Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung

An atrial natriuretic factor (ANF) receptor from rat lung was solubilized with Lubrol-PX and purified by sequential chromatographic steps on GTP-agarose, DEAE-Sephacel, phenyl-agarose, and wheat germ agglutinin-agarose. The ANF receptor was enriched 19,000-fold. The purified receptor has a binding profile and properties that correspond to the affinity and specificity found in membranes and crude detergent extracts. Polyacrylamide gel electrophoresis of the purified preparation in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of one major protein band with a molecular mass of 120,000 daltons. When purified preparations were incubated with 125I-ANF, then cross-linked with disuccinimidyl suberate, the 120,000-dalton protein was specifically radiolabeled. This high affinity binding site for ANF co-purified with particulate guanylate cyclase. Particulate guanylate cyclase was purified to a specific activity of 19 mumol cyclic GMP produced/min/mg of protein utilizing Mn-GTP as substrate. This represented a 15,000-fold purification compared to the initial lung membrane preparation with Lubrol-PX. Gel permeation high performance liquid chromatography and glycerol density gradient sedimentation studies of the purified preparation also resulted in co-migration of specific ANF binding and guanylate cyclase activities. The co-purification of these activities suggests that both ANF binding and guanylate cyclase activities reside in the same macromolecular complex. Presumably ANF binding occurs at the external membrane surface and cyclic GMP synthesis at the internal membrane surface of this transmembrane glycoprotein.     

ATP-regulated module (ARM) of the atrial natriuretic factor receptor guanylate cyclase

ATP is an obligatory agent for the atrial natriuretic factor (ANF) and the type C natriuretic peptide (CNP) signaling of their respective receptor guanylate cyclases, ANF-RGC and CNP-RGC. Through a common mechanism, it binds to a defined ARM domain of the cyclase, activates the cyclase and transduces the signal into generation of the second messenger cyclic GMP. In this presentation, the authors review the ATP-regulated transduction mechanism and refine the previously simulated three-dimensional ARM model (Duda T, Yadav P, Jankowska A, Venkataraman V, Sharma RK. Three dimensional atomic model and experimental validation for the ATP-regulated module (ARM) of the atrial natriuretic factor receptor guanylate cyclase. Mol Cell Biochem 2000;214:7-14; reviewed in: Sharma RK, Yadav P, Duda T. Allosteric regulatory step and configuration of the ATP-binding pocket in atrial natriuretic factor receptor guanylate cyclase transduction mechanism. Can J Physiol Pharmacol 2001;79: 682-91; Sharma RK. Evolution of the membrane guanylate cyclase transduction system. Mol Cell Biochem 2002;230:3-30). The model depicts the ATP-binding dependent configurational changes in the ARM and supports the concept that in the first step, ATP partially activates the cyclase and primes it for the subsequent transduction steps, resulting in full activation of the cyclase.     

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.     

HeLa cells contain the atrial natriuretic peptide receptor with guanylate cyclase activity

Receptors for atrial natriuretic peptide (ANP) are heterogeneous: an approximately 140-kDa receptor exhibits ANP-stimulated guanylate cyclase activity whereas an approximately 65-kDa receptor is thought to act only as a clearance-storage protein. We have used photoaffinity labeling techniques to show that the human cell line, HeLa, contains predominantly the approximately 140-kDa ANP receptor. In contrast, several other cell lines contain primarily the approximately 65-kDa receptor. In HeLa cells, ANP bound specifically to high affinity binding sites (Kd approximately 2 nM) and stimulated a rapid, dose-dependent accumulation of cGMP. These cell lines can thus provide useful models to study the multiple mechanisms of ANP action.     

Calcium reveals different mechanisms of guanylate cyclase activation by atrial natriuretic factor and ATP in rat lung membranes.

CaCl2 inhibited ATP-stimulated guanylate cyclase activity, but had little effect on basal and atrial natriuretic factor-stimulated guanylate cyclase activity in rat lung membranes. LaCl3 had similar effects as CaCl2 on basal and stimulated guanylate cyclase activity. LiCl and other monovalent salts inhibited ATP-stimulated guanylate cyclase activity more than basal enzyme activity. However, atrial natriuretic factor somehow stabilized the enzyme against the inhibitory effect of LiCl. These results suggest that ATP and atrial natriuretic factor activate the enzyme through different mechanisms. Since the effect of calcium on guanylate cyclase activity is different from that of monovalent salts and can be mimicked by lanthanum, it may be mediated by a specific calcium binding site or binding protein.     

