Ca²⁺ Modulation of ANF-RGC: New Signaling Paradigm Interlocked with Blood Pressure Regulation

Abstract

ANF-RGC is the prototype receptor membrane guanylate cyclase being both the receptor and the signal transducer of the most hypotensive hormones, ANF and BNP. It is a single trans-membrane protein. After binding these hormones at the extracellular domain, ANF-RGC at its intracellular domain signals the activation of the C-terminal catalytic module and accelerates the production of the second messenger, cyclic GMP, which controls blood pressure, cardiac vasculature, and fluid secretion. At present this is the sole transduction mechanism and the physiological function of ANF-RGC. Through comprehensive studies involving biochemistry, immunohistochemistry, and blood pressure measurements in mice with targeted gene deletions, the present study demonstrates a new signaling model of ANF-RGC that also controls blood pressure. In this model (1) ANF-RGC is not the transducer of ANF and BNP; (2) its extracellular domain is not used for signaling; and (3) the signal-flow is not downstream from the extracellular domain to the core catalytic domain. Instead, the signal is the intracellular Ca²⁺, which is translated at the site of its reception, at the core catalytic domain of ANF-RGC. A model for this Ca²⁺ signal transduction is diagrammed. It captures Ca²⁺ through its Ca²⁺ sensor myristoylated neurocalcin δ and up-regulates ANF-RGC activity with a K_1/2 of 0.5 μM. The neurocalcin δ-modulated domain resides in the ⁸⁴⁹DIVGFTALSAESTPMQVV⁸⁶⁶ segment of ANF-RGC, which is a part of the core catalytic domain. Thereby, ANF-RGC is primed to receive, transmit and translate the Ca²⁺ signals into the generation of cyclic GMP at a rapid rate. The study defines a new paradigm of the membrane guanylate cyclase signaling, which is linked to the physiology of cardiac vasculature regulation and possibly also to fluid secretion.

Introduction

The discovery of ANF-RGC (Atrial Natriuretic Factor Receptor Guanylate Cyclase), the first member of the membrane guanylate cycles family, was a landmark event in the field of cellular signaling^1–9. It established a new field of membrane guanylate cyclases. With the inclusion of two other members, CNP-RGC, the receptor of C-type natriuretic peptide (CNP)^{10, 11} and STa-RGC, the receptor of heat stable enterotoxin, guanylin and uroguanylin^{12, 13}, it demonstrated that the membrane guanylate cyclase is a surface receptor family. The sequential developments in the field disclosed three branches of the family: (1) the original, surface receptor; (2) the Ca²⁺-modulated, ROS-GC, with two members, ROS-GC1 and ROS-GC2; and (3) the odorant (uroguanylin) surface receptor and Ca²⁺-modulated with one member, ONE-GC⁸. ROS-GC primarily exists in the vision-linked sensory neurons. There it is a central component of phototransduction. It is also present in the olfactory bulb neurons but its physiological linkage with olfaction has not been established¹⁴. ONE-GC is the olfactory receptor of the odorant uroguanylin^{15, 16} and, indirectly, of atmospheric CO₂^{17, 18}.

Common structural traits of the membrane guanylate cyclase family are that its members are single transmembrane-spanning proteins, composed of modular blocks⁸. Functionally, they are homodimeric. In each monomeric subunit, the transmembrane module divides the protein into two roughly equal portions, extracellular and intracellular. Their core catalytic domains are conserved, all residing in the intracellular region of their respective cyclases.

A striking topographical difference on the orientation of the core catalytic domain between the subfamilies of the surface receptor and the ROS-GC and ONE-GC exists. This is caused by the C-terminal extension (CTE) tails of ROS-GC and ONE-GC, which are absent in the surface receptor. Thus, in the ROS-GC and ONE-GC subfamilies the catalytic domain flows into CTE, which is not the case with the surface receptor subfamily.

The core catalytic domain of ROS-GC and ONE-GC is modulated differently than that of surface receptor subfamily members. ROS-GC and ONE-GC sense Ca²⁺ signals via their Ca²⁺ sensor domains, which reside in the intracellular region on the N- and C-terminal sides. There are four Ca²⁺ sensors of ROS-GC1: GCAP1, GCAP2, neurocalcin δ and S100B, each bound to its respective domain¹⁹. Importantly, the GCAP2 and S100B domains overlap and reside on CTE and the GCAP1-binding domain is located N-terminally to the catalytic domain. ONE-GC is modulated by three Ca²⁺ sensors: GCAP1, neurocalcin δ and hippocalcin^20–22. Their targeted domains reside on the core catalytic domain of ONE-GC^20–22. Intriguingly, GCAP1-modulated Ca²⁺ signal stimulates ONE-GC activity²⁰, in contrast to ROS-GC1 where it inhibits the cyclase activity.

There is one prominent difference between the transduction mechanisms of ROS-GC and ONE-GC. While ROS-GC is solely modulated by the Ca²⁺ signals generated inside the sensory neurons, ONE-GC transduction mechanism is more complex. Being a uroguanylin receptor, it generates the uroguanylin signal at its extracellular domain and via its Ca²⁺ sensors amplifies the signal at its intracellular domain²³. Additionally, ONE-GC is also modulated in a Ca²⁺-independent fashion. It transduces atmospheric CO₂ signal via carbonic anhydrase enzyme¹⁷. The enzyme generates bicarbonate which, in turn, binds and stimulates the ONE-GC core catalytic domain, aa880-1028¹⁸.

Noting these mechanistic complexities and similarities, these authors proposed a unified signaling theme of the ROS-GC and ONE-GC subfamilies where “Ca²⁺-sensors and ROS-GC are interlocked sensory transduction elements”¹⁹. The present study extends this theme to ANF-RGC. It discloses new ANF-RGC transduction mechanism. In this mechanism, the receptor domain where ANF and BNP bind is not involved in signaling. In fact, the extracellular domain, trans-membrane and the ATP-regulated domain are bypassed and the core catalytic domain is directly stimulated and generates cyclic GMP. The signal for the stimulation is Ca²⁺. In mice, the disabling of the Ca²⁺ signaling mechanism through genetic modification leads to hypertension. The findings define a new signal transduction model of the membrane guanylate cyclase family and link it with blood pressure regulation.

