Atrial Natriuretic Factor Receptor Guanylate Cyclase Research Articles

Ca2+-Sensor Neurocalcin δ and Hormone ANF Modulate ANF-RGC Activity by Diverse Pathways: Role of the Signaling Helix Domain.

Prototype member of the membrane guanylate cyclase family, ANF-RGC (Atrial Natriuretic Factor Receptor Guanylate Cyclase), is the physiological signal transducer of two most hypotensive hormones ANF and BNP, and of the intracellular free Ca2+. Both the hormonal and the Ca2+-modulated signals operate through a common second messenger, cyclic GMP; yet, their operational modes are divergent. The hormonal pathways originate at the extracellular domain of the guanylate cyclase; and through a cascade of structural changes in its successive domains activate the C-terminal catalytic domain (CCD). In contrast, the Ca2+ signal operating via its sensor, myristoylated neurocalcin δ both originates and is translated directly at the CCD. Through a detailed sequential deletion and expression analyses, the present study examines the role of the signaling helix domain (SHD) in these two transduction pathways. SHD is a conserved 35-amino acid helical region of the guanylate cyclase, composed of five heptads, each meant to tune and transmit the hormonal signals to the CCD for their translation and generation of cyclic GMP. Its structure is homo-dimeric and the molecular docking analyses point out to the possibility of antiparallel arrangement of the helices. Contrary to the hormonal signaling, SHD has no role in regulation of the Ca2+- modulated pathway. The findings establish and define in molecular terms the presence of two distinct non-overlapping transduction modes of ANF-RGC, and for the first time demonstrate how differently they operate, and, yet generate cyclic GMP utilizing common CCD machinery.

Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, transduces two independent signals, ANF and Ca2+

Atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), was the first discovered member of the mammalian membrane guanylate cyclase family. The hallmark feature of the family is that a single protein contains both the site for recognition of the regulatory signal and the ability to transduce it into the production of the second messenger, cyclic GMP. For over two decades, the family has been classified into two subfamilies, the hormone receptor subfamily with ANF-RGC being its paramount member, and the Ca2+ modulated subfamily, which includes the rod outer segment guanylate cyclases, ROS-GC1 and 2, and the olfactory neuroepithelial guanylate cyclase. ANF-RGC is the receptor and the signal transducer of the most hypotensive hormones, ANF– and B-type natriuretic peptide (BNP). After binding these hormones at the extracellular domain it, at its intracellular domain, signals activation of the C-terminal catalytic module and accelerates the production of cyclic GMP. Cyclic GMP then serves the second messenger role in biological responses of ANF and BNP such as natriuresis, diuresis, vasorelaxation, and anti-proliferation. Very recently another modus operandus for ANF-RGC was revealed. Its crux is that ANF-RGC activity is also regulated by Ca2+. The Ca2+ sensor neurocalcin d mediates this signaling mechanism. Strikingly, the Ca2+ and ANF signaling mechanisms employ separate structural motifs of ANF-RGC in modulating its core catalytic domain in accelerating the production of cyclic GMP. In this review the biochemistry and physiology of these mechanisms with emphasis on cardiovascular regulation will be discussed.

ATP allosteric activation of atrial natriuretic factor receptor guanylate cyclase

Atrial natriuretic factor receptor guanylate cyclase (ANF-RGC) is the receptor and the signal transducer of two natriuretic peptide hormones: atrial natriuretic factor and brain natriuretic peptide. It is a single transmembrane-spanning protein. It binds these hormones at its extracellular domain and activates its intracellular catalytic domain. This results in the accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature and fluid secretion. ATP is obligatory for the transduction of this hormonal signal. Two models of ATP action have been proposed. In Model 1, it is a direct allosteric transducer. It binds to the defined regulatory domain (ATP-regulated module) juxtaposed to the C-terminal side of the transmembrane domain of ANF-RGC, induces a cascade of temporal and spatial changes and activates the catalytic module residing at the C-terminus of the cyclase. In Model 2, before ATP can exhibit its allosteric effect, ANF-RGC must first be phosphorylated by an as yet unidentified protein kinase. This initial step is obligatory in atrial natriuretic factor signaling of ANF-RGC. Until now, none of these models has been directly validated because it has not been possible to segregate the allosteric and the phosphorylation effects of ATP in ANF-RGC activation. The present study accomplishes this aim through a novel probe, staurosporine. This unequivocally validates Model 1 and settles the over two-decade long debate on the role of ATP in ANF-RGC signaling. In addition, the present study demonstrates that the mechanisms of allosteric modification of ANF-RGC by staurosporine and adenylyl-imidodiphosphate, a nonhydrolyzable analog of ATP, are almost (or totally) identical.

