CORD6-linked mutations in ROS-GC1, which occur at adjacent positions, result in vastly different biochemical phenotypes, and they are connected with very specific molecular defects in the Ca(2+) switching components of the cyclase. These defects, in turn, are proposed to have a profound effect on both the machinery of phototransduction and the retinal synapse. The study for the first time defines the biochemistry of CORD6 pathology in precise molecular terms.     

A second calcium regulator of rod outer segment membrane guanylate cyclase, ROS-GC1: neurocalcin.

ROS-GC represents a membrane guanylate cyclase subfamily whose distinctive feature is that it transduces diverse intracellularly generated Ca(2+) signals into the production of the second messenger cyclic GMP. An intriguing feature of the first subfamily member, ROS-GC1, is that it is both stimulated and inhibited by these signals. The inhibitory signals are processed by the cyclase activating proteins, GCAPs. The only known stimulatory signal is by the Ca(2+)-dependent guanylate cyclase activating protein, CD-GCAP. There are two GCAPs, 1 and 2, which link the cyclase with phototransduction, and one CD-GCAP, which is predicted to link ROS-GC1 with its retinal synaptic activity. Individual switches for these GCAPs and CD-GCAP have been respectively defined as CRM1, CRM3, and CRM2. This report defines the identity of a new ROS-GC1 regulator: neurocalcin. A surprising feature of the regulator is that it structurally is a GCAP but functionally behaves as a CD-GCAP. Recombinant neurocalcin stimulates ROS-GC1 in a dose-dependent fashion; the stimulation is Ca(2+)-dependent with an EC(50) of 20 microM; and the modulated domain resides at the C-terminal segment, between amino acids 731 and 1054. Previously, the residence of CRM2 has also been defined in this segment of the cyclase. However, the present study shows that the neurocalcin-regulated domain is distinct from CRM2. This is now designated as CRM4. Thus, the signal transduction mechanisms of neurocalcin and CD-GCAP are different, occurring through different modules of ROS-GC1. Neurocalcin signaling of ROS-GC1 is highly specific. It does not influence the activity of its second subfamily member, ROS-GC2, and of the other retinal guanylate cyclase, atrial natriuretic factor-receptor guanylate cyclase. In conclusion, the findings extend the concept of ROS-GC1's sensing diverse Ca(2+) signals, reveal the identity of its unexpected new Ca(2+) regulator, and show that the regulator acts through its specific cyclase domain. This represents an additional transduction mechanism of Ca(2+) signaling via ROS-GC1.     

The calcium-sensor guanylate cyclase activating protein type 2 specific site in rod outer segment membrane guanylate cyclase type 1

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.     

Structural, biochemical, and functional characterization of the calcium sensor neurocalcin delta in the inner retinal neurons and its linkage with the rod outer segment membrane guanylate cyclase transduction system

This study documents the detailed biochemical, structural, and functional identity of a novel Ca(2+)-modulated membrane guanylate cyclase transduction system in the inner retinal neurons. The guanylate cyclase is the previously characterized ROS-GC1 from the photoreceptor outer segments (PROS), and its new modulator is neurocalcin delta. At the membrane, the myristoylated form of neurocalcin delta senses submicromolar increments in free Ca(2+), binds to its specific ROS-GC1 domain, and stimulates the cyclase. Neurocalcin delta is not present in PROS, indicating the absence of the pathway in the outer segments and the dissociation of its linkage with phototransduction. Thus, the pathway is linked specifically with the visual transduction machinery in the secondary neurons of the retina. With the inclusion of this pathway, the findings broaden the understanding of the existing mechanisms showing how ROS-GC1 is able to receive and transduce diverse Ca(2+) signals into the cell-specific generation of second-messenger cyclic GMP in the retinal neurons.     

Regulatory modes of rod outer segment membrane guanylate cyclase differ in catalytic efficiency and Ca(2+)-sensitivity.