Molecular cloning and expression of murine guanylate cyclase/atrial natriuretic factor receptor cDNA

The potent diuretic and natriuretic peptide hormone atrial natriuretic factor (ANF), with vasodilatory activity also stimulates steroidogenic responsiveness in Leydig cells. The actions of ANF are mediated by its interaction with specific cell surface receptors and the membrane-bound form of guanylate cyclase represents an atrial natriuretic factor receptor (ANF-R). To understand the mechanism of ANF action in testicular steroidogenesis and to identify guanylate cyclase/ANF-R that is expressed in the Leydig cells, the primary structure of murine guanylate cyclase/ANF-R has been deduced from its cDNA sequence. A cDNA library constructed from poly(A+) RNA of murine Leydig tumor (MA-10) cell line was screened for the membrane-bound form of ANF-R/guanylate cyclase sequences by hybridization with a rat brain guanylate cyclase/ANF-R cDNA probe. The amino acid sequence deduced from the cDNA shows that murine guanylate cyclase/ANF-R cDNA consists of 1057 amino acids with 21 amino acids comprising the transmembrane domain which separates an extracellular ligand-binding domain (469 amino acid residues) and an intracellular guanylate cyclase domain (567 amino acid residues). Upon transfection of the murine guanylate cyclase/ANF-R cDNA in COS-7 cells, the expressed protein showed specific binding to 125I-ANF, stimulation of guanylate cyclase activity and production of intracellular cGMP in response to ANF. The expression of guanylate cyclase/ANF-R cDNA transfected in rat Leydig tumor cells stimulated the production of testosterone and intracellular cGMP after treatment with ANF. The results presented herein directly show that ANF can regulate the testicular steroidogenic responsiveness in addition to its known regulatory role in the control of cardiovascular homeostasis.     

Allosteric modification, the primary ATP activation mechanism of atrial natriuretic factor receptor guanylate cyclase

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 transmembrane-spanning protein. 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 its second messenger, cyclic GMP, which controls blood pressure, cardiac vasculature, and fluid secretion. ATP is obligatory for the posttransmembrane dynamic events leading to ANF-RGC activation. It functions through the ATP-regulated module, ARM (KHD) domain, of ANF-RGC. In the current over a decade held model "phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A" [Potter, L. R., and Hunter, T. (1998) Mol. Cell. Biol. 18, 2164-2172]. The presented study challenges this concept. It demonstrates that, instead, ATP allosteric modification of ARM is the primary signaling step of ANF-GC activation. In this two-step new dynamic model, ATP in the first step binds ARM. This triggers in it a chain of transduction events, which cause its allosteric modification. The modification partially activates (about 50%) ANF-RGC and, concomitantly, also prepares the ARM for the second successive step. In this second step, ARM is phosphorylated and ANF-RGC achieves additional (～50%) full catalytic activation. The study defines a new paradigm of the ANF-RGC signaling mechanism.     

Amiloride increases the sensitivity of particulate guanylate cyclase to atrial natriuretic factor

The natriuretic agent amiloride induces a shift of the dose-response curve of particulate guanylate cyclase to atrial natriuretic factor (ANF) to the left. The ANF concentration for half-maximal activation of guanylate cyclase is shifted from 20 to 3 nM in the presence of 100 microM amiloride. This effect is observed with GTP*Mn2+, but not with GTP*Mg2+ as substrate. Amiloride derivatives, which inhibit a specific Na+-channel, also shift the dose-response curve to the left. These data suggest that some of the effects of amiloride may be mediated by an increased sensitivity of particulate guanylate cyclase to ANF.     

Stimulation of guanylate cyclase by atrial natriuretic factor in isolated human glomeruli

A 23 amino acid synthetic peptide fragment of atrial natriuretic factor (ANF) stimulated guanylate cyclase activity in isolated human glomeruli in a concentration- and time-dependent manner. ANF activated particulate guanylate cyclase whereas it had no effect on soluble guanylate cyclase. These results demonstrate that the glomerulus is a target structure for ANF in humans. They also suggest that ANF-induced increase in glomerular filtration rate is due to a direct effect of this peptide on the glomerular cells mediated by activation of glomerular guanylate cyclase.     

