Membrane Guanylate Cyclase Research Articles

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.

Plasma and Urine Levels of Uroguanylin, a New Natriuretic Peptide, in Nephrotic Syndrome

Uroguanylin, a new natriuretic peptide originally isolated from urine, stimulates the membrane guanylate cyclase C receptor. No information, however, is available on the plasma and urine levels of uroguanylin in nephrotic syndrome (NS), the state associated with sodium and water retention. Using a sensitive radioimmunoassay, we measured the plasma and urine concentrations of immunoreactive (ir-)uroguanylin in NS patients and compared them with those of patients with non-nephrotic glomerulonephritis. Plasma ir-uroguanylin, blood pressure and the cardiothoracic ratio were higher, and urine excretion of ir-uroguanylin was lower in the NS patients. Plasma ir-uroguanylin in the NS patients significantly decreased during remission as compared with findings on admission. There was a significant inverse correlation between the concentration of plasma ir-uroguanylin and that of serum total protein or albumin. Moreover, fluid retention in the NS patients was correlated with the changes in plasma ir-uroguanylin between admission and remission, indicative that the plasma concentration increases with the severity of the nephrotic state. Taking into account its potent natriuretic effect, these findings suggest that uroguanylin may function in the pathophysiological mechanism in NS.

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.

Natriuretic and kaliuretic activities of guanylin and uroguanylin in the isolated perfused rat kidney.

Guanylin and uroguanylin are novel peptides that activate membrane guanylate cyclases found in the kidney and intestine. We compared the effects of these peptides in the isolated perfused rat kidney. Both peptides are natriuretic and kaliuretic in this preparation. Uroguanylin (0.19-1.9 microM) increased glomerular filtration rate from 0.77 +/- 0.07 to 1.34 +/- 0.3 ml . g-1 . min-1 at the highest concentration. A maximal increase in Na+ excretion was achieved at 0. 66 microM uroguanylin, with a reduction in fractional Na+ reabsorption from 78.7 +/- 1.7 to 58.8 +/- 4.4%. The highest dose of uroguanylin increased kaliuresis by 50%. Osmolar clearance doubled at the highest concentration of uroguanylin tested (P < 0.05). Guanylin also elicited a natriuresis and kaliuresis but appeared to be less potent than uroguanylin. The highest concentration of guanylin (1.3 microM) decreased fractional Na+ reabsorption from 73. 9 +/- 2.4 to 64.5 +/- 4.0%, but lower doses were ineffective. Guanylin stimulated urine K+ excretion at the lowest concentration tested (0.33 microM) without any effect on Na+ excretion. These peptides may influence salt and water homeostasis by biological effects in the kidney that are mediated by the intracellular second messenger, cGMP.

Constitutive Activation of Photoreceptor Guanylate Cyclase by Y99C Mutant of GCAP-1: POSSIBLE ROLE IN CAUSING HUMAN AUTOSOMAL DOMINANT CONE DEGENERATION

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

Identification of effector binding sites on S100 beta: studies with guanylate cyclase and p80, a retinal phosphoprotein.

S100 beta is a calcium-binding protein, which regulates the activities of several enzymes and inhibits the phosphorylation of a variety of protein kinase C substrates in a calcium-dependent manner. The protein was recently found to activate a retinal membrane guanylate cyclase, and in this paper, we report that it inhibits the phosphorylation of an 80 kDa retinal protein (p80). Structurally, S100 beta consists of two EF-hands connected by a hinge region. In view of its small size, wide distribution in a variety of tissues, and regulation of many different proteins, it is of interest to identify the sites on the protein that interact with the effectors, and to determine if the same sites are responsible for interaction with different effectors. We addressed these questions with the use of synthetic peptides with sequences corresponding to different regions of S100 beta and testing their effects on the protein's activation of guanylate cyclase, and inhibition of p80 phosphorylation. Peptides with sequences corresponding to effector interaction sites were anticipated to either block or simulate the effects of S100 beta. The results show that two regions of S100 beta interact with effectors: the C-terminal region of Thr81-Glu91 and the hinge region of Leu32-Leu40. The synthetic peptide containing the latter sequence blocked the S100 beta activation of guanylate cyclase and inhibition of p80 phosphorylation, while the peptide containing the former sequence blocked cyclase activation and simulated S100 beta in inhibiting p80 phosphorylation. By determining the effects of including or excluding dithiothreitol in the assays, we observed that the cysteine residue in the C-terminal region of S100 beta (Cys84) participates in the regulation of guanylate cyclase but not of p80 phosphorylation. We conclude from these results that the C-terminal and hinge regions of S100 beta are important in the regulation of effector proteins and that Cys84 is essential for interaction with only specific effectors.