In rod phototransduction, cyclic GMP synthesis by membrane bound guanylate cyclase ROS-GC1 is under Ca(2+)-dependent negative feedback control mediated by guanylate cyclase-activating proteins, GCAP-1 and GCAP-2. The cellular concentration of GCAP-1 and GCAP-2 approximately sums to the cellular concentration of a functional ROS-GC1 dimer. Both GCAPs increase the catalytic efficiency (kcat/Km) of ROS-GC1. However, the presence of a myristoyl group in GCAP-1 has a strong impact on the regulation of ROS-GC1, this is in contrast to GCAP-2. Catalytic efficiency of ROS-GC1 increases 25-fold when it is reconstituted with myristoylated GCAP-1, but only by a factor of 3.4 with nonmyristoylated GCAP-1. In contrast to GCAP1, myristoylation of GCAP-2 has only a minor effect on kcat/Km. The increase with both myristoylated and nonmyristoylated GCAP-2 is 10 to 13-fold. GCAPs also confer different Ca(2+)-sensitivities to ROS-GC1. Activation of the cyclase by GCAP-1 is half-maximal at 707 nM free [Ca(2+)], while that by GCAP-2 is at 100 nM. The findings show that differences in catalytic efficiency and Ca(2+)-sensitivity of ROS-GC1 are conferred by GCAP-1 and GCAP-2. The results further indicate the concerted operation of two 'GCAP modes' that would extend the dynamic range of cyclase regulation within the physiological range of free cytoplasmic Ca(2+) in photoreceptor cells.     

Impairment of the rod outer segment membrane guanylate cyclase dimerization in a cone-rod dystrophy results in defective calcium signaling

Rod outer segment membrane guanylate cyclase1 (ROS-GC1) is the original member of the membrane guanylate cyclase subfamily whose distinctive feature is that it transduces diverse intracellularly generated Ca(2+) signals in the sensory neurons. In the vertebrate retinal neurons, ROS-GC1 is pivotal for the operations of phototransduction and, most likely, of the synaptic activity. The phototransduction- and the synapse-linked domains are separate, and they are located in the intracellular region of ROS-GC1. These domains sense Ca(2+) signals via Ca(2+)-binding proteins. These proteins are ROS-GC activating proteins, GCAPs. GCAPs control ROS-GC1 activity through two opposing regulatory modes. In one mode, at nanomolar concentrations of Ca(2+), the GCAPs activate the cyclase and as the Ca(2+) concentrations rise, the cyclase is progressively inhibited. This mode operates in phototransduction via two GCAPs: 1 and 2. The second mode occurs at micromolar concentrations of Ca(2+) via S100beta. Here, the rise of Ca(2+) concentrations progressively stimulates the enzyme. This mode is linked with the retinal synaptic activity. In both modes, the final step in Ca(2+) signal transduction involves ROS-GC dimerization, which causes the cyclase activation. The identity of the dimerization domain is not known. A heterozygous, triple mutation -E786D, R787C, T788M- in ROS-GC1 has been connected with autosomal cone-rod dystrophy in a British family. The present study shows the biochemical consequences of this mutation on the phototransduction- and the synapse-linked components of the cyclase. (1) It severely damages the intrinsic cyclase activity. (2) It significantly raises the GCAP1- and GCAP2-dependent maximal velocity of the cyclase, but this compensation, however, is not sufficient to override the basal cyclase activity. (3) It converts the cyclase into a form that only marginally responds to S100beta. The mutant produces insufficient amounts of the cyclic GMP needed to drive the machinery of phototransduction and of the retinal synapse at an optimum level. The underlying cause of the breakdown of both types of machinery is that, in contrast to the native ROS-GC1, the mutant cyclase is unable to change from its monomeric to the dimeric form, the form required for the functional integrity of the enzyme. The study defines the CORD in molecular terms, at a most basic level identifies a region that is critical in its dimer formation, and, thus, discloses a single unifying mechanistic theme underlying the complex pathology of the disease.     