Allosteric regulatory step and configuration of the ATP-binding pocket in atrial natriuretic factor receptor guanylate cyclase transduction mechanism

The atrial natriuretic factor (ANF) signal transduction mechanism consists of the transformation of the signal information into the production of cyclic GMP. The binding of ANF to its receptor, which is also a guanylate cyclase, generates the signal. This cyclase has been termed atrial natriuretic factor receptor guanylate cyclase, ANF-RGC. ANF-RGC is a single transmembrane-spanning protein. The ANF receptor domain resides in the extracellular region of the protein, and the catalytic domain is located in the intracellular region at the C-terminus of the protein. Thus, the signal is relayed progressively from the receptor domain to the catalytic domain, where it is converted into the formation of cyclic GMP. The first transduction step is the direct binding of ATP with ANF-RGC. This causes allosteric regulation of the enzyme and primes it for the activation of its catalytic moiety. The partial structural motif of the ATP binding domain in ANF-RGC has been elucidated, and it has been named ATP regulatory module (ARM). In this presentation, we provide a brief review of the ATP-regulated transduction mechanism and the ARM model. The model depicts a configuration of the ATP-binding pocket that has been experimentally validated, and the model shows that the ATP-dependent transduction process is a two- (or more) step event. The first step involves the binding of ATP with its ARM. This partially activates the cyclase and prepares it for the subsequent steps, which are consistent with its being phosphorylated and attaining the fully activated state.     

Interaction of atrial natriuretic peptide-stimulated guanylate cyclase and vasopressin-stimulated calcium signaling pathways in the glomerular mesangial cell

Receptors for atrial natriuretic peptide (ANP) have been demonstrated in renal mesangial cells as well as other cell types in the glomerulus. The biochemical basis for the effects of ANP on glomerular hemodynamics remains undefined. Using cultured rat glomerular mesangial cells, we demonstrated a concentration-dependent stimulation of cGMP production in intact cells, and of guanylate cyclase in membranes. Despite the presence of a guanylate cyclase response, ANP had no inhibitory effect on basal inositol trisphosphate production nor on basal cytosolic calcium. Arginine vasopressin stimulated IP3 production, caused a rise in cytosolic calcium as measured using the calcium-sensitive fluorescent probe Indo-1, and caused mesangial cell contraction. ANP caused a slight but significant enhancement of vasopressin-stimulated IP3 production, but had no effect on the cytosolic calcium response nor on the contractile response. 8-Bromo-cGMP likewise had no effect on the generation of the calcium signal. These results indicate that the effects of ANP on glomerular hemodynamics are not mediated by an alteration in the generation of the calcium signal in mesangial cells. In contrast, addition of calcium inhibited ANP stimulated guanylate cyclase activity.     

Atrial natriuretic factor stimulates luteal guanylate cyclase

Effect of a synthetic atrial natriuretic peptide, rat atriopeptin II (rAP-II) on the formation of cyclic nucleotides and progesterone production in Percoll-purified rat luteal cells was investigated. Incubation of luteal cells with varying concentrations of rAP-II resulted in a dose-related stimulation of intracellular cyclic GMP content; maximum stimulation being achieved with 10 nM rAP-II. The increase in cyclic GMP formation was extremely rapid and a 12-fold increase in the cyclic GMP content over basal level was attained within 5 min of incubation of the cells with 10 nM rAP-II. In the presence of phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine, both basal and rAP-II-stimulated levels of cyclic GMP were increased approximately 10 times, but the magnitude of stimulation remained similar in the presence or absence of the inhibitor. The atrial peptide at the concentration of 1-100 nM, however, had no effect on either basal or gonadotropin-stimulated progesterone production and cyclic AMP formation by the luteal cells. Furthermore, the increase in the level of cellular cyclic GMP content of rAP-II was demonstrated to result from a selective activation of particulate guanylate cyclase.     

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.     

Regulation of guanylate cyclase activity by atrial natriuretic factor and protein kinase C

Summary The putative second messenger of certain atrial natriuretic factor (ANF) signal transductions is cyclic GMP. Recently, we purified a 180-kDa protein, apparently containing both ANF receptor and guanylate cyclase activities, and hypothesized that this is one of the cyclic GMP transmembrane signal transducers. The enzyme is ubiquitous and appears to be conserved. Utilizing the 180-kDa membrane guanylate cyclase, we now show that the 180-kDa guanylate cyclase is regulated in opposing fashions by two receptor signals—ANF stimulating it and protein kinase C inhibiting it. Furthermore, protein kinase C phosphorylates the 180-kDa enzyme. This suggests a novel switch on and switch off mechanism of the cyclic GMP signal transduction. Switch off represents the phosphorylation while switch on the dephosphorylation of the enzyme.     