EXPERIMENTAL PROCEDURES

Expression in COS cells

COS cells maintained in DMEM medium supplemented with 10% fetal bovine serum and antibiotics were transfected with ANF-RGC cDNA using calcium phosphate co-precipitation technique²⁴. 64 hr after transfection cells were washed with 50mM Tris-HCl pH 7.4/10 mM Mg²⁺ buffer, homogenized and the particulate fraction pelleted by centrifugation.

ANF-RGC soluble construct aa 788-1029

A full-length ANF-RGC cDNA in pcDNA3 expression vector was used for PCR amplification. The amplified fragment coding for ANF-RGC region aa 788-1029 was cloned into pFastBac vector (Bac-to-Bac Baculovirus expression system, Invitrogen system) yielding 6-His tag at N-terminus. The plasmid was sequenced to confirm its identity. Using DH10Bac cells the recombinant bacmid was generated and transfected into Sf-9 cells to produce recombinant baculoviruses. For protein expression, suspension of Sf-9 cells was infected at a rate of 6–10 MOI at a cell density of ~1×10⁶ cells/ml. Cells were harvested 70–80 hrs after infection lysed and the protein was purified on a Ni-NTA column and through FPLC on Superdex 75 column.

Guanylate cyclase activity assay

The membrane fraction was incubated on ice-bath with or without neurocalcin δ in the assay system containing 10 mM theophylline, 15 mM phosphocreatine, 20 μg creatine kinase and 50 mM Tris-HCl, pH 7.5. Appropriate Ca²⁺ concentrations were adjusted with pre-calibrated Ca²⁺/EGTA solutions of a Ca²⁺ buffer kit (Molecular Probes/Invitrogen). The total assay volume was 25 μl. The reaction was initiated by addition of the substrate solution (4 mM MgCl₂ and 1mM GTP, final concentration) and maintained by incubation at 37 °C for 10 min. The reaction was terminated by the addition of 225 μl of 50 mM sodium acetate buffer, pH 6.2 followed by heating on a boiling water bath for 3 min. The amount of cyclic GMP formed was determined by radioimmunoassay²⁵.

Expression and purification of neurocalcin δ

Myristoylated neurocalcin δ was expressed and purified according to the protocol described previously²⁶. Nonmyristoylated neurocalcin δ was expressed and purified following the same protocol except that the cells expressing neurocalcin δ were not co-transfected with N-myristoyltransferase and myristic acid was not added to the culture.

Antibodies

Antibodies against ANF-RGC and neurocalcin δ were raised in rabbits. Their specificities were described previously^{20, 26}. The antibodies were affinity purified. PDE2A antibody was purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Secondary antibodies conjugated to a fluorescent dye (DyLight 488 and DyLight 549) were purchased from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA.

Protein quantification for determination of ANF-RGC catalytic efficiency

The antigen for ANF-RGC antibody (ANF-RGC fragment aa 486-661) was expressed and purified as described previously²⁷. Its concentration was determined using purified bovine serum albumin standards. Aliquots of the antigen (10 - 0.1 ng) were diluted in a Laemmli SDS sample buffer and loaded next to 50 μg (total protein) of COS cells membranes expressing ANF-RGC. After electrophoresis in 7% SDS-polyacrylamide gel the proteins were transferred onto PVDF membrane. The membrane was blocked overnight in blocking solution (2% BSA in Tris-buffered saline containing Tween-20), immunostained with anti-ANF-RGC polyclonal antibody and developed using a Pierce/ThermoFisher Scientific SuperSignal reagent kit and goat anti rabbit peroxidase conjugates. The images were collected by exposing the membrane to X-ray Kodak film. The intensities of the signals were quantified from the calibration curve produced by the antigen standards.

Immunohistochemistry

Mice were sacrificed by lethal injection of ketamine/xylazine (the protocol approved by the Salus University IUCAC) and perfused through the heart, first with a standard Tris-buffered saline (TBS) and then with freshly prepared 4% paraformaldehyde in TBS. The adrenal glands were removed and fixed for 1–4 hours in 4% paraformaldehyde with TBS at 4°C, cryoprotected in 30% sucrose overnight at 4°C and cut into 20 μm sections using Hacker-Bright OTF5000 microtome cryostat (HACKER Instruments and Industries Inc., Winnsboro, SC). The sections were washed with TBS, blocked in 10% normal donkey serum in TBS/0.5% Triton X-100 (TBST) for 1hr at room temperature, washed with TBST, incubated with respective antibody in blocking solution overnight at 4°C, washed with TBST, incubated with DyLight (488 or 549) conjugated donkey anti-rabbit (or as necessary donkey anti-goat) antibody (200:1) for 1 hr and again washed with TTBS. Images were acquired using an inverted Olympus IX81 microscope/FV1000 Spectral laser confocal system, and analyzed using Olympus FluoView FV10-ASW software. Digital images were processed using Adobe Photoshop software.

Genetically modified mice

Care of the experimental animals conformed to the protocols approved by the IACUC at Salus University and was in strict compliance with the NIH guidelines.

Neurocalcin δ^+/− (NCδ^+/−)

Two mouse genomic fragments of neurocalcin δ were amplified by PCR: first, from intron 14 to codon for V¹² (exon 15); second, within intron 15. The genomic distance between these two fragments was ~1000 bp. They were cloned individually into the multiple cloning sites, separated by the PGK-neo cassette of the pPNT vector. The linear DNA consisting of both neurocalcin δ fragments separated by the PGK-neo cassette was released from the vector and electroporated into mouse ES cells. The clones with homologous recombination were injected into C57BL/6 blastocytes. Male chimera was bred to C57BL/6 females. NCδ^+/− heterozygotes were obtained. The attempts (one-year) to breed homozygous NCδ^−/− mice have to date been unsuccessful.