Ca(2+) sensor S100beta-modulated sites of membrane guanylate cyclase in the photoreceptor-bipolar synapse

This study documents the identity of a calcium- regulated membrane guanylate cyclase transduction system in the photoreceptor-bipolar synaptic region. The guanylate cyclase is the previously characterized ROS-GC1 from the rod outer segments and its modulator is S100beta. S100beta senses increments in free Ca(2+) and stimulates the cyclase. Specificity of photoreceptor guanylate cyclase activation by S100beta is validated by the identification of two S100beta-regulatory sites. A combination of peptide competition, surface plasmon resonance binding and deletion mutation studies has been used to show that these sites are specific for S100beta and not for another regulator of ROS-GC1, guanylate cyclase-activating protein 1. One site comprises amino acids (aa) Gly962-Asn981, the other, aa Ile1030-Gln1041. The former represents the binding site. The latter binds S100beta only marginally, yet it is critical for control of maximal cyclase activity. The findings provide evidence for a new cyclic GMP transduction system in synaptic layers and thereby extend existing concepts of how a membrane-bound guanylate cyclase is regulated by small Ca(2+)-sensor proteins.     

Ca2+-modulated membrane guanylate cyclase in the testes

To date, the calcium-regulated membrane guanylate cyclase Rod Outer Segment Guanylate Cyclase type 1 (ROS-GC1) transduction system in addition to photoreceptors is known to be expressed in three other types of neuronal cells: in the pinealocytes, mitral cells of the olfactory bulb and the gustatory epithelium of tongue. Very recent studies from our laboratory show that expression of ROS-GC1 is not restricted to the neuronal cells; the male gonads and the spermatozoa also express ROS-GC1. In this presentation, the authors review the existing information on the localization and function of guanylate cyclase with special emphasis on Ca²⁺-modulated membrane guanylate cyclase, ROS-GC1, in the testes. The role of ROS-GC1 and its Ca²⁺-sensing modulators in the processes of spermatogenesis and fertilization are discussed.     

A novel calcium-regulated membrane guanylate cyclase transduction system in the olfactory neuroepithelium

This report defines the identity of a calcium-regulated membrane guanylate cyclase transduction system in the cilia of olfactory sensory neurons, which is the site of odorant transduction. The membrane fraction of the neuroepithelial layer of the rat exhibited Ca(2+)-dependent guanylate cyclase activity, which was eliminated by the addition of EGTA. This indicated that the cyclase did not represent a rod outer segment guanylate cyclase (ROS-GC), which is inhibited by free Ca(2+). This interpretation was supported by studies with the Ca(2+) binding proteins, GCAPs (guanylate cyclase activating proteins), which stimulate photoreceptor ROS-GC in the absence of Ca(2+). They did not stimulate the olfactory neuroepithelial membrane guanylate cyclase. The olfactory neuroepithelium contained a Ca(2+) binding protein, neurocalcin, which stimulated the cyclase in a Ca(2+)-dependent fashion. The cyclase was cloned from the neuroepithelium and was found to be identical in structure to that of the previously cloned cyclase termed GC-D. The cyclase was expressed in a heterologous cell system, and was reconstituted with its Ca(2+)-dependent activity in the presence of recombinant neurocalcin. The reconstituted cyclase mimicked the native enzyme. Immunocytochemical studies showed that the guanylate cyclase coexists with neurocalcin in the apical region of the cilia. Deletion analysis showed that the neurocalcin-regulated domain resides at the C-terminal region of the cyclase. The findings establish the biochemical, molecular, and functional identity of a novel Ca(2+)-dependent membrane guanylate cyclase transduction system in the cilia of the olfactory epithelium, suggesting a mechanism of the olfactory neuroepithelial guanylate cyclase regulation fundamentally distinct from the phototransduction-linked ROS-GC.     

