The kinase homology domain of receptor guanylyl cyclase C: ATP binding and identification of an adenine nucleotide sensitive site

The role of the kinase homology domain (KHD) in receptor guanylyl cyclases is to regulate the activity of the catalytic guanylyl cyclase domain. The KHD lacks many of the amino acids required for phosphotransfer activity and, therefore, is not expected to possess kinase activity. Guanylyl cyclase activity of the receptor guanylyl cyclase C (GC-C) is modulated by ATP, and computational modeling showed that the KHD can adopt a structure similar to protein kinases, suggesting that the KