Outer Segment Membrane Guanylate Cyclase Research Articles

ROS-GC interlocked Ca(2+)-sensor S100B protein signaling in cone photoreceptors: review.

Photoreceptor rod outer segment membrane guanylate cyclase (ROS-GC) is central to visual transduction; it generates cyclic GMP, the second messenger of the photon signal. Photoexcited rhodopsin initiates a biochemical cascade that leads to a drop in the intracellular level of cyclic GMP and closure of cyclic nucleotide gated ion channels. Recovery of the photoresponse requires resynthesis of cyclic GMP, typically by a pair of ROS-GCs, 1 and 2. In rods, ROS-GCs exist as complexes with guanylate cyclase activating proteins (GCAPs), which are Ca2+-sensing elements. There is a light-induced fall in intracellular Ca2+. As Ca2+ dissociates from GCAPs in the 20–200 nM range, ROS-GC activity rises to quicken the photoresponse recovery. GCAPs then progressively turn down ROS-GC activity as Ca2+ and cyclic GMP levels return to baseline. To date, GCAPs mediate the only known mechanism of ROS-GC regulation in the photoreceptors. However, in mammalian cone outer segments, cone synapses and ON bipolar cells, another Ca2+ sensor protein, S100B, complexes with ROS-GC1 and senses the Ca2+ signal with a K1/2 of 400 nM. Unlike GCAPs, S100B stimulates ROS-GC activity when Ca2+ is bound. Thus, the ROS-GC system in cones functions as a Ca2+ bimodal switch; with rising intracellular Ca2+, its activity is first turned down by GCAPs and then turned up by S100B. This presentation provides a historical perspective on the role of S100B in the photoreceptors, offers a pictorial model for the “bimodal” operation of the ROS-GC switch and projects future tasks that are needed to understand its operation. Some accounts of this review have been adopted from the original publications of these authors.

Differential Ca2+ 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.

S100B Serves as a Ca2+ 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 Ca²⁺ signal transduction switch. Lowering [Ca²⁺]_i from 200 to 20 nM progressively turns it “ON” as does raising [Ca²⁺]_i from 500 to 5000 nM. The mode operating at lower [Ca²⁺]_i plays a vital role in phototransduction in both rods and cones. The physiological function of the mode operating at elevated [Ca²⁺]_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 Ca²⁺-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 Ca²⁺ 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 [Ca²⁺]_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 [Ca²⁺]_i in cone outer segments. The findings define a new cone-specific Ca²⁺-dependent feature of photoreceptors and expand our understanding of the operational principles of phototransduction machinery.

Rod outer segment membrane guanylate cyclase type 1 (ROS-GC1) calcium-modulated transduction system in the sperm

Evaluation of the presence of a Ca(2+)-regulated membrane guanylate cyclase signal transudation system in the spermatozoa. Experimental study. Research university laboratory. Human sperm obtained from healthy donors who met the criteria of the World Health Organization for normozoospermia and bovine semen collected from bulls of proven fertility. Radioimmunoassay and immunohistochemistry of human and bovine spermatozoa. The membrane guanylate cyclase activity and the presence of membrane guanylate cyclase transduction machinery components in the spermatozoa. The identity of a Ca(2+)-modulated membrane guanylate cyclase transduction machinery in human and bovine spermatozoa has been documented. The machinery is both inhibited and stimulated within nanomolar to semimicromolar range of free Ca(2+). The transduction component of this machinery is the rod outer segment membrane guanylate cyclase type 1 (ROS-GC1). The enzyme coexists with three Ca(2+)-dependent modulators: guanylate cyclase activating protein type 1 (GCAP1), S100B and neurocalcin delta. ROS-GC1 and its modulators are present in the heads and tails of both species' spermatozoa. The coexpression of ROS-GC1 and its activators in spermatozoa suggests that the Ca(2+)-modulated ROS-GC1 transduction system may be a part of the fertilization machinery.

