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.     

Novel frequenin-modulated Ca2+-signaling membrane guanylate cyclase (ROS-GC) transduction pathway in bovine hippocampus

Frequenin is a member of the neuronal Ca²⁺ sensor protein family, implicated in being the modulator of the neurotransmitter release, potassium channels, phosphatidylinositol signaling pathway and the Ca²⁺-dependent exocytosis of dense-core granules in the PC12 cells. Frequenin exhibits these biological activities through its Ca²⁺ myristoyl switch, yet the switch is functionally inactive. These structural and functional traits of frequenin have been derived through the use of recombinant frequenin. In the present study, frequenin (BovFrq) native to the bovine hippocampus has been purified, sequenced for its 9 internal fragments, cloned, and studied. The findings show that structure of the BovFrq is identical to its form present in chicken, rat, mouse and human, indicating its evolutionary conservation. Its Ca²⁺ myristoyl switch is active in the hippocampus. And, BovFrq physically interacts and turns on yet undisclosed ONE-GC-like ROS-GC membrane guanylate cyclase transduction machinery in the hippocampal neurons. This makes BovFrq a new Ca²⁺-sensor modulator of a novel ROS-GC transduction machinery. The study demonstrates the presence and mechanistic features of this cyclic GMP signaling pathway in the hippocampal neurons, and also provides one more support for the evolving concept where the Ca²⁺-modulated membrane guanylate cyclase transduction machinery in its variant forms is a central operational component of all neurons.     

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 Ca²⁺ 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 Ca²⁺ via two Ca²⁺-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 Ca²⁺-dependent modulation. This feature is not dependent upon the ability of frog GCAP1 to bind Ca²⁺ because unmyristoylated GCAP1 mutants which do not bind Ca²⁺, 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.     

Photoreceptor Guanylate Cyclases: A Review

Almost three decades of research in the field of photoreceptor guanylate cyclases are discussed in this review. Primarily, it focuses on the members of membrane-bound guanylate cyclases found in the outer segments of vertebrate rods. These cyclases represent a new guanylate cyclase subfamily, termed ROS-GC, which distinguishes itself from the peptide receptor guanylate cyclase family that it is not extracellularly regulated. It is regulated, instead, by the intracellularly-generated Ca²⁺ signals. A remarkable feature of this regulation is that ROS-GC is a transduction switch for both the low and high Ca²⁺ signals. The low Ca²⁺ signal transduction pathway is linked to phototransduction, but the physiological relevance of the high Ca²⁺ signal transduction pathway is not yet clear; it may be linked to neuronal synaptic activity. The review is divided into eight sections. In Section I, the field of guanylate cyclase is introduced and the scope of the review is briefly explained; Section II covers a brief history of the investigations and ideas surrounding the discovery of rod guanylate cyclase. The first five subsections of Section III review the experimental efforts to quantify the guanylate cyclase activity of rods, including in vitro and in situ biochemistry, and also the work done since 1988 in which guanylate cyclase activity has been determined. In the remaining three subsections an analytical evaluation of the Ca²⁺ modulation of the rod guanylate cyclase activity related to phototransduction is presented. Section IV deals with the issues of a biochemical nature: isolation and purification, subcellular localization and functional properties of rod guanylate cyclase. Section V summarizes work on the cloning of the guanylate cyclases, analysis of their primary structures, and determination of their location with in situ hybridization. Section VI summarizes studies on the regulation of guanylate cyclases, with a focus on guanylate cyclases activating proteins. In Section VII, the evidence about the localization and functional role of guanylate cyclases in other retinal cells, especially in on-bipolar cells, in which guanylate cyclase most likely plays a critical role in electrical signaling, is discussed. The review concludes with Section VIII, with remarks about the future directions of research on retinal guanylate cyclases.     

Ca2+-modulated vision-linked ROS-GC guanylate cyclase transduction machinery

Vertebrate phototransduction depends on the reciprocal relationship between two-second messengers, cyclic GMP and Ca²⁺. The concentration of both is reciprocally regulated including the dynamic synthesis of cyclic GMP by a membrane bound guanylate cyclase. Different from hormone receptor guanylate cyclases, the cyclases operating in phototransduction are regulated by the intracellular Ca²⁺-concentration via small Ca²⁺-binding proteins. Based on the site of their expression and their Ca²⁺ modulation, this sub-branch of the cyclase family was named sensory guanylate cyclases, of which the retina specific forms are named ROS-GCs (rod outer segment guanylate cyclases). This review focuses on the structure and function of the ROS-GC subfamily present in the mammalian retinal neurons: photoreceptors and inner layers of the retinal neurons. Portions and excerpts of the review are from a previous chapter (Curr Top Biochem Res 6:111–144, 2004).     

Membrane guanylate cyclase is a beautiful signal transduction machine: overview

This article is a sequel to the four earlier comprehensive reviews which covered the field of membrane guanylate cyclase from its origin to the year 2002 (Sharma in Mol Cell Biochem 230:3–30, 2002) and then to the year 2004 (Duda et al. in Peptides 26:969–984, 2005); and of the Ca²⁺-modulated membrane guanylate cyclase to the year 1997 (Pugh et al. in Biosci Rep 17:429–473, 1997) and then to 2004 (Sharma et al. in Curr Top Biochem Res 6:111–144, 2004). This article contains three parts. The first part is “Historical”; it is brief, general, and freely borrowed from the earlier reviews, covering the field from its origin to the year 2004 (Sharma in Mol Cell Biochem, 230:3–30, 2002; Duda et al. in Peptides 26:969–984, 2005). The second part focuses on the “Ca²⁺-modulated ROS-GC membrane guanylate cyclase subfamily”. It is divided into two sections. Section “Historical” and covers the area from its inception to the year 2004. It is also freely borrowed from an earlier review (Sharma et al. in Curr Top Biochem Res 6:111–144, 2004). Section “Ca²⁺-modulated ROS-GC membrane guanylate cyclase subfamily” covers the area from the year 2004 to May 2009. The objective is to focus on the chronological development, recognize major contributions of the original investigators, correct misplaced facts, and project on the future trend of the field of mammalian membrane guanylate cyclase. The third portion covers the present status and concludes with future directions in the field.     

Distinct ONE-GC transduction modes and motifs of the odorants: Uroguanylin and CO2

In a subset of the olfactory sensory neurons ONE-GC^$ membrane guanylate cyclase is a central component of two odorant-dependent cyclic GMP signaling pathways. These odorants are uroguanylin and CO₂. The present study was designed to decipher the biochemical and molecular differences between these two odorant signaling mechanisms. The study shows (1) in contrast to uroguanylin, CO₂ transduction mechanism is Ca²⁺-independent. (2) CO₂ transduction site, like that of uroguanylin-neurocalcin δ, resides in the core catalytic domain, aa 880-1028, of ONE-GC. (3) The site, however, does not overlap the signature neurocalcin δ signal transduction domain, ⁹⁰⁸LSEPIE⁹¹³. Finally, (4) this study negates the prevailing concept that CO₂ uniquely signals ONE-GC activity (Sun et al. [19]; Guo et al. [21]). It demonstrates that it also signals the activation of photoreceptor membrane guanylate cyclase ROS-GC1. These results show an additional new transduction mechanism of the membrane guanylate cyclases and broaden our understanding of the molecular mechanisms by which different odorants using a single guanylate cyclase can regulate diverse cyclic GMP signaling pathways.     

Expression level and activity profile of membrane bound guanylate cyclase type 2 in rod outer segments

Rod and cone cells of the mammalian retina harbor two types of a membrane bound guanylate cyclase (GC), rod outer segment guanylate cyclase type 1 (ROS-GC1) and ROS-GC2. Both enzymes are regulated by small Ca²⁺-binding proteins named GC-activating proteins that operate as Ca²⁺ sensors and enable cyclases to respond to changes of intracellular Ca²⁺after illumination. We determined the expression level of ROS-GC2 in bovine ROS preparations and compared it with the level of ROS-GC1 in ROSs. The molar ratio of a ROS-GC2 dimer to rhodopsin was 1 : 13 200. The amount of ROS-GC1 was 25-fold higher than the amount of ROS-GC2. Heterologously expressed ROS-GC2 was differentially activated by GC-activating protein 1 and 2 at low free Ca²⁺ concentrations. Mutants of GC-activating protein 2 modulated ROS-GC2 in a manner different from their action on ROS-GC1 indicating that the Ca²⁺ sensitivity of the Ca²⁺ sensor is controlled by the mode of target–sensor interaction.     