Membrane preparation

Adrenal glands were removed from the wild type (control) or the genetically modified mice. The tissues were powdered using pistol ant mortar under liquid nitrogen and homogenized in a buffer consisting of 250 mM sucrose, 10 mM Tris-HCl pH 7.4, 1 mM EGTA (or 10 μM Ca²⁺), containing a protease inhibitors cocktail, centrifuged at 1,000g and then at 100,000g to pellet the membrane fraction. This fraction was suspended in 50 mM Tris-HCl pH 7.4/10 mM MgCl₂ buffer and used for guanylate cyclase activity assay.

Blood pressure measurements

Systolic blood pressures of neurocalcin δ gene-targeted mice (NCδ^+/−) and their isogenic controls (wild type: NCδ^+/+) were measured every day for one week by the noninvasive computerized tail-cuff method with CODA (Kent Scientific) according to the manufacturer’s protocol. An average blood pressure level of 10 sessions per day was calculated for analysis after 3 days of mice training. The mice were maintained on normal chow and drinking water available ad libitum.

Statistical analysis

The blood pressure data are expressed as mean ± SD. Differences between the two groups analyzed, NCδ^+/+ (wt, control) and NCδ^+/−, were compared using Student’s t test. A P value of <0.05 was considered significant.

RESULTS

ANF-RGC is a bimodal transduction switch

ANF-RGC is the receptor and the transducer of the signals generated by the peptide hormones ANF and BNP⁹. Binding of these hormones at the ANF-RGC extracellular domain signals activation of the C-terminal catalytic module and accelerates the production of their second messenger, cyclic GMP, which controls blood pressure, cardiac vasculature, and fluid secretion. The mechanism of ANF-RGC signal transduction is that ligand binding to its receptor domain triggers a chain of structural changes. These changes are carried through the trans-membrane to the intracellular domain where, by ATP-dependent changes within the ARM domain they are processed further. Ultimately, they are sensed by the core catalytic module, which translates them into the production of cyclic GMP⁹. This is the traditional and the only established ANF-RGC signal transduction mechanism.

The myristoylated (myr) form of neurocalcin δ (NCδ) is a Ca²⁺-sensor protein of ROS-GC1 signaling^{19, 26}. In the course of mapping its domain on ROS-GC1 to which it binds and transmits Ca²⁺ signal for its translation into the generation of cyclic GMP, these authors made a remarkable observation. This was that it binds directly to the core catalytic domain and, thereby, activates ROS-GC1²⁸. Protein database search indicated sequence conservation of the catalytic domain in the membrane guanylate cyclase family. Therefore, to test whether ANF-RGC is also linked with its Ca²⁺ signaling²⁰, recombinant ANF-RGC expressed in COS cells was exposed to varying concentrations of Ca²⁺ in the presence of 2 μM of myr-NCδ. The results, presented in figure 1A, show that Ca²⁺ in a dose-dependent fashion with an EC₅₀ of 0.5 μM stimulated ANF-RGC activity, V_max occurring at ~ 1 μM Ca²⁺. Thus, Ca²⁺ via its sensor NCδ signals ANF-RGC activation. As expected, side-by-side experiment showed that ANF in the presence of ATP stimulated ANF-RGC with an EC₅₀ of 1 nM (Fig. 1B). These results demonstrate that ANF-RGC is a bimodal signal transduction switch; one mode transducing the ANF signal and the other, the Ca²⁺ signal into the production of cyclic GMP.

Figure 1. ANF-RGC is responsive to ANF and Ca²⁺ signals.

COS cells were transfected with ANF-RGC cDNA. 60 hr after transfection the membrane fraction was prepared and assayed for guanylate cyclase activity in the presence of increasing concentrations of Ca²⁺ and 2 μM recombinant myristoylated neurocalcin δ (A) or indicated concentrations of ANF and 0.8 mM ATP. Ca²⁺ or EGTA were not added to the assay mixture. (B). The experiments were done in triplicate and repeated two times for reproducibility. The results presented are average ± SD from these experiments. The EC₅₀ values were determined graphically.

To determine the liaison between these two signaling modes, the membranes of COS cells expressing ANF-RGC were exposed first to 1 μM Ca²⁺ in the presence of 2 μM myr-NCδ and then to increasing concentrations, ranging from 10⁻¹¹ M to 10⁻⁶ M, of ANF and constant 0.8 mM ATP. Myr-NCδ caused 3.2-fold stimulation from 31.6 to 102 pmol cyclic GMP min⁻¹ (mg prot)⁻¹ and ANF an additional 4.5-fold stimulation to 454 pmol cyclic GMP min⁻¹ (mg prot)⁻¹, amounting to a total of 7.7-fold stimulation of ANF-RGC activity (Fig. 2A). Thus, the results demonstrate that the NCδ and ANF effects are additive. To further substantiate this conclusion, membranes of COS cells expressing ANF-RGC were exposed to 2 μM NCδ, 10⁻⁹ or 10⁻⁷ M ANF, 0.8 mM ATP and increasing concentrations of Ca²⁺ and the cyclase activity was assessed. At low, ~10 nM Ca²⁺, the ANF-RGC activity was 70 ± 5 pmol cyclic GMP min⁻¹(mg protein)⁻¹ in the presence 10⁻⁹ M ANF and 109 ± 9 pmol cyclic GMP min⁻¹(mg protein)⁻¹ in the presence 10⁻⁷ M ANF (Fig. 2B). These activities are comparable to those achieved by ANF-RGC in the presence of only 10⁻⁹ or 10⁻⁷ M ANF (compare figure 1A) and consistent with the fact that Ca²⁺-free NCδ does not stimulate ANF-RGC (compare figure 1B). Thus, the cyclase activity at low Ca²⁺ concentration is regulated exclusively by ANF. Increasing concentration of Ca²⁺ in the assay mixture resulted in increased cyclase activity at both ANF concentrations. Because NCδ concentration was constant, 2 μM, these results indicate that increasing amount of Ca²⁺-bound NCδ in the assay mixture resulted in increased ANF-RGC activity. The half-maximal ANF-RGC activation was achieved at ~0.4 μM Ca²⁺ and maximal, at ~1 μM. Based on these results it is concluded that the two modes of ANF-RGC signaling, hormonal ANF and NCδ-mediated Ca²⁺, are processed by individual mechanisms. Because deletion of the extracellular domain of ANF-RGC has no influence on the NCδ-modulated Ca²⁺ signaling but is critical for the ANF signaling²⁰, it is concluded that the two signaling modes are generated through different domains of ANF-RGC: ANF by the extracellular domain and Ca²⁺ by the intracellular domain.