Negatively calcium-modulated membrane guanylate cyclase signaling system in the rat olfactory bulb

The mechanism by which the individual odor signals are translated into the perception of smell in the brain is unknown. The signal processing occurs in the olfactory system which has three major components: olfactory neuroepithelium, olfactory bulb, and olfactory cortex. The neuroepithelial layer is composed of ciliated sensory neurons interspersed among supportive cells. The sensory neurons are the sites of odor transduction, a process that converts the odor signal into an electrical signal. The electrical signal is subsequently received by the neurons of the olfactory bulb, which process the signal and then relay it to the olfactory cortex in the brain. Apart from information about certain biochemical steps of odor transduction, there is almost no knowledge about the means by which the olfactory bulb and cortical neurons process this information. Through biochemical, functional, and immunohistochemical approaches, this study shows the presence of a Ca(2+)-modulated membrane guanylate cyclase (mGC) transduction system in the bulb portion of the olfactory system. The mGC is ROS-GC1. This is coexpressed with its specific modulator, guanylate cyclase activating protein type 1 (GCAP1), in the mitral cells. Thus, a new facet of the Ca(2+)-modulated GCAP1--ROS-GC1 signaling system, which, until now, was believed to be unique to phototransduction, has been revealed. The findings suggest a novel role for this system in the polarization and depolarization phenomena of mitral cells and also contradict the existing belief that no mGC besides GC-D exists in the olfactory neurons.     

Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism

Ca²⁺-modulated rod outer segment membrane guanylate cyclase (ROS-GC1) has been cloned and reconstituted to show that it is regulated by two processes: one inhibitory, the other stimulatory. The inhibitory process is consistent with its linkage to phototransduction; the physiology of the stimulatory process is probably linked to neuronal transmission. In both regulatory processes, calcium modulation of the cyclase takes place through the calcium binding proteins; guanylate cyclase activating proteins (GCAP1 and GCAP2) in the case of the phototransduction process and calcium-dependent GCAP (CD-GCAP) in the case of the stimulatory process. The cyclase domains involved in the two processes are located at two different sites on the ROS-GC1 intracellular region. The GCAP1-modulated domain resides within the aa 447-730 segment of ROS-GC1 and the CD-GCAP-modulated domain resides within the aa 731-1054 segment. In the present study the GCAP2-dependent Ca²⁺ modulation of the cyclase activity has been reconstituted using recombinant forms of GCAP2 and ROS-GC1, and its mutants. The results indicate that consistent to phototransduction, GCAP2 at low Ca²⁺ concentration (10 nM) maximally stimulates the cyclase activity of the wild-type and its mutants: ext- (deleted aa 8-408); kin- (deleted aa 447-730) and hybrid consisting of the ext, transmembrane and kin domains of ANF-RGC and the C-terminal domain, aa 731-1054, of ROS-GC1. In all cases, it inhibits the cyclase activity with an IC₅₀ of about 140 nM. A previous study has shown that under identical conditions the kin- and the hybrid mutant are at best only minimally stimulated. Thus, the GCAP1 and GCAP2 signal transduction mechanisms are different, occurring through different modules of ROS-GC1. These findings also demonstrate that the intracellular region of ROS-GC1 is composed of multiple modules, each designed to mediate a particular calcium-specific signalling pathway.     

GCAP1 rescues rod photoreceptor response in GCAP1/GCAP2 knockout mice

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.     

657WTAPELL663 motif of the photoreceptor ROS-GC1: a general phototransduction switch

This study documents the identity of an intriguing transduction mechanism of the [Ca(2+)](i) signals by the photoreceptor ROS-GC1. Despite their distal residences and operational modes in phototransduction, the two GCAPs transmit and activate ROS-GC1 through a common Ca(2+) transmitter switch (Ca(2+)TS). A combination of immunoprecipitation, fluorescent spectroscopy, mutational analyses and reconstitution studies has been used to demonstrate that the structure of this switch is (657)WTAPELL(663). The two Ca(2+) signaling GCAP pathways converge in Ca(2+)TS, get transduced, activate ROS-GC1, generate the LIGHT signal second messenger cyclic GMP and yet functionally perform divergent operations of the phototransduction machinery. The findings define a new Ca(2+)-modulated photoreceptor ROS-GC transduction model; it is depicted and discussed for its application to processing the different shades of LIGHT.     