Calcium ion effects on guanylate cyclase of brain.

Soluble guanylate cyclase activity of brain is stimulated by Ca²⁺ in the presence of low concentrations of Mn²⁺. Unlike Ca²⁺ stimulation of adenylate cyclase, the effect does not depend upon interaction of guanylate cyclase with a specific high-affinity Ca²⁺-binding protein. In the presence of Mg²⁺, Ca²⁺ inhibits soluble guanylate cyclase as well as the particulate enzyme. The concept that stimulation of brain cells results in increased cyclic GMP concentration secondary to Ca²⁺ influx merits additional critical study.     

Ultracytochemistry as a tool for the study of the cellular and subcellular localization of membrane-bound guanylate cyclase (GC) activity. Applicability to both receptor-activated and receptor-independent GC activity

Membrane-bound guanylate cyclase activity was detected by ultracytochemistry at the electron microscope level in several mammalian tissues. The technique used in these studies allows the detection of active enzyme at the membrane site where it is located. In a few cases, such as normal and regenerating peripheral nerves and placenta, membrane-bound guanylate cyclase could be detected in the absence of stimulators of enzyme activity. However, in the majority of these studies membrane-bound guanylate cyclase was investigated following stimulation with natriuretic peptides, guanylin, or the Ca²⁺ sensor proteins, S100B and S100A1. In general, membrane-bound guanylate cyclase was localized to plasma membranes, in accordance with the functional role of this enzyme. Yet, in secretory cells the enzyme activity was localized on intracellular membranes, suggesting a role of membrane-bound guanylate cyclase in secretory processes. Finally, S100B and S100A1 were found to colocalize with membrane-bound guanylate cyclase on photoreceptor disc membranes and to stimulate enzyme activity at these sites in dark-adapted retinas in a Ca²⁺-dependent manner. The results of these analyses are discussed in relation to the proposed functional role(s) of this enzyme.     

Phorbol myristate acetate stimulation of lymphocyte guanylate cyclase

Human lymphocyte guanylate cyclase activities are increased in a dose-dependent fashion by incubation of intact cells with phorbol myristate acetate, a tumor promoter and lymphocyte mitogen. Increased activity is detectable after 1 minute, and peak membrane-bound and soluble forms of guanylate cyclase occur after 10- and 30-minute exposure to phorbol myristate acetate, respectively. The soluble form is stimulated much more than the membrane form. Enzyme activities measured in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺ are elevated to similar degrees. Comparisons of phorbol and a series of its diesters revealed a good correlation between the capacities for guanylate cyclase stimulation, lymphocyte mitogenesis, and tumor promotion.     

Involvement of rhodopsin and ATP in the activation of membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC) by GC-activating proteins (GCAPs): a new model for ROS-GC activation and its link to retinal diseases

Membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC), a key enzyme for the recovery of photoreceptors to the dark state, has a topology identical to and cytoplasmic domains homologous to those of peptide-regulated GCs. However, under the prevailing concept, its activation mechanism is significantly different from those of peptide-regulated GCs: GC-activating proteins (GCAPs) function as the sole activator of ROS-GC in a Ca²⁺-sensitive manner, and neither reception of an outside signal by the extracellular domain (ECD) nor ATP binding to the kinase homology domain (KHD) is required for its activation. We have recently shown that ATP pre-binding to the KHD in ROS-GC drastically enhances its GCAP-stimulated activity, and that rhodopsin illumination, as the outside signal, is required for the ATP pre-binding. These results indicate that illuminated rhodopsin is involved in ROS-GC activation in two ways: to initiate ATP binding to ROS-GC for preparation of its activation and to reduce [Ca²⁺] through activation of cGMP phosphodiesterase. These two signal pathways are activated in a parallel and proportional manner and finally converge for strong activation of ROS-GC by Ca²⁺-free GCAPs. These results also suggest that the ECD receives the signal for ATP binding from illuminated rhodopsin. The ECD is projected into the intradiscal space, i.e., an intradiscal domain(s) of rhodopsin is also involved in the signal transfer. Many retinal disease-linked mutations are found in these intradiscal domains; however, their consequences are often unclear. This model will also provide novel insights into causal relationship between these mutations and certain retinal diseases.     