Figure 2. The ANF and Ca²⁺ stimulatory effects on the activity of ANF-RGC are additive.

(A) Membranes of COS cells expressing ANF-RGC were preincubated with 1 μM Ca²⁺ and 2 μM recombinant myristoylated neurocalcin δ (NCδ). This was followed by incubation with indicated concentrations of ANF and 0.8 mM ATP and the guanylate cyclase activity was assayed as described in the Experimental Procedures. (B) Membranes of COS cells expressing ANF-RGC were incubated with 10⁻⁹ or 10⁻⁷ M ANF in the presence of 0.8 mM ATP, 2 μM NCδ and increasing concentrations of Ca²⁺. The guanylate cyclase activity was assessed. The experiments were repeated two times with different preparation of transfected cells. The results shown are from one experiment done in triplicate. The ANF EC₅₀ values were determined graphically.

Myristoylated form of NCδ is necessary for effective Ca²⁺ signaling of ANF-RGC

NCδ belongs to the family of neuronal calcium sensor proteins (NCS). Characteristic feature of majority, but not all, of these proteins is that they are myristoylated at their N-termini and the myristoylation is important for their cellular functions.

To determine if myristoylation is required for NCδ to transduce the Ca²⁺ signal for ANF-RGC activation, its myristoylated and nonmyristoylated forms were expressed, purified and individually incubated in the presence of 1 μM Ca²⁺ with membranes of COS cells expressing recombinant ANF-RGC. The results are shown in figure 3. The nonmyristoylated form caused only partial stimulation of ANF-RGC: from 24 to 43 pmol cyclic GMP min⁻¹(mg prot)⁻¹ (Fig. 3: open circles). In contrast, the myristoylated form robustly stimulated ANF-RGC: from 24 to 108 pmol cyclic GMP min⁻¹(mg prot)⁻¹ (Fig. 3: closed circles). The EC₅₀ values of the both forms were identical, 0.5 μM, indicating their similar affinities for ANF-RGC. In the absence of Ca²⁺ [presence of 1 mM EGTA], both the myristoylated and nonmyristoylated forms caused no stimulation of ANF-RGC (not shown). The conclusion, therefore, is that the myristoylated form of NCδ is the transducer of Ca²⁺ signal in the activation of ANF-RGC.

Figure 3. Myristoylated neurocalcin δ effectively transmits the Ca²⁺ signal to ANF-RGC.

Membranes of COS cells expressing ANF-RGC were exposed to 1 μM Ca²⁺ and increasing concentrations of neurocalcin δ in its myristoylated (myr NCδ) or nonmyristoylated (nonmyr NCδ) form. The experiment was done in triplicate and repeated four times with separate membrane or neurocalcin d preparations. The results shown (mean ± SD) are from one experiment.

In addition to increasing the V_max, myristoylation of NCδ influences other enzymatic properties of ANF-RGC. It lowers its K_M for GTP from 0.86 mM to 0.37 mM and raises its enzymatic efficiency, k_cat, from 6.5 ± 0.3 cyclic GMP sec⁻¹ to 41.4 ± 0.5 cyclic GMP sec⁻¹.

Core catalytic region of ANF-RGC, amino acids V⁸⁵¹-V⁸⁶⁶, binds myristoylated NCδ

Guided by the information that the NCδ binding site on ROS-GC1, aa V⁸⁹²-D⁹¹², and on ONE-GC, aa M⁸⁸⁰-L⁹²¹, resides on the core catalytic domain^{16, 28}, and this domain is conserved in all members of the membrane guanylate cyclase family, it was tested whether the corresponding domain in ANF-RGC is the target site of NCδ. Two approaches were used. In first, the ANF-RGC fragment, aa 788-1029, encompassing the core catalytic domain, I⁸²⁰-G¹⁰²⁹, was expressed as a soluble protein and purified to homogeneity. The expressed protein was biologically active and possessed intrinsic guanylate cyclase activity of 18 ± 4 pmol cyclic GMP min⁻¹ mg prot⁻¹. That the fragment is Ca²⁺ modulated via NCδ was verified by its dose-dependent response to varying concentrations of NCδ in the presence of 1 μM Ca²⁺. Myr-NCδ stimulated its guanylate cyclase activity in a dose-dependent fashion with an EC₅₀ of 0.7 μM (Fig. 4A), the value comparable to 0.5 μM estimated for the full-length ANF-RGC. These results demonstrated that the NCδ signaling site resides in this fragment, which contains the core catalytic domain of ANF-RGC.

Figure 4.

A. Neurocalcin δ interacts with the catalytic domain of ANF-RGC. The ANF-RGC fragment aa 788-1029 was expressed in SF-9 cells and purified to homogeneity. The expressed protein was assayed guanylate cyclase activity in the presence of 1 μM Ca²⁺ and indicated concentrations of myristoylated neurocalcin δ. B. Site of neurocalcin δ interaction with ANF-RGC – functional interference. ANF-RGC expressed in COS cells was exposed first to 2 μM myristoylated neurocalcin δ and 1 μM Ca²⁺ than to increasing concentrations (up to 0.5 mM) of peptide covering ANF-RGC sequence aa D⁸⁴⁹-V⁸⁶⁶ or control scrambled peptide, which had the same as D⁸⁴⁹-V⁸⁶⁶ peptide amino acid composition but random sequence. The experiments were done in triplicate and repeated two times. The results presented are mean ± SD of these experiments. The IC₅₀ values were determined graphically.