S100B serves as a Ca(2+) sensor for ROS-GC1 guanylate cyclase in cones but not in rods of the murine retina

Rod outer segment membrane guanylate cyclase (ROS-GC1) is a bimodal Ca(2+) signal transduction switch. Lowering [Ca(2+)](i) from 200 to 20 nM progressively turns it "ON" as does raising [Ca(2+)](i) from 500 to 5000 nM. The mode operating at lower [Ca(2+)](i) plays a vital role in phototransduction in both rods and cones. The physiological function of the mode operating at elevated [Ca(2+)](i) is not known. Through comprehensive studies on mice involving gene deletions, biochemistry, immunohistochemistry, electroretinograms and single cell recordings, the present study demonstrates that the Ca(2+)-sensor S100B coexists with and is physiologically linked to ROS-GC1 in cones but not in rods. It up-regulates ROS-GC1 activity with a K(1/2) for Ca(2+) greater than 500 nM and modulates the transmission of neural signals to cone ON-bipolar cells. Furthermore, a possibility is raised that under pathological conditions where [Ca(2+)](i) levels rise to and perhaps even enter the micromolar range, the S100B signaling switch will be turned "ON" causing an explosive production of CNG channel opening and further rise in [Ca(2+)](i) in cone outer segments. The findings define a new cone-specific Ca(2+)-dependent feature of photoreceptors and expand our understanding of the operational principles of phototransduction machinery.     

Neurocalcin delta modulation of ROS-GC1, a new model of Ca(2+) signaling

ROS-GC1 membrane guanylate cyclase is a Ca(2+) bimodal signal transduction switch. It is turned "off" by a rise in free Ca(2+) from nanomolar to the semicromolar range in the photoreceptor outer segments and the olfactory bulb neurons; by a similar rise in the bipolar and ganglion retinal neurons it is turned "on". These opposite operational modes of the switch are specified by its Ca(2+) sensing devices, respectively termed GCAPs and CD-GCAPs. Neurocalcin delta is a CD-GCAP. In the present study, the neurocalcin delta-modulated site, V(837)-L(858), in ROS-GC1 has been mapped. The location and properties of this site are unique. It resides within the core domain of the catalytic module and does not require the alpha-helical dimerization domain structural element (amino acids 767-811) for activating the catalytic module. Contrary to the current beliefs, the catalytic module is intrinsically active; it is directly regulated by the neurocalcin delta-modulated Ca(2+) signal and is dimeric in nature. A fold recognition based model of the catalytic domain of ROS-GC1 was built, and neurocalcin delta docking simulations were carried out to define the three-dimensional features of the interacting domains of the two molecules. These findings define a new transduction model for the Ca(2+) signaling of ROS-GC1.     

p19 detected in the rat retina and pineal gland is a guanylyl cyclase-activating protein (GCAP)

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.     

Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy

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.     

Light inhibition of bovine retinal rod guanylyl cyclase mediated by beta gamma-transducin

Photoreceptor guanylyl cyclase (ROS-GC), converting GTP into cGMP and pyrophosphate, is a key enzyme in the regulation of the visual transduction cascade. ROS-GC requires GC-activating proteins (GCAPs) and low free [Ca] for full activity. We found that when choline or potassium were the major cations present, light caused a 70% inhibition of stimulated ROS-GC in native unstripped membranes. In the presence of sodium ions, however, no inhibition was observed. ROS-GC activity of ROS membranes, stripped of transducin and other components, was not affected by light when reconstituted with GCAP1 only. However, when stripped ROS membranes were reconstituted with both GCAP1 and either transducin (T alpha beta gamma) or the T beta gamma-subunits, the inhibition of ROS-GC by light was restored. The T alpha-subunit alone was ineffective. These results suggest that under saturating light conditions, ROS-GC may be regulated by T beta gamma and cations, providing a possible mechanism of desensitization and light adaptation.