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

Guanylate cyclase activating proteins (GCAPs) serve as neuronal Ca²⁺-sensor proteins in vertebrate rod and cone photoreceptor cells. Zebrafish express in their retina a variety of six different GCAPs, of which four are specific for cone cells. One isoform, zGCAP4, is mainly expressed in double cones and long single cones. We cloned the zGCAP4 gene, purified non-myristoylated and myristoylated forms of the protein after heterologous expression in Escherichia coli and studied its properties: zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish retina, showing half-maximal activation at 520–570 nM free Ca²⁺ concentration. Furthermore, the Ca²⁺-sensitive activation properties of non-myristoylated and myristoylated zGCAP4 were similar, indicating no influence of the myristoyl moiety on Ca²⁺-sensor function. Myristoylated zGCAP4 showed low affinity for membranes and did not exhibit a Ca²⁺–myristoyl switch, a feature typical of some but not all neuronal Ca²⁺-sensor proteins. However, tryptophan fluorescence studies and Ca²⁺-dependent differences in protease accessibility revealed Ca²⁺-induced conformational changes in myristoylated and non-myristoylated zGCAP4, indicating the operation as a Ca²⁺ sensor. Thus, expression and biochemical properties of zGCAP4 are in agreement with its function as an efficient Ca²⁺-sensitive regulator of guanylate cyclase activity in cone vision.     

GUANYLATE CYCLASES IN CNS: ENZYMATIC CHARACTERISTICS OF SOLUBLE AND PARTICULATE ENZYMES FROM MOUSE CEREBELLUM AND RETINA

Guanylate cyclase activity is present in both soluble and particulate fractions of homogenates of mouse cerebellum and retina. Soluble guanylate cyclases in cerebellum and retina have an apparent K_m for GTP of approx 40 and 70 μM, respectively; are stimulated by Ca²⁺ and Mg²⁺ in the presence of low Mn²⁺; and do not respond to NaN₃, NH₂OH or detergent. The particulate guanylate cyclase found in brain has an apparent K_m GTP of 237 7mu;M, is not stimulated by Ca²⁺ or Mg²⁺ in the presence of low Mn²⁺, but is stimulated by NaN₃, NH₂OH, and detergent. In particulate fractions of normal retina, guanylate cyclase has two apparent K_m GTP values (42 and 225 μM); has higher activity at low concentrations of Mn²⁺ (0.5 mM) than at high concentrations (5.0 mM); is inhibited by Ca²⁺; and does not respond to NaN₃, NH₂OH, or detergent. Retinas essentially devoid of photoreceptor cells (from mice with photoreceptor dystrophy) have soluble guanylate cyclase activity which is similar to that in normal retina, but have only 4% as much particulate guanylate cyclase activity. This residual particulate guanylate cyclase has an apparent K_m GTP value of 392 μM and other properties similar to particulate guanylate cyclase from brain. These data indicate the presence of three distinguishable guanylate cyclases in CNS: (1) a soluble enzyme present in both brain and retina: (2) a particulate enzyme which is also present in brain and in the inner or neural retina: and (3) another particulate enzyme which is apparently unique and confined to retinal photoreceptor cells.     

WTAPELL motif of the photoreceptor ROS-GC1: A general phototransduction switch

This study documents the identity of an intriguing transduction mechanism of the [Ca²⁺]_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 Ca²⁺ transmitter switch (Ca²⁺TS). A combination of immunoprecipitation, fluorescent spectroscopy, mutational analyses and reconstitution studies has been used to demonstrate that the structure of this switch is ⁶⁵⁷WTAPELL⁶⁶³. The two Ca²⁺ signaling GCAP pathways converge in Ca²⁺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 Ca²⁺-modulated photoreceptor ROS-GC transduction model; it is depicted and discussed for its application to processing the different shades of LIGHT.     