In the second approach, the NCδ binding site was precisely mapped through peptide competition analysis. Advantage was taken from the knowledge that the experimentally validated sites on ROS-GC1 and ONE-GC are located within the aa segments V⁸³⁷-L⁸⁵⁸ and V⁹⁰⁰-L⁹²¹, respectively and the segments have, among themselves, 100% sequence conservation. The corresponding site on ANF-RGC, ⁸⁴⁹DIVGFTALSAESTPMQVVTLLMQ⁸⁷¹, has 70% sequence conservation in comparison with ROS-GC1 and ONE-GC. An ANF-RGC sequence-specific peptide ⁸⁴⁹DIVGFTALSAESTPMQVV⁸⁶⁶ was synthesized and used in a functional interference experiment. A scrambled peptide VDASAIVMFVGLPSQTET was used as control in this experiment.

COS cell membranes expressing ANF-RGC were incubated with 2 μM myr-NCδ, increasing concentrations of the peptide and 1 μM Ca²⁺ (Fig. 4B). The peptide caused almost complete inhibition of the NCδ-stimulated ANF-RGC activity at 200 μM with an IC₅₀ value of 80 μM. Under the same conditions the scrambled peptide did not exhibit any inhibitory effect. These results demonstrate that the ANF-RGC region ⁸⁴⁹DIVGFTALSAESTPMQVV⁸⁶⁶ mediates NCδ-dependent Ca²⁺ stimulation of ANF-RGC activity. This region is a part of the core catalytic domain and common to the corresponding sites in other membrane guanylate cyclases²⁸, it has a secondary structure of helix-loop-helix and is acidic in nature with a pI of 3.37.

Interactive NCδ dimer with ANF-RGC dimer constitutes the functional Ca²⁺ signal transduction unit

The present concept based on the complementary biochemical and homology based modeling studies indicates that the secondary structure of the functional form of all membrane guanylate cyclases is homodimeric^28–30. The contact points for their homo-dimeric formation reside (1) in their extracellular domain³¹, in the intracellular domains of (2) highly conserved dimerization domain³² and (3) core catalytic core domain²⁸. The X-ray crystallographic studies have demonstrated that NCδ also exists as a dimer³³. Thus the theoretical prediction was that the Ca²⁺-modulated functional unit is the interactive NCδ dimer and ANF-RGC dimer.

To assess this prediction experimentally, monomeric and dimeric forms of myristoylated NCδ were separated by FPLC and were used for reconstitution experiment with ANF-RGC. The dimeric form stimulated ANF-RGC 5-fold above the basal activity, from 24 to 120 pmol cGMP min⁻¹ (mg prot)⁻¹ (Fig. 5). Three separate experiments yielded calculated Hill coefficient values for the stimulation of ANF-RGC as 1.08 ± 0.21, 0.91 ± 0.29 and 0.9 ± 0.23. The stimulation by the monomeric form was only marginal, from 24 to 38 pmol cGMP min⁻¹ (mg prot)⁻¹ (Fig. 5). These results, thus, demonstrate and validate the prediction that the functional Ca²⁺ signal transduction unit is composed of one NCδ dimer and one ANF-RGC dimer.

Figure 5. Dimer is the functional entity of neurocalcin δ.

Myristoylated neurocalcin δ was expressed and purified by FPLC as described in “Experimental Procedures”. The monomeric and dimeric fractions were collected. These were individually used to assess their stimulatory effect on ANF-RGC activity expressed in COS cells. The experiment was done in triplicate and repeated four times with different neurocalcin δ preparations and ANF-RGC expressions. The results shown are from one experiment.

The functional Ca²⁺ signal transduction unit is present in the mouse adrenal gland

To grasp the physiological relevance of the above biochemical findings, these authors followed their preliminary study where they observed first through Western blot analysis and then through immunohistochemistry the co-presence of both elements of the functional transduction unit: NCδ and ANF-RGC in the glomerulosa cells of the mouse adrenal gland²⁰.

The first question was: does the mouse adrenal gland contain Ca²⁺-modulated membrane guanylate cyclase activity? The rational was founded on the earlier studies, which had shown that two vital endocrine glands containing functional ANF-RGC transduction system were adrenal and kidney^{7, 34, 35}. Mouse adrenal gland was chosen for these studies. The gland was homogenized in the presence of 1 mM EGTA or 10 μM Ca²⁺ and the particulate fractions were prepared. Each preparation was analyzed for guanylate cyclase activity in the presence of 1 mM EGTA or 1 μM Ca²⁺. The membranes isolated in the absence of Ca²⁺ (1 mM EGTA) exhibited comparable cyclase activity regardless of the presence or absence of Ca²⁺ in the assay mixture. The activities were 59 ± 5 and 66 ± 5 pmol cyclic GMP min⁻¹ (mg prot)⁻¹ in the absence and presence of Ca²⁺, respectively (Fig. 6: panel “−Ca²⁺”). However, the membranes isolated in the presence of Ca²⁺ exhibited Ca²⁺-dependent activity; in the absence of Ca²⁺ in the assay mixture it was 70 ± 5 pmol cyclic GMP min⁻¹ (mg prot)⁻¹, the activity comparable to the activity in membranes isolated in the absence of Ca²⁺, but in its presence the activity was 210 ± 13 pmol cyclic GMP min⁻¹ (mg prot)⁻¹ (Fig. 6: panel “+Ca²⁺”). These results demonstrate that the adrenal gland houses functional Ca²⁺-dependent ANF-RGC signal transduction machinery.

Figure 6. The ANF-RGC activity in mouse adrenal gland is Ca²⁺-dependent.

Particulate fractions of mouse adrenal gland were isolated in the presence of 1 mM EGTA (panel “−Ca^2+”) or 10 μM Ca²⁺ (panel “+Ca²⁺”) as described in the “Experimental Procedures” section. The membranes were assayed for guanylate cyclase activity in the presence of 1 mM EGTA or 1 μM Ca^2+. The experiment was repeated three times with separate membrane preparations. The results are mean ± SD of these experiments.