Calcium‐Modulated Rod Outer Segment Membrane Guanylate Cyclase Type 1 Transduction Machinery in the Testes

The importance of the second messengers, Ca(2+) and cyclic GMP, for the process of fertilization is well established; the mechanisms for their intracellular regulations in the testes are, however, poorly understood. This study documents the biochemical, molecular, and functional identity of a Ca(2+)-modulated membrane guanylate cyclase transduction machinery in bovine testes. The machinery is both inhibited and stimulated by free Ca(2+) levels. The Ca(2+)-sensor component of the inhibitory mode of the machinery is GCAP1 (guanylate cyclase activating protein type 1) and for the stimulatory mode is S100B. The transduction component is a Ca(2+)-driven rod outer segment membrane guanylate cyclase type 1, ROS-GC1. The cyclase is predominantly expressed in spermatogenic cells. GCAP1 expression is restricted to a small population of spermatogonia, whereas S100B is present in the majority of spermatocytes and spermatids. The expression of GCAP1 and S100B in spermatocytes and spermatids is mutually exclusive.

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 δ 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.

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.

Structure and Ca2+ regulation of frog photoreceptor guanylate cyclase, ROS-GC1.

Rod outer segment membrane guanylate cyclase (ROS-GC) is a critical component of the vertebrate phototransduction machinery. In response to photoillumination, it senses a decline in free Ca(2+) levels from 500 to below 100 nM, becomes activated, and replenishes the depleted cyclic GMP pool to restore the dark state of the photoreceptor cell. It exists in two forms, ROS-GC1 and ROS-GC2. In outer segments, ROS-GCs sense fluctuations in Ca(2+) via two Ca(2+)-binding proteins, which have been termed GCAP1 and GCAP2. In the present study we report on the cloning of two ROS-GCs from the frog retinal cDNA library. These cyclases are the structural and functional counterparts of the mammalian ROS-GC1 and ROS-GC2. There is, however, an important difference between the regulation of mammalian and frog ROS-GC1: In contrast to the mammalian, the frog form does not require the myristoylated form of GCAP1 for its Ca(2+)-dependent modulation. This feature is not dependent upon the ability of frog GCAP1 to bind Ca(2+) because unmyristoylated GCAP1 mutants which do not bind Ca(2+), activate frog ROS-GC1. The findings establish frog as a suitable phototransduction model and show a facet of frog ROS-GC signaling, which is not shared by the mammalian form.

Rod outer segment membrane guanylate cyclase type 1-linked stimulatory and inhibitory calcium signaling systems in the pineal gland: biochemical, molecular, and immunohistochemical evidence.

Recent evidence indicates the presence of a novel alpha(2D/A)-adrenergic receptor (alpha(2D/A)-AR) linked membrane guanylate cyclase signal transduction system in the pineal gland. This system operates via a Ca(2+)-driven rod outer segment membrane guanylate cyclase (ROS-GC). In the present study, this transduction system has been characterized via molecular, immunohistochemical, and biochemical approaches. The two main components of the system are ROS-GC1 and its Ca(2+) regulator, S100B. Both components coexist in pinealocytes where the signaling component alpha(2D/A)-AR also resides. The presence of ROS-GC2 was not detected in the pineal gland. Thus, transduction components involved in processing alpha(2D/A)-AR-mediated signals are Ca(2+), S100B, and ROS-GC1. During this investigation, an intriguing observation was made. In certain pinealocytes, ROS-GC1 coexisted with its other Ca(2+) modulator, guanylate cyclase activating protein type 1 (GCAP1). In these pinealocytes, S100B was not present. The other GCAP protein, GCAP2, which is also a known modulator of ROS-GC in photoreceptors, was not present in the pineal gland. The results establish the identity of an alpha(2D/A)-AR-linked ROS-GC1 transduction system in pinealocytes. Furthermore, the findings show that ROS-GC1, in a separate subpopulation of pinealocytes, is associated with an opposite Ca(2+) signaling pathway, which is similar to phototransduction in retina. Thus, like photoreceptors, pinealocytes sense both positive and negative Ca(2+) signals, where ROS-GC1 plays a pivotal role; however, unlike photoreceptors, the pinealocyte is devoid of the ROS-GC2/GCAP2 signal transduction system.

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.