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.     

Activation of the renal cortical and hepatic guanylate cyclase-guanosine 3′,5′-monophosphate systems by nitrosoureas divalent cation requirements and relationship to thiol reactivity

Streptozotocin, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and N-methyl nitrosourea, compounds with both oncogenic and cytotoxic properties, increased guanylate cyclase activity in the 100 000 × g soluble fractions of rat renal cortex and liver 35- to 65-fold over basal values. Particulate enzyme activities of these tissues were increased 2- to 4-fold by a maximally effective concentration of the nitrosoureas. In the presence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, maximally effective concentrations of these nitrosoureas increased cyclic GMP accumulation of hepatic and renal cortical slices to peak levels 7- to 10-fold over control in 30 min. By contrast, with the structurally related carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) peak increases occurred in 5–10 min and were 40- to 70-fold over control levels in renal cortex and liver, respectively. Unlike the Ca²⁺-dependent actions of cholinergic stimuli on cyclic GMP, the nitrosoureas and MNNG increased cyclic GMP in either the presence or absence of extracellular Ca²⁺. Moreover, while basal soluble guanylate cyclase of renal cortex was highly Mn²⁺-dependent and decreased 85% when either Mg²⁺ or Ca²⁺ was employed as sole divalent cation in reaction mixtures, the actions of nitrosoureas on enzyme activity were well expressed with either Mn²⁺ or Mg²⁺, but not with Ca²⁺, as sole divalent cation. Improved utilization of Mg²⁺ by guanylate cyclase in the presence of nitrosoureas would favor enhanced enzyme activity under cellular conditions where Mg²⁺ is abundant. In the presence of maximally stimulatory concentrations of streptozotocin or BCNU, high concentrations of Mg²⁺ or Mn²⁺ further increased soluble guanylate cyclase, suggesting important differences in metal and nitrosourea stimulation of enzyme activity.Preincubation of supernatant fractions with nitrosoureas plus dithiothreitol inhibited the action of the N-nitroso compounds to increase renal cortical guanylate cyclase. Glutathione and cysteine were also inhibitory, but less effective than dithiothreitol. Initial incubation of nitrosoureas with dithiothreitol in buffer alone similarly suppressed the subsequent action of the N-nitroso compounds on guanylate cyclase, and implicated direct chemical interactions. Prior incubation of renal cortical supernatant fractions with the SH blockers N-ethylmaleimide or maleimide significantly suppressed guanylate cyclase activation mediated by streptozotocin or BCNU. Direct drug interactions seemed unlikely, since effects of the inhibitors were optimally expressed by initial exposure of the supernatant fraction of tissue to the SH blockers and were not potentiated by a 30 min preincubation of the SH blockers and nitrosoureas in buffer alone.Thus, nitrosoureas activate and alter the metal requirements of soluble guanylate cyclase and increase cellular cyclic GMP in the presence or absence of extracellular Ca²⁺. Activation of soluble guanylate cyclase by nitrosoureas may involve an interaction of these agents with tissue SH groups, and possibly SH to SS transformation. Stimulation of the guanylate cyclase system by nitrosoureas could be related to the oncogenic actions of these agents.     

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.     

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.     

Cell Cycle-associated Changes of Guanylate Cyclase Activity in Synchronized Tetrahymena: a Possible Involvement of Calmodulin in its Regulation1

Guanylate cyclase activity decreased during the division phase of heat-shock synchronized Tetrahymena pyriformis, strain GL. However, when Ca²⁺ was removed by EGTA to negate the effects of the Ca²⁺-binding protein (calmodulin), which is required for the full activity of guanylate cyclase in this organism, no significant change in the enzymatic activity was observed throughout the cell cycle. On the other hand, the reduced guanylate cyclase activity at division phase was associated with a decreased level of calmodulin content. These results suggest that fluctuations in guanylate cyclase activity during the cell cycle would be dependent on the concentration of calmodulin.