The second question was: Is the Ca²⁺-dependent ANF-RGC signal transduction machinery expressed in the mouse adrenocortical glomerulosa cells NCδ-modulated? This problem was approached through immunocytochemistry. Because both antibodies at hand, against ANF-RGC and NCδ, were raised in rabbits, using them for co-immunostaining experiments was not possible. Hence, the goal was achieved indirectly. It has been established that the cyclic GMP-stimulated phosphodiesterase PDE2A is a specific marker for the glomerulosa cells³⁶. Therefore, the co-presence of ANF-RGC and PDE2A was determined first, then, of NCδ and PDE2A, and from them, the co-presence of ANF-RGC, NCδ and PDE2A was assessed.

Figure 7A “ANF-RGC” shows that the glomerulosa cells in this section exhibit an intense signal (green) generated with the ANF-RGC antibody. Figure 7A “PDE2A” shows the same section with the red signal generated with the PDE2A antibody. The “merged” image of the two signals generating yellow fluorescence shows that the immunostaining of ANF-RGC and PDE2A overlap, demonstrating that these two proteins are co-present in the glomerulosa cells of the adrenal gland. Similarly, double staining with NCδ and PDE2A antibodies (Fig. 7B) demonstrates that NCδ and PDE2A are also co-present in these cells. It is, thus concluded that ANF-RGC and NCδ together with PDE2A are present in the same adrenocortical glomerulosa cells.

Figure 7. Neurocalcin δ is expressed in the same as ANF-RGC and PDE2A mouse adrenocortical zona glomerulosa cells.

Cryosections of the mouse adrenal gland were immuno-stained with ANF-RGC and PDE2A antibodies (A) or neurocalcin δ and PDE2A antibodies (B) as described in “Experimental Procedures”. The right-hand panels (“Merged”) present the composite images of ANF-RGC and PDE2 or neurocalcin δ and PDE2 staining and document that both ANF-RGC and neurocalcin δ are co-expressed with PDE2A.

NCδ is the Ca²⁺-sensor modulator of ANF-RGC in the adrenocortical glomerulosa cells

To demonstrate that the ANF-RGC Ca²⁺ signal transduction unit present in the mouse adrenocortical glomerulosa cells is functional, two independent approaches were used. First, a mouse model with deletion of one copy of the NCδ gene, NCδ^+/−, was constructed. It was reasoned that if NCδ is indeed the Ca²⁺-sensor modulator of ANF-RGC, in the adrenal glands of these mice the NCδ-modulated Ca²⁺ signaling pathway should be half as active as in those of the wild type mice (wt; NCδ^+/+). To test this prediction, the particulate fractions of the adrenal glands from wild type and NCδ^+/− mice were prepared in the presence and absence of Ca²⁺ and the experiment was performed according to the protocol described earlier. Similar to the results presented in figure 6, the guanylate cyclase activity in membranes isolated in the absence of Ca²⁺ was about 65 ± 8 pmol cyclic GMP min⁻¹ (mg prot)⁻¹ for the wt and NCδ^+/− mice and the activity was not affected by the presence or absence of Ca²⁺ in the assay mixture (Fig. 8A). However, the cyclase activity in membranes isolated in the presence of Ca²⁺ was strongly dependent on the mice genotype and Ca²⁺ in the assay mixture. When assessed in the absence of Ca²⁺, the activity was 70 ± 8 pmol cyclic GMP min⁻¹ (mg prot)⁻¹ for the wt and NCδ^+/− mice but when assessed in the presence of 1 μM Ca²⁺ the activity was 223 ± 20 pmol cyclic GMP min⁻¹ (mg prot)⁻¹ for the wild type mice and 135 ± 10 pmol cyclic GMP min⁻¹ (mg prot)⁻¹ for the NCδ^+/− mice. These results, as predicted, demonstrate that the Ca²⁺-dependent NCδ-modulated ANF-RGC signaling pathway in the mice with one copy of NCδ gene deleted (NCδ^+/−) is functionally half as active as in the wild type mice. To further validate that the lowering of the Ca²⁺-dependent cyclase activity in the adrenal gland membranes of NCδ^+/− mice is the exclusive consequence of lower NCδ expression, 2 μM exogenous NCδ was added to the NCδ^+/+ and NCδ^+/− membranes (isolated in the presence of Ca²⁺) and the cyclase activity was assessed in the presence of 1 μM Ca²⁺. The cyclase activity in the NCδ^+/+ adrenal membranes increased only minimally, from 220 to 279 ± 21 pmol cyclic GMP min-1 (mg protein)-1 but in the NCδ^+/− membranes, from 133 to 284 ± 24 (Fig. 8B). Thus, the activity achieved was practically the same for both types of membranes. These results demonstrate that addition of exogenous NCδ to the NCδ^+/− adrenal gland membranes restores the guanylate cyclase activity and brings it to the level of activity in the NCδ^+/+ membranes. The slight activity increase in the NCδ^+/+ membranes can be explained by a partial loss of the native NCδ during the membrane preparation procedure.

Figure 8. Neurocalcin δ modulates ANF-RGC activity in mouse adrenal gland.

(A) Adrenal glands were removed from the wild type (WT) and neurocalcin δ^+/− (NCδ^+/−) mice and their particulate fractions were isolated in the presence of 1 mM EGTA (panels “−Ca²⁺” in “WT membranes” and “NCδ^+/− membranes” sections of the figure) or 10 μM Ca²⁺ (panels “+Ca²⁺” in both sections of the figure). These were assayed for guanylate cyclase activity in the presence of 1 mM EGTA or 1 μM Ca²⁺. (B) 2 μM myr-NCδ was added to the adrenal gland membranes isolated in the presence of 10 μM Ca²⁺ from the wild type (“WT memb.”) or NCδ^+/− (“NCδ^+/− memb.”) mice and the guanylate cyclase activity was assessed in the presence of 1 μM Ca²⁺. The experiments were repeated two times with separate membrane preparations. The results are mean ± SD of these experiments.