Mapping Functional Domains of the Guanylate Cyclase Regulator Protein, GCAP-2

Guanylate cyclase regulator protein (GCAP)-2 is a Ca2+-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca2+-sensitive manner. GCAP-2 activates RetGC at free Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca2+ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca2+-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe78 and Asp113 determines whether GCAP-2 activates outer segment RetGC in low or high Ca2+ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca2+ concentrations. 2) The amino acid sequence of GCAP-2 between Lys29 and Phe48 that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca2+ and inhibition at high Ca2+ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val171-Asn189, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca2+-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.

Crystal structure of recombinant bovine neurocalcin.

The crystal structure of calcium-bound unmyristoylated bovine neurocalcin from Escherichia coli has been determined at 2.4 A resolution. The three-dimensional structure reveals a highly compact structure consisting of: (i) two pairs of calcium-binding EF-hands (EF1-EF2 and EF3-EF4); (ii) a calcium ion bound at EF2, EF3 and EF4 sites; and (iii) an EF1-hand that is disabled from calcium-binding due to a Cys-Pro sequence in the Ca2+-binding loop. The crystal structure of neurocalcin resembles photoreceptor recoverin in overall topology, however its EF2- and EF4-hands differ. Recently, neurocalcin in the calcium-bound state has been shown to stimulate mammalian rod outer segment membrane guanylate cyclase. A possible site for cyclase activity based on the three-dimensional structure is discussed.

Functional consequences of a rod outer segment membrane guanylate cyclase (ROS-GC1) gene mutation linked with Leber's congenital amaurosis.

ROS-GC1 is the original member of the subfamily of membrane guanylate cyclases with two Ca2+ switches, which have been defined as CRM1 and CRM2. These are separately located within the intracellular domain of the cyclase. CRM1 switches on the enzyme at nanomolar concentrations of Ca2+ and is linked with phototransduction; the other stimulates at micromolar Ca2+ concentrations and is predicted to be linked with retinal synaptic activity. Ca2+ acts indirectly via Ca2+-binding proteins, GCAP1 and CD-GCAP. GCAP1 is a modulator of the CRM1 switch, and CD-GCAP turns on the CRM2 switch. A Leber's congenital amaurosis, termed LCA1, involves F514S point mutation in ROS-GC1. The present study shows that the mutation severely damages its intrinsic cyclase activity and inactivates its CRM1 switch but does not affect the CRM2 switch. In addition, on the basis of the established modulatory features of ROS-GC1, it is predicted that, in two other forms of LCA1 involving deletion of nt 460C or 693C, there is a frameshift in ROS-GC1 gene, which results in the nonexpression of the cyclase. For the first time, the findings define the linkage of distinct molecular forms of LCA to ROS-GC1 in precise biochemical terms; they also explain the reasons for the insufficient production of cyclic GMP in photoreceptors to sustain phototransduction, which ultimately leads to the degeneration of the photoreceptors.

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.

Rod outer segment membrane guanylate cyclase type 1 (ROS-GC1) gene: structure, organization and regulation by phorbol ester, a protein kinase C activator.

At present there are two recognized members of the ROS-GC subfamily of membrane guanylate cyclases. They are ROS-GC1 and ROS-GC2. A distinctive feature of this family is that its members are not switched on by the extracellular peptide hormones; instead, they are modulated by intracellular Ca2+ signals, consistent to their linkage with phototransduction. An intriguing feature of ROS-GC1, which distinguishes it from ROS-GC2, is that it has two Ca2+ switches. One switch inhibits the enzyme at micromolar concentrations of Ca2+, as in phototransduction; the other, stimulates. The stimulatory switch, most likely, is linked to retinal synaptic activity. Thus, ROS-GC1 is linked to both phototransduction and the synaptic activity. The present study describes (1) the almost complete structural identity of 18.5 kb ROS-GC1 gene; (2) its structural organization: the gene is composed of 20 exons and 19 introns with classical GT/AG boundaries; (3) the activity of the ROS-GC1 promoter assayed through luciferase reporter in COS cells; and (4) induction of the gene by phorbol ester, a protein kinase C (PKC) activator. The co-presence of PKC and ROS-GC1 in photoreceptors suggests that regulation of the ROS-GC1 gene by PKC might be a physiologically relevant phenomenon.