The increase of cGMP by atrial natriuretic factor correlates with the distribution of particulate guanylate cyclase

We have demonstrated previously that atrial natriuretic factor (ANF) augments urinary, plasma and kidney cGMP levels but has no significant effect upon cAMP. Using cGMP as a marker, we searched for specific target sites involved in the action of ANF in the dog kidney, and observed no change of cGMP in the proximal tubules, a 2-fold increase over basal levels in the thick loop of Henle and a 3-fold elevation in the collecting duct. The most striking action on cGMP occurred in the glomeruli with a rise of up to 50-fold being evident at 1-2 min. after the addition of ANF. The results obtained in the absence or presence of a phosphodiesterase inhibitor support the notion that the effects of ANF were exerted at the level of guanylate cyclase stimulation rather than cGMP phosphodiesterase inhibition. The action of sodium nitroprusside (SNP), a direct stimulator of soluble guanylate cyclase, differed from that of ANF. The ability of the factor to enhance cGMP levels was correlated with the distribution of particulate guanylate cyclase. This study identifies the glomeruli and the distal part of the nephron as specific targets of ANF and implicates particulate guanylate cyclase as the enzyme targetted for the expression of its action.     

Comparison of particulate guanylate cyclase in cells with and without atrial natriuretic peptide receptor binding activity

Summary A line of kidney cells (PK,) which does not possess measurable ANP binding but has an active particulate guanylate cyclase has been identified. The physical characteristics of this enzyme were compared with those of particulate guanylate cyclase and ANP receptors isolated from rat lung. Although receptor and enzyme appear to reside on the same protein in the lung while the cyclase from PK₁ cells does not possess ANP binding activity, these proteins exhibit identical physical characteristics. Guanylate cyclase from PK₁ cells and rat lung and ANP receptor from lung co-eluted during gel filtration chromatography, with a Stokes radius of 6.1 nm. Also, these activities co-migrated through sucrose density gradients with S_20,w values of 10.4 to 10.9. Using these parameters, a molecular weight of about 270 kD was estimated for all three activities. Furthermore, these enzyme activities exhibited similar mobilities in isoelectric focusing gels, with a pI of 6.1. Thus, although particulate guanylate cyclase from lung presumably possesses receptor binding activity, it is physically identical to a form of this enzyme associated with no measurable binding activity. Possible explanations for these observations are discussed.     

Characterization and regulation by protein kinase C of renal glomerular atrial natriuretic peptide receptor-coupled guanylate cyclase

The nature and regulation of atrial natriuretic peptide (ANP)-sensitive guanylate cyclase in rat renal glomerular membranes was examined. By affinity crosslinking techniques, three bands with apparent molecular masses of 180, 130 and 64 kDa were specifically labeled with [125I]ANP. A specific antibody to the 180 kDa membrane guanylate cyclase of rat adrenocortical carcinoma recognized a 180 kDa band on Western blot analysis of solubilized, GTP-affinity purified glomerular membrane proteins. The same antibody completely inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. Partially purified protein kinase C inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. It is concluded that a 180 kDa ANP-sensitive guanylate cyclase is present in glomerular membranes, and that this enzyme is inhibited directly by protein kinase C.     

Proteolytic cleavage of atrial natriuretic factor receptor in bovine adrenal membranes by endogenous metalloendopeptidase. Effects on guanylate cyclase activity and ligand-binding specificity.