In the second approach, Ca²⁺-dependent reconstitution of the transduction system was conducted using the heterologous system of COS cells. The cells were co-transfected with ANF-RGC and NCδ cDNAs. Their membranes were isolated in the presence and absence of Ca²⁺ and assayed for guanylate cyclase activity without and with Ca²⁺ in the assay mixture. The results are shown in figure 9. The cyclase activity remained at the basal level in the membranes isolated without Ca²⁺ but it increased 3.5-fold in membranes isolated and assayed in a buffer containing Ca²⁺. Membranes of COS cells transfected with ANF-RGC cDNA alone exhibited cyclase activity of 25 ± 4 pmol cyclic GMP min⁻¹ (mg prot)⁻¹ and the activity was unaffected by the presence or absence of Ca²⁺ during their preparation or activity assay. Because the only difference between the co-transfected cells and those transfected with ANF-RGC cDNA only was the expression of NCδ in the co-transfected cells, these results demonstrate that in living cells NCδ exhibits Ca²⁺-dependent association with the membranes and transmits the Ca²⁺ signal to ANF-RGC.

Figure 9. Ca²⁺-neurocalcin δ modulation of ANF-RGC activity is reconstituted in co-transfected COS cells.

COS cells were co-transfected with ANF-RGC and neurocalcin δ cDNAs. On the third day post-transfection their membranes were isolated and assayed for guanylate cyclase activity as described in legends to figures 8 and 10. The experiment was repeated two times. The results presented (mean ± SD) are from one experiment.

Together, the results of these two approaches establish that the Ca²⁺ modulation of ANF-RGC is not only an in vitro phenomenon but it also occurs in vivo both in mouse adrenal gland and in reconstituted system of transfected cells, and NCδ is the transducer of the Ca²⁺ signal.

NCδ-modulated Ca²⁺ signaling ANF-RGC transduction system modulates blood pressure

The primary role of ANF-RGC in the adrenal gland is to offset the renin-angiotensin system and inhibit aldosterone synthesis, thus, to lower blood pressure^37–39. It does so by responding to the two most hypotensive peptide hormones, ANF and BNP, and producing their second messenger cyclic GMP. Cyclic GMP then elicits the natriuretic, diuretic, vasorelaxant, and anti-proliferative effects programmed by the ANF and BNP signals. The obvious question, therefore, was: Is the Ca²⁺-neurocalcin δ-ANF-RGC transduction system also involved in rgulation of blood pressure. To answer it, the systolic blood pressure of the NCδ^+/− and of the isogenic control (wild type; NCδ^+/+) was measured.

After three days of mock measuremnt sessions for training purposes, the systolic blood pressure was determined to be 92 ± 6 mm Hg for the wild type mice and 127 ± 9 mm Hg for the NCδ^+/− mice (Fig. 10). The increase was statistically highly significant (P<0.005). These results clearly show that the deletion of one gene copy of neurocalcin δ leads to significant increase of blood pressure. Thus, it is concluded that the Ca²⁺-neurocalcin δ-ANF-RGC transduction system is a physiological modulator of the blood pressure.

Figure 10. Systolic blood pressure in NCδ gene-targeted mice.

The mice used were NCδ 2-copy (+/+) wild type allele (control) and NCδ 1-copy (+/−) gene-disrupted heterozygous allele. The mice were fed a normal-salt diet. Systolic blood pressure was measured every day for one week by the noninvasive computerized tail-cuff method. An average blood pressure level of 10 sessions a day was calculated for analysis after 3 days of training. Bars indicate means ± SD values for the representative genotypes. n describes the number of mice analyzed for each genotype. ** indicates that the P value was <0.005.

DISCUSSION

Coinciding with time periods of observations that the hormone-dependent guanylate cyclase which is independent of the soluble form exists in the mammalian cells^{25, 40, 41}, the discovery of the ANF was announced⁴². ANF regulated sodium excretion, water balance and blood pressure^{43, 44}. A hint that there is a link between ANF actions and the particulate form of guanylate cyclase emerged with the findings that ANF stimulated membrane guanylate cyclase activities in the rat tissues^{45, 46}. The linkage was firmed with the findings that ANF-RGC is the receptor of ANF and is also its signal transducer^1–8. This marked the beginning of the membrane guanylate cyclase field and also its entry into cardio-vasculature research.

More than 3-decades of research involving ANF and ANF-RGC gene deletions and biochemical studies^{8, 47} has now established that abnormalities in the ANF-GC transduction pathway cause many cardio-vascular complications, including hypertension and heart failure. The ANF/ANF-RGC gene-deleted mice possess salt-sensitive and salt-insensitive hypertension. These defects in cardio-vasculature occur because ANF-RGC does not properly transduce the ANF signal in the generation of cyclic GMP, the second messenger of ANF. Cyclic GMP is a major regulator of blood pressure. For these reasons, it is critical to understand the basic mechanism/s by which ANF-RGC signal transduction machinery operates. Many gaps remain, yet important strides have been made in this direction. The current, and (note) the only, model is that ANF-RGC signal transduction is only meant to process the hormonal signals of ANF and BNP, the signals generated in the domains at the extracellular region of ANF-RGC. These signals are then processed through ATP-dependent two-step activation process⁴⁸. The model is: the ANF signal originates by the binding of one molecule of ANF to the extracellular domain of ANF-RGC. The binding modifies the juxtamembrane region where the disulfide ⁴²³C-C⁴³² structural motif is a key element in this modification. The signal twists the trans-membrane domain, induces structural changes in the ARM domain, and prepares it for the ATP activation. Step 1: ATP binding to the ARM domain leads to a cascade of temporal and spatial changes. They involve (1) shift in ATP binding pocket position by 3–4 Å and rotation of its floor by 15°; G⁵⁰⁵ acts as a critical PIVOT for both the shift and the rotation; (2) movement by 2–7 Å but not the rotation of its β4 and β5 strands and its loop; and (3) movement of its αEF helix by 2–5 Å. This movement exposes its hydrophobic motif, ⁶⁶⁹WTAPELL⁶⁷⁵, which facilitates its direct (or indirect) interaction with the catalytic module resulting in its partial, about 50%, activation. Step 2: The six phosphorylation sites are brought from their buried to the exposed state. Through ATP and a hypothetical protein kinase they get phosphorylated, and the full activation (additional 50%) of ANF-RGC is achieved. Concomitantly, phosphorylation converts the ATP binding site from high to low affinity, ATP dissociates and ANF-RGC returns to its ground state. A conspicuous feature of this model is that the trajectory of the pathway originating at the extracellular domain to the core catalytic domain, the site of signal translation into the production of cyclic GMP, is downstream (Fig. 11).