Atrial natriuretic factor-receptor guanylate cyclase signal transduction mechanism

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC), like the other members of the membrane guanylate cyclase family, is a single transmembrane-spanning protein. The transmembrane domain separates the protein into two regions, extracellular and intracellular. The extracellular region contains the ANF-binding domain and the intracellular region the catalytic domain located at the C-terminus of the protein. Preceding the catalytic domain, the intracellular region is comprised of the following functional domains: juxtaposed 40 amino acids to the transmembrane domain is the ATP-regulated module (ARM) domain [also termed the kinase homology domain (KHD)], and the putative dimerization domain. The ANF-RGC signaling is initiated by hormone, ANF, binding to its extracellular binding site. The binding signal is transduced through the transmembrane domain to the intracellular portion where ATP binding to the ARM domain partially activates the cyclase and prepares it for subsequent steps involving phosphorylation and attaining the fully activated state. This chapter reviews the signaling modules of ANF-RGC.

ATP signaling site in the ARM domain of atrial natriuretic factor receptor guanylate cyclase

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single transmembrane spanning modular protein. It binds ANF to its extracellular module and activates its intracellular catalytic module located at its carboxyl end. This results in the accelerated production of cyclic GMP, which acts as a critical second messenger in decreasing blood pressure. Two mechanistic models have been proposed for the ANF signaling of ANF-RGC. One is ATP-dependent and the other ATP-independent. In the former, ATP works through the ARM (ATP-regulated transduction module) of ANF-RGC. This model has recently been challenged [Antos et al. (2005) J Biol Chem 280:26928-26932] in support of the ATP-independent model. The present in-depth study analyzes the major principles of this challenge and concludes that the challenge lacks merit. The study then moves on to dissect the ATP mechanism of ANF signaling of ANF-RGC. It shows that the ATP photoaffinity probe, [gamma(32)P]-8-azido-ATP, reacts with Cys(634) residue in the ATP-binding pocket of ARM, and also signals the ANF-dependent activation of ANF-RGC. The target site of the 8-azido (nitrene) group is between the Cys(634) and Val(635) bond of the ATP-binding pocket. Thus, the study experimentally validates the ARM model-predicted role of Val(635) in the folding pattern of the ATP-binding pocket. And, it also identifies another residue Cys(634) that along with eight already identified residues is a part of the fold around the adenine ring of the ATP pocket. This information establishes the direct role of ATP in ANF signal transduction model of ANF-RGC, and provides a significant advancement on the mechanism by which the ATP-dependent transduction model operates.

Three dimensional atomic model and experimental validation for the ATP-Regulated Module (ARM) of the atrial natriuretic factor receptor guanylate cyclase.

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single chain transmembrane-spanning protein, containing both ANF binding and catalytic activities. ANF binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening transduction step, which is regulated by the binding of ATP to the cyclase. The partial structural motif of the ATP binding domain of the cyclase has been elucidated and has been termed ATP Regulatory Module (ARM). The crystal structures of the tyrosine kinase domains of the human insulin receptor and haematopoietic cell kinase were used to derive a homology-based model of the ARM domain of ANF-RGC. The model identifies the precise configuration of the ATP-binding pocket in the ARM domain, accurately represents its ATP-dependent features, and shows that the ATP-dependent transduction phenomenon is a two-step mechanism. In the first step, ATP binds to its pocket and changes its configuration; in the second step, via an unknown protein kinase, it phosphorylates the cyclase for its full activation.

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

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

The glycine residue of ATP regulatory module in receptor guanylate cyclases that is essential in natriuretic factor signaling

Atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP)-activated guanylate cyclases are single-chain transmembrane-spanning proteins, containing both ligand binding and catalytic activities. In both proteins, ligand binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an ATP-dependent step. ATP directly binds to a defined ATP-regulatory module (ARM) sequence motif in the cyclases and through ARM bridges the events of ligand binding and signal transduction. These ARM sequence motifs are respectively represented by Gly 503-Xa-Gly 505-Xa-Xa-Xa-Gly 509 and Gly 499-Xa-Xa-Xa-Gly 503 in the case of ANF receptor guanylate cyclase (ANF-RGC) and CNP receptor guanylate cyclase (CNP-RGC). Through genetic remodeling techniques, we now show that ARM-Gly 505 in ANF-RGC and the corresponding ARM-Gly 499 in CNP-RGC are critical for ANF and CNP signaling, and other ARM-Gly residues have minimal effect in the respective signaling processes.