The α 2D/A-adrenergic receptor-linked membrane guanylate cyclase: a new signal transduction system in the pineal gland

In the pineal gland, the membrane guanylate cyclase activity was specifically stimulated by α 2D/A-adrenergic receptor (α 2D/A-AR) agonists. The agonists, however, did not stimulate the cyclase activity in the cell-free membranes. It was possible to stimulate the cyclase in cell-free membranes by the addition of the pineal soluble fraction, but this stimulation was Ca 2+-dependent and α 2D/A-agonist-independent. It was also possible to achieve Ca 2+-dependent stimulation of the cyclase by the direct addition of CD-GCAP to the isolated pineal membranes. CD-GCAP is a Ca 2+-binding protein and is a specific activator of one of the two members of the ROS-GC subfamily of membrane guanylate cyclases, ROS-GC1. The soluble fraction of the pineal gland stimulated recombinant ROS-GC1 in a Ca 2+-dependent fashion. The direct presence of both ROS-GC1 and CD-GCAP in the pineal was established by molecular cloning/PCR studies. The findings demonstrate the existence of a novel signal transduction mechanism – the linkage of the α 2D/A-AR signaling system with ROS-GC1 transduction system, occurring through intracellular Ca 2+ via CD-GCAP.

Calcium Modulated Signaling Site in Type 2 Rod Outer Segment Membrane Guanylate Cyclase (ROS-GC2)

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

Insulin resistance and the regulation of vascular tone: is insulin a vasodilator?

Insulin resistance is a key element in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM). Insulin resistance is usually assessed by measurements of insulin-induced glucose uptake, mainly by skeletal muscle, using the euglycemic hyperinsulinemic clamp technique. This involves a constant insulin infusion which stimulates glucose uptake. A variable glucose infusion is regularly adjusted to maintain a constant blood glucose level, i.e. to compensate for tissue glucose uptake. The amount of glucose infused at a given insulin concentration under steady state conditions is used as an index of insulin sensitivity. Insulin increases glucose extraction by skeletal muscle cells, but may also enhance glucose delivery via increased blood flow. This hypothesis was proposed in 1990 by Baron’s group (1). Their experimental design involved catheterization of the femoral artery and vein. Measurements of leg blood flow (by thermodilution) and of the arteriovenous glucose difference were performed, and the leg glucose uptake was determined as the product of these two parameters. It came somewhat as a surprise to find that insulin-induced glucose uptake was associated with a striking increase in leg blood flow; the insulin dose‐responses of blood flow and glucose extraction were very similar. These findings raised the possibility that insulin is a vasodilator hormone. In addition, the effect of insulin on leg blood flow was blunted in obese insulin-resistant patients, suggesting that vascular (in)sensitivity is a component of insulin resistance. The mechanism of insulin-induced vasodilation has now become a hotly debated issue. Is there a direct effect of insulin on the vascular wall, or does insulin act indirectly via a metabolic intermediate? Is blood flow (dys)regulation really involved in the pathogenesis of insulin resistance? Attention has focused on the regulation of endothelium-dependent vasodilation. In response to neurotransmitters such as acetylcholine, endothelial cells produce the vasodilator nitric oxide (NO), which acts in a paracrine manner on underlying smooth muscle cells to regulate contraction. NO activates a membrane guanylate cyclase, resulting in increased cGMP levels and activation of cGMP-dependent protein kinases (2). Using the leg perfusion system, the Baron group found that administration of metacholine (an acetylcholine analog) caused an expected dosedependent increase in leg blood flow. Simultaneous administration of insulin had a strong potentiating effect on the response to metacholine. Furthermore, the administration of L-NMMA, a specific inhibitor of NO synthase, abolished insulin-induced vasodilation (3). This finding was confirmed by others using a forearm perfusion technique (4). The response to metacholine was severely blunted in patients with insulin resistance or NIDDM, whereas the response to sodium nitroprusside, a direct activator of guanylate cyclase, was unaffected (5). These findings strongly suggest that insulin resistance is associated with impaired endothelium-dependent vasodilation. Similarly, the insulin resistance associated with insulin-dependent diabetes mellitus (IDDM) has also been attributed to impaired insulin-dependent NO-mediated vasodilation (6).