Figure 11.

(A) Schematic representation of the structural topography of ANF-RGC. ANF-RGC is a single transmembrane spanning homodimer protein. The dashed lines on the right show the defined boundaries of its segments: LS, leader sequence; ExtD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain. The functional domains housed in ICD, their designated names and the amino acid residues constituting their boundaries are indicated at the left: JMD, juxtamembrane domain; ARM, the ATP regulated module; SHD-signaling helix domain; CCD-core catalytic domain. The site targeted by neurocalcin δ (NCΔ) (encircled) is located within CCD. (B) The signaling pathways of ANF and of NCδ are independent. The trajectory of the ANF pathway is shown in red dashed arrow. From its origin at the ExtD, it passes through the structural domains of TM, ARM and SHD in its course to CCD. In contrast, the trajectory of the NCδ pathway (shown in blue dashed arrow) is within the CCD. The CCD exists as an antiparallel homodimer.

The ongoing studies by the present authors provide physiological support to this model (unpublished studies). The mice lacking the ANF-RGC gene encoded ⁶⁶⁹WTAPELL⁶⁷⁵ motif are hypertensive. The systolic blood pressure of the ⁶⁶⁹WTAPELL⁶⁷⁵-KO mice is 147 ± 4 Hg whereas for the isogenic wt-mice it is 99 ± 9 Hg.

The present comprehensive study defines a new paradigm of ANF-RGC signal transduction. Here, (1) ANF-RGC is the transducer of Ca²⁺ signal; (2) this pathway is independent of the ANF and BNP signal transduction, thus, does not utilize the extracellular domain of ANF-RGC; (3) the trajectory of the pathway is not downstream. It originates and acts directly on the core catalytic domain (Fig. 11), providing a novel mechanism of signal transduction in common with the sensory neuron-linked membrane guanylate cyclases: ROS-GC1 and ONE-GC; (4) Like the physiology of ANF and BNP signaling modes, the Ca²⁺ signaling mode is present in the glomerulosa cells of the adrenal cortex; and importantly, like those modes, regulates blood pressure in mice. The absence of those modes leads to hypertension.

There are two functional and independent structural compartments of ANF-RGC. One compartment processes the signals of ANF and BNP and the other, of intracellularly generated Ca²⁺ (Fig. 11). The transduction mechanisms of these two types of signals are fundamentally different yet both modulate blood pressure and the absence of either of them causes hypertension in the genetically targeted mice.

Ca²⁺ Signal Transduction Model

Based on facts provided by this study, a preliminary stepwise Ca²⁺ signal transduction is envisioned. Ground state: Dimer form of myr-NCδ is bound to a dimer of ANF-RGC. It constitutes the Ca²⁺ sensor element of ANF-RGC and it binds to the ⁸⁴⁹DIVGFTALSAESTPMQVV⁸⁶⁶ domain of ANF-RGC. The presence of the Ca²⁺ sensor element keeps the basic enzymatic efficiency of ANF-RGC at the threshold level in the production of cyclic GMP from GTP. Activation and activated state. A rise of Ca²⁺ to the semimicromolar range is sensed by NCδ; Ca²⁺ with K_1/2 of 0.5 μM binds myr-NCδ, which undergoes a Ca²⁺-dependent configurational changes. These, in turn, cause a structural change in the NCδ binding domain of ANF-RGC, which is also a part of the core catalytic domain. This increases the catalytic efficiency of ANF-RGC by more than 6-fold: k_cat, from 6.5 to 41 cyclic GMP sec⁻¹ and results in the accelerated production of cyclic GMP.

This model opens up important avenues of future cardio-vascular research. It is envisioned that in the adrenal glomerulosa cells, cyclic GMP formed through the Ca²⁺-dependent mechanism, acts as a second messenger, offsets, at least partially, the rennin-angiotensin system, inhibits aldosterone synthesis and lowers the blood pressure. Significantly, the 0.4–0.5 μM Ca²⁺ concentration that, through NCδ, causes half-maximal activation of ANF-RGC (Fig. 1A and 2B) is within the range of cytoplasmic Ca²⁺ concentration in glomerulosa cells synthesizing aldosterone⁴⁹. Whether, in these, the operation of the ANF-RGC-Ca²⁺-NCδ system precedes the operation of the ANF/ANF-RGC system or is concomitant with it remains to be determined.

The model may also aid in explaining the contractile and relaxation properties of vascular smooth muscle cells (VSMC). The magic signaling molecule in these cells is Ca²⁺. Volumes of documented evidence indicate that in VSMC the pulsated rise of Ca²⁺ causes their contraction and its fall, relaxation^50–52. It is also well established that in these cells cyclic GMP-generated through ANF-RGC transduction system causes relaxation⁵³. The present study demonstrates, however, that Ca²⁺ directly through ANF-RGC can generate cyclic GMP and cause relaxation of VSMC. Thus, the intracellular rise of Ca²⁺ in VSMC can cause both their contraction and relaxation. In accordance with the presently proposed Ca²⁺-relaxation related hypothesis, the present study demonstrates that deletion of the Ca²⁺ sensor, NCδ, from the ANF-RGC transduction system causes hypertension in the mice.

Ca²⁺-NCδ-modulated ANF-RGC transduction pathway is a new regulator of mouse blood pressure. The task for future research is to identify the source of Ca²⁺ that turns on the Ca²⁺ sensor element of the ANF-RGC transduction machinery.

Abbreviations

ANF

atrial natriuretic factor

BNP

B-type natriuretic peptide

CNP

C-type natriuretic peptide

ANF-RGC

atrial natriuretic factor receptor guanylate cyclase

ARM

ATP regulatory module

EGTA

ethylene glycol tetraacetic acid

GCAP

guanylate cyclase activating protein

ONE-GC

olfactory neuroepithelial guanylate cyclase

ROS-GC

rod outer segment guanylate cyclase