Structural and Biochemical Identity of Retinal Rod Outer Segment Membrane Guanylate Cyclase

Recent molecular cloning reports show that there are at least three membrane guanylate cyclases in vertebrate retina: (1) atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), (2) C-type natriuretic peptide receptor guanylate cyclase (CNP-RGC), and (3) "retinal guanylate cyclase" (RetGC). The specific cellular localization of the first two cyclases is unknown, but RetGC is apparently localized in photoreceptor cells, suggesting that it participates in visual transduction. With the overall objective of identifying the guanylate cyclase that is linked to phototransduction, we compared the structural and regulatory properties of the biochemically characterized 112 kDa bovine rod outer segment membrane guanylate cyclase (ROS-GC) with those of RetGC, ANF-RGC and CNP-RGC. The N-terminal and two internal peptide sequences of purified ROS-GC had about 90% similarity with the corresponding sequences of the RetGC; the sequence identity with natriuretic peptide receptor cyclases was about 30%. A 19 amino acid long sequence from a tryptic peptide of ROS-GC had no corresponding sequence in the other three cyclases. ROS-GC was inhibited by ATP but ANF-RGC and CNP-RGC were activated by ATP in the presence of the respective peptide hormones. These results suggest that ROS-GC represents a new subtype of the membrane guanylate cyclase family that is structurally and biochemically distinct from the other retinal cyclases.

Cloning and expression of an ATP-regulated human retina C-type natriuretic factor receptor guanylate cyclase.

The natriuretic factors are structurally related polypeptide hormones that regulate the hemodynamics of the physiological processes of diuresis, water balance, and blood pressure. Presumably, these hormones act through the activation of guanylate cyclases which are also the specific receptors of these hormones. Two such structurally similar cell surface receptors are known; the ligand for one is atrial natriuretic factor (ANF) and for the other is C-type natriuretic peptide (CNP). Studies with ANF receptor guanylate cyclase (ANF-RGC) have indicated that its ligand binding site is extracellular and the catalytic site is intracellular, but the mere ligand binding to the receptor domain does not activate the cytosolic catalytic domain. An intervening ATP-mediated event is obligatory: ATP binds to a defined ATP-regulated module (ARM) sequence and bridges the events of ligand binding and signal transduction. The mechanism of CNP signaling is not known, although CNP in intact cells transfected with CNP receptor guanylate cyclase (CNP-RGC) stimulates the formation of cyclic GMP. Furthermore, there is no prior evidence of the presence of CNP signal transduction system in retina, although the presence of ANF-RGC has been documented. We now report the molecular cloning and expression of CNP-RGC from human retina and show that ATP is obligatory in CNP signaling also.(ABSTRACT TRUNCATED AT 250 WORDS)

Core sequence of ATP regulatory module in receptor guanylate cyclases

Atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP)-activated guanylate cyclases are single-chain transmembrane-spanning proteins, containing both ligand binding and catalytic activities. In both proteins, ligand binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Studies with ANF receptor guanylate cyclase (ANF-RGC) have indicated that obligatory in this activation process is an ATP-dependent step. ATP directly binds to the cyelase and bridges the events of ligand binding and signal transduction. A defined ATP-regulated module (ARM) sequence (Gly 503-Arg-Gly-Ser-Asn-Tyr-Gly 509) in the cyclase is critical in the ATP-mediated event. Through genetic remodeling techniques, we have now identified the core ARM sequence that is essential in both ANF and CNP signaling. This sequence is Gly-Xa-Xa-Xa-Gly, represented by Gly 505-Ser-Asn-Tyr-Gly 509 in the case of ANF-RGC ARM and by Gly 499-Ser-Ser-Tyr-Gly 503 in the CNP receptor guanylate cyclase ARM.

Interaction of atrial natriuretic factor and endothelin-1 signals through receptor guanylate cyclase in pulmonary artery endothelial cells.

The endothelial cell has a unique intrinsic feature: it produces a most potent vasopressor peptide hormone, endothelin (ET-1), yet it also contains a signaling system of an equally potent hypotensive hormone, atrial natriuretic factor (ANF). This raises two related curious questions: does the endothelial cell also contain an ET-1 signaling system? If yes, how do the two systems interact with each other? The present investigation was undertaken to determine such a possibility. Bovine pulmonary artery endothelial (BPAE) cells were chosen as a model system. Identity of the ANF receptor guanylate cyclase was probed with a specific polyclonal antibody to the 180 kDa membrane guanylate cyclase (mGC) ANF receptor. A Western-blot analysis of GTP-affinity-purified endothelial cell membrane proteins recognized a 180 kDa band; the same antibody inhibited the ANF-stimulated guanylate cyclase activity; the ANF-dependent rise of cyclic GMP in the intact cells was dose-dependent. By affinity cross-linking technique, a predominant 55 kDa membrane protein band was specifically labeled with [125I]ET-1. ET-1 treatment of the cells showed a migration of the protein kinase C (PKC) activity from cytosol to the plasma membrane; ET-1 inhibited the ANF-dependent production of cyclic GMP in a dose-dependent fashion with an EC50 of 100 nM. This inhibitory effect was duplicated by phorbol 12-myristate 13-acetate (PMA), a known PKC-activator. The EC50 of PMA was 5 nM. A PKC inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H-7), blocked the PMA-dependent attenuation of ANF-dependent cyclic GMP formation. These results demonstrate that the 180 kDa mGC-coupled ANF and ET-1 signaling systems coexist in endothelial cells and that the ET-1 signal negates the ANF-dependent guanylate cyclase activity and cyclic GMP formation. Furthermore, these results support the paracrine and/or autocrine role of ET-1.