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.

Differential Activation of Rod Outer Segment Membrane Guanylate Cyclases, ROS-GC1 and ROS-GC2, by CD-GCAP and Identification of the Signaling Domain

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

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.

Signaling pathways for guanylin and uroguanylin in the digestive, renal, central nervous, reproductive, and lymphoid systems.

Guanylin and uroguanylin are peptides that stimulate membrane guanylate cyclases (GC) and regulate intestinal and renal function via cGMP. Complementary DNAs were isolated encoding opossum preproguanylin and a 279-amino acid portion of a receptor-guanylate cyclase expressed in opossum kidney (OK) cells (GC-OK). The tissue expression of messenger RNA transcripts for these signaling molecules were then compared. Northern and/or reverse transcription-PCR assays revealed that guanylin, uroguanylin, and GC-OK messenger RNAs are expressed in tissues within the digestive, renal, central nervous, reproductive, and lymphoid organ systems. Receptor autoradiography localized the receptors for uroguanylin and guanylin to renal proximal tubules and seminiferous tubules of testis. Synthetic guanylin and uroguanylin peptides activated the receptor-GCs in opossum kidney cortex and in cultured OK cells eliciting increased intracellular cGMP. Expression of agonist and receptor-GC signaling molecules provides a pathway for paracrine and/or autocrine regulation of cellular functions via cGMP in the digestive, renal, central nervous, reproductive, and lymphoid/immune organ systems. Uroguanylin also links the intestine and kidney in a potential endocrine axis that activates tubular receptor-GCs and influences renal function.

The bovine guanylate cyclase GC-E gene and 5′ flanking region

The gene encoding the bovine guanylate cyclase isoform E ( GC-E) was isolated as a single 18 kb genomic clone and shown to have 20 exons and 19 introns. Comparison of the structure of the GC-E gene with structures of other membrane guanylate cyclase genes indicates that the GC-E is most closely related to the subfamily of sensory guanylate cyclases. Comparison of the GC-E structure with that of the more distantly related guanylate cyclase isoform A ( GC-A) gene shows the most divergence in the extracellular and C-terminal regions, but general conservation of introns and exons in the intracellular kinase-like and catalytic domains. RT-PCR from several bovine tissues shows that GC-E is expressed only in the retina. Consistent with this pattern of expression, elements for the retinal-specific transcription factors RET-1, RET-2 and Tα-1 are located in the 5′ flanking promoter region.

Synthesis, solution structure, binding activity, and cGMP activation of human guanylin and its disulfide isomer

Guanylin is a recently isolated peptide consisting of 15 amino acid residues with four cysteines, which may form two intramolecular disulfide bridges, and stimulates intestinal membrane guanylate cyclase. The position of the disulfide linkages of guanylin was predicted from its structural similarity to a heat stable enterotoxin which is thought to be responsible for secretory diarrhoea. Both guanylin, with disulfide positions 4–12 and 7–15, and its disulfide isomer, with disulfides positions 4–15 and 7–12, were chemically synthesized by the solid-phase method and purified. Two specific disulfides were selectively formed and confirmed by sequencing, mass spectrometry and high-performance liquid chromatography in combination with enzymatic cleavage. The structure of both isomers has been investigated in solution by 1H nuclear magnetic resonance spectroscopy. Guanylin exists as a mixture of two stable conformations which have compact spiral structures, from comparison with literature data. In contrast, the disulfide isomer of guanylin shows only a single conformation with an elongated curved plate-like structure. Binding assays were performed using labelled guanylin with membranes obtained from rat jejunum. Both disulfide isomers were investigated by the cGMP assay. Both binding and cGMP assays indicated that the relevant form of disulfide bridges in the intact guanylin was as predicted.

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.

Inhibition of human neutrophil beta2-integrin-dependent adherence by hyperbaric O2.