Structural and Functional Characterization of a Second Subfamily Member of the Calcium-Modulated Bovine Rod Outer Segment Membrane Guanylate Cyclase, ROS-GC2

A native bovine calcium-modulated rod outer segment membrane guanylate cyclase (ROS-GC) has been cloned and reconstituted to show its linkage consistent to the process of phototransduction. In the present study, a second form of the membrane guanylate cyclase has been cloned from the bovine retina. This cyclase shares a high sequence identity with ROS-GC, is specifically expressed in the bovine retina, and, like ROS-GC, is modulated in low Ca2+by a calmodulin-like Ca2+-binding protein, termed GCAP2. For this reason, this cyclase has now been named ROS-GC2 and the previously described ROS-GC as ROS-GC1. The tail end of ROS-GC2 contains a stretch of five amino acids, a structural feature unique to itself. These findings support the existence of a calcium-modulated subfamily of ROS-GC and indicate that ROS-GC2 embodies a five amino acid signature element at its tail end.

Distinct inhibitory ATP-regulated modulatory domain (ARMi) in membrane guanylate cyclases.

Depending upon the cofactors Mg2+ or Mn2+, ATP stimulates or inhibits the signal transduction activities of the natriuretic factor receptor guanylate cyclases, ANF-RGC and CNP-RGC: there is stimulation in the presence of Mg2+ and inhibition in the presence of Mn2+. A defined core ATP-regulated modulatory (ARM) sequence motif within the intracellular 'kinase-like' domain of the cyclases is critical for stimulation, but the mechanism of the inhibitory transduction process is not known. In addition, ATP inhibits the basal cyclase activity of a rod outer segment membrane guanylate cyclase (ROS-GC). The mechanism of this inhibitory transduction process is also not known. These issues have been addressed in the present investigation through a program of deletion mutagenesis/expression studies of the cyclases. The study shows that the ATP-mediated inhibitory transduction processes of the natriuretic factor receptor cyclases and of ROS-GC are identical. The ATP-regulated inhibitory domain of all these cyclases resides within the C-terminal segment of the cyclase. This domain is in a different location from the one representing the ATP-stimulatory ARM. The identification of the inhibitory domain in the C-terminal segment of the cyclase indicates that this segment is composed of two separate domains: one representing a catalytic cyclase domain and the other an ATP-regulated inhibitory (ARMi) domain. These findings establish a novel ATP-mediated inhibitory transduction mechanism of the membrane guanylate cyclases which is distinct from that of its counterpart, the stimulatory ATP-mediated hormonal signal transduction mechanism. Thus, they define a new paradigm of guanylate cyclase-linked signal transduction pathways.

Molecular characterization of S100A1-S100B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclase.

In contrast to the membrane guanylate cyclases which are stimulated by extracellular ligands, rod outer segment membrane guanylate cyclase (ROS-GC) activity is modulated intracellularly by calcium in two ways: one, where it is inhibited, and the other, where it is stimulated. The former way is linked to the phototransduction, and physiology of the second is unknown. In both ways calcium modulation of the cyclase occurs through the calcium binding proteins: through guanylate cyclase activating proteins (GCAPs) in the case of phototransduction, and through the recently discovered calcium-dependent GCAP (CD-GCAP) in the case of the other way. The kinase-like domain of ROS-GC is critical for the phototransduction-linked process. The present study shows the expression of alpha and beta chains of S100A1-S100B protein in the bovine retina and demonstrates that this protein stimulates ROS-GC activity in a dose-dependent fashion, that the stimulation is calcium dependent with an EC50 of 17 microM, and that the kinase-like domain is not involved in the calcium-modulated cyclase activation. Instead the involved domain resides at the C-terminal segment, between amino acids 731 and 1054. Thus, this S100A1-S100B protein-mediated calcium-modulated signal transduction mechanism is novel. Furthermore, this study provides the molecular understanding of the two transduction processes mediated by the same ROS-GC, one linked to the low and the other to the high calcium levels.