Atrial natriuretic factor (ANF) is a peptide hormone from the heart atrium with potent natriuretic and vasorelaxant activities. The natriuretic activity of ANF is, in part, mediated through the adrenal gland, where binding of ANF to the 130-kDa ANF receptor causes suppression of aldosterone secretion. Incubation of bovine adrenal membranes at pH < 5.6 caused a rapid and spontaneous cleavage of the 130-kDa ANF receptor, yielding a 65-kDa polypeptide that could be detected by photoaffinity labeling by 125I-labeled N alpha 4-azidobenzoyl-ANF(4-28) followed by SDS/PAGE under reducing conditions. Within 20 min of incubation at pH 4.0, essentially all the 130-kDa receptor was converted to a 65-kDa ANF binding protein. This cleavage reaction was completely inhibited by inclusion of 5 mM EDTA. When SDS/PAGE was carried out under non-reducing conditions, the apparent size of the ANF receptor remained unchanged at 130 kDa, indicating that the 65-kDa ANF-binding fragment was still linked to the remaining part(s) of the receptor polypeptide through a disulfide bond(s). The disappearance of the 130-kDa receptor was accompanied by a parallel decrease in guanylate cyclase activity in the membranes. Inclusion of EDTA in the incubation not only prevented cleavage of the 130-kDa receptor, but also protected guanylate cyclase activity, indicating that proteolysis, but not the physical effects of the acidic pH, causes inactivation of guanylate cyclase. The 130-kDa ANF receptor in adrenal membranes was competitively protected from photoaffinity labeling by ANF(1-28) or ANF(4-28), but not by atriopeptin I [ANF(5-25)] or C-ANF [des-(18-22)-ANF(4-23)-NH2]. On the contrary, the 65-kDa ANF-binding fragment generated after incubation at pH 4.0 was protected from labeling by any of the above peptides, indicating broader binding specificity. After incubation in the presence of EDTA, the 130-kDa ANF receptor, which was protected from proteolysis, retained binding specificity identical to that of the 130-kDa receptor in untreated membranes. The results indicate that the broadening of selectivity is caused by cleavage, but not by the physical effect of acidic pH. Spontaneous proteolysis of ANF receptor by an endogenous metalloendopeptidase, occurring with concomitant inactivation of guanylate cyclase activity and broadening of ligand-binding selectivity, may be responsible for the generation of low-molecular-mass receptors found in the adrenal gland and other target organs of ANF. The proteolytic process may play a role in desensitization or down-regulation of the ANF receptor.     

Evidence for particulate guanylate cyclase in rat kidney after stimulation by atrial natriuretic factor. An ultracytochemical study

Summary Cytochemical localization of particulate guanylate cyclase (GC) in rat kidney, after stimulation with atrial natriuretic factor (ANF), was studied by electron microscopy. In the renal corpuscle GC reaction product was localized on podocytes. Other segments of the nephron that showed ultracytochemical evidence of GC activity were the proximal convoluted tubule, the thick ascending limb of the loop of Henle and the collecting tubule. All GC positivity was associated with plasma membranes. Samples incubated in basal conditions (without ANF) did not reveal any GC reaction product. These results indicate that ANF is a strong activator of particulate GC. Our data also suggests that, through the enzyme, ANF acts directly on epithelial cells of tubules where Na⁺ reabsorption occurs. This is in agreement with the hypothesis that ANF has a direct tubular effect on natriuresis.     

Atrial natriuretic factor receptor guanylate cyclase signaling: new ATP-regulated transduction motif

ANF-RGC membrane guanylate cyclase is the receptor for the hypotensive peptide hormones, atrial natriuretic factor (ANF) and type B natriuretic peptide (BNP). It is a single transmembrane spanning protein. Binding the hormone to the extracellular domain activates its intracellular catalytic domain. This results in accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature, and fluid secretion. ATP is the obligatory transducer of the ANF signal. It works through its ATP regulated module, ARM, which is juxtaposed to the C-terminal side of the transmembrane domain. Upon interaction, ATP induces a cascade of temporal and spatial changes in the ARM, which, finally, result in activation of the catalytic module. Although the exact nature and the details of these changes are not known, some of these have been stereographed in the simulated three-dimensional model of the ARM and validated biochemically. Through comprehensive techniques of steady state, time-resolved tryptophan fluorescence and Forster Resonance Energy Transfer (FRET), site-directed and deletion-mutagenesis, and reconstitution, the present study validates and explains the mechanism of the model-based predicted transduction role of the ARM’s structural motif, ⁶⁶⁹WTAPELL⁶⁷⁵. This motif is critical in the ATP-dependent ANF signaling. Molecular modeling shows that ATP binding exposes the ⁶⁶⁹WTAPELL⁶⁷⁵ motif, the exposure, in turn, facilitates its interaction and activation of the catalytic module. These principles of the model have been experimentally validated. This knowledge brings us a step closer to our understanding of the mechanism by which the ATP-dependent spatial changes within the ARM cause ANF signaling of ANF-RGC.