Animal and clinical investigations have reported that exposure to hyperbaric O(2) improved the outcome of some reperfusion injuries. Animal studies have suggested that this may be due to an inhibition of leukocyte adherence to injured endothelium. This investigation tested the hypothesis that exposure to hyperbaric O(2) would inhibit beta2-integrin-dependent adherence of human neutrophils. Subjects were exposed to O(2) at partial pressures of up to 3 atmospheres absolute (ATA; 1 ATA = 0.1 MPa) for 45 min, and neutrophil binding to nylon columns and to fibrinogen-coated surfaces was measured. Exposure to O(2) at 2.8 or 3.0 ATA inhibited beta2-integrin-dependent neutrophil adherence but had no effect on the cell-surface expression of beta2-integrins, respiratory burst in response to phorbol ester, or non-beta2-integrin-dependent adherence to plastic plates coated with a fibronectin-like protein. beta2-Integrin adherence was restored by incubating blood with 8-bromoguanosine 3',5'-cyclic monophosphate (cGMP) and hyperbaric O(2) inhibited synthesis of cGMP by neutrophils stimulated with N-formyl-Met-Leu-Phe (FMLP). In studies of cell fractions, the activity of membrane guanylate cyclase was found to be increased by incubation with FMLP as well as by atrial natriuretic peptide (ANP) plus ATP. Hyperbaric O(2) had no effect on the basal activity of soluble or membrane-bound guanylate cyclase. However, hyperbaric O(2) inhibited the function of both the extracellular binding domain of membrane guanylate cyclase as well as intracellular catalytic activity. There are approximately 7,300 membrane guanylate cyclase molecules per cell, based on binding studies with ANP, with a dissociation constant of approximately 450 pM. Hyperbaric O(2) inhibits the function of human neutrophil beta2-integrins by a process linked to impaired synthesis of cGMP.

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.

The Photoreceptor Guanylate Cyclase Is an Autophosphorylating Protein Kinase

The photoreceptor membrane guanylate cyclase is a member of a family of proteins with a set of four structural motifs: an extracellular ligand binding domain, a transmembrane domain, an intracellular protein kinase-like domain, and an intracellular catalytic domain. Purified preparations of the photoreceptor guanylate cyclase have allowed us to explore the function of the protein kinase-like domain. ATP enhances the guanylate cyclase activity 2-fold in membranes stripped of peripheral proteins. The stimulation can be mimicked by ATPgammaS (adenosine 5'-O-(3-thiotriphosphate)), AMPPNP (5'-adenylyl beta,gamma-imidodiphosphate), and ADP, but not AMP. While this effect is lost by solubilizing guanylate cyclase, ATP binds the purified, solubilized enzyme in a site distinct from the catalytic GTP site as shown by specific labeling with 8-N3[alpha-32P]ATP. The enzyme has a protein kinase activity that is Mg2+-dependent and autophosphorylates serine residues. Myelin basic protein serves as a substrate for the kinase and enables further characterization of the kinase properties. The Km for ATP is 81 microM. The kinase activity is unaffected by calcium, cyclic nucleotides, and phorbol 12-myristate 13-acetate/L-alpha-phosphatidylserine/Ca2+ and is inhibited by high concentrations of staurosporine. These properties are distinct from other Ser/Thr kinases identified in rod outer segment preparations including protein kinase A, protein kinase C, and rhodopsin kinase. The observations offer the first biochemical evidence that a member of the receptor guanylate cyclase family has intrinsic protein kinase activity.

PGE2 and PGI2 inhibit ET-1 secretion from endothelial cells by stimulating particulate guanylate cyclase.

Prostaglandins (PG)E2 and prostacyclin (PGI2) can cause vasodilation in selective vascular beds and could act in part by inhibiting the production of the vasoconstrictor endothelin-1 (ET-1). We recently reported that these prostanoids inhibit ET-1 production/secretion from cultured endothelial cells via the generation of guanosine 3'-5'-cyclic monophosphate (cGMP). It is unclear whether this results from the stimulation of the particulate (membrane) of soluble (cytosolic) form of guanylate cyclase, and whether these effects are through an intermediate, such as nitric oxide. PGE2 and PGI2 each caused a three- to fourfold increase in both membrane and whole bovine aortic endothelial cell guanylate cyclase activity. The stimulations were significantly reversed (80-90%) by the compound LY-83583, an antagonist to cGMP generation, but were unaffected by methylene blue (MB), an inhibitor of nitric oxide-induced soluble guanylate cyclase. In contrast, the prostaglandins did not generate cGMP in cytosolic fractions. The prostaglandins inhibited ET-1 secretion from the intact cells, which was significantly prevented by LY-83583, but not by MB. Neither prostaglandin stimulated NO synthase activity, an indicator of nitric oxide generation. We conclude that PGE2 and PGI2 are likely to inhibit ET-1 secretion through the activation of the particulate guanylate cyclase, identifying a novel mechanism by which the prostanoids signal in the endothelial cell.