Genetically tailored atrial natriuretic factor-dependent guanylate cyclase Immunological and functional identity with 180 kDa membrane guanylate cyclase and ATP signaling site

Biochemical and immunological studies have established that one of the signal transducers of atrial natriuretic factor (ANF) is a 180 kDa membrane guanylate cyclase (180 kDa mGC), which is also an ANF receptor; obligatory in the transduction process is an intervening ATP-regulated step, but its mechanism is not known. GCα is a newly discovered member of the guanylate cyclase family whose activity is independent of the known natriuretic peptides, and the enzyme is not an ANF receptor. The genetically tailored GCα, GCα-DmutGln 338 Leu 364, however, is not only a guanylate cyclase but also an ANF receptor and is structurally and functionally identical to the cloned wild-type ANF receptor guanylate cyclase, GC-A. We now report that the ANF-dependent guanylate cyclase activity in the particulate fractions of cells transfected with GCα-DmutGln 338 Leu 364 was inhibited by the 180 kDa mGC polyclonal antibody, and with this antibody probe it was possible to purify the 130 kDa expressed receptor; the hormone-dependent cyclase activity of this receptor was exclusively dependent upon ATP; and through site-directed mutational studies with GCα mutants, the signaling sequence that defines ATP binding site was identified. We thus conclude that 180 kDa mGC and the mutant protein are immunologically similar; both proteins are linked to the ANF signal in the generation of cyclic GMP synthesis; and in both the ligand binding and catalytic activities are bridged through a defined ATP binding module.

Ontogeny of Atrial Natriuretic Factor Receptors and Cyclic GMP Response in Rabbit Renal Glomeruli

Atrial natriuretic factor (ANF) has been identified in fetal and newborn mammals, and considerable data regarding fetal ANF metabolism are available. However, there is limited information concerning ANF receptors or receptor ontogenesis in developing mammals. We measured ANF receptor binding capacity, affinity, and ANF-induced cyclic GMP (cGMP) generation in isolated renal glomeruli from fetal (29 d gestation, term = 31 d), newborn (3 d), juvenile (28 d), and adult rabbits. The (mean +/- SEM) glomerular receptor binding capacity values for ANF in fetal and newborn animals (10 +/- 1 and 12 +/- 3 fmol/mg protein) were similar and significantly lower than the values for juvenile and adult animals (30 +/- 8 and 74 +/- 15 fmol/mg protein, respectively). In contrast, there were no significant differences in ANF receptor affinity values or dose-dependent increases in ANF-stimulated cGMP generation among the age groups studied. In competition studies, we observed effective displacement of 125I-ANF by C-ANF4-23, a ring-deleted ANF analogue, in adult, juvenile, and newborn glomeruli; however, C-ANF displaced only about 50% of the 125I-ANF in fetal tissue. The addition of C-ANF did not elicit cGMP generation, nor did C-ANF affect ANF-induced cGMP generation in fetal, newborn, or adult glomeruli. These results indicate that 1) the ANF receptor-guanylate cyclase system is intact in 29-d fetal rabbit glomeruli, and 2) the ANF-induced cGMP formation is similar in fetal and adult animals, whereas receptor binding capacity is relatively higher in adult glomeruli. These results suggest a higher proportion of nonguanylate cyclase-coupled ANF receptors in the mature rabbit.

Dual regulation of atrial natriuretic factor-dependent guanylate cyclase activity by ATP.

The 'second messenger' of certain atrial natriuretic factor (ANF) signals is cyclic GMP. One type of ANF receptor linked to the synthesis of cyclic GMP is a transmembrane protein which contains both the ANF-binding and guanylate cyclase activities. The consensus is that the maximal activity of this guanylate cyclase is observed in the presence of ATP. We now show that depending upon the cofactors Mg2+ or Mn2+, ATP stimulates or inhibits the ANF-dependent guanylate cyclase activity in the testicular plasma membranes: stimulation in the presence of Mg2+ and inhibition in the presence of Mn2+. With Mg2+ as cofactor neither ATP nor ANF stimulate the cyclase activity--it is only when the two are together that the enzyme is activated. Furthermore, this investigation for the first time demonstrates binding of ATP to the ANF receptor guanylate cyclase, suggesting that ATP-mediated responses could occur by direct ATP binding to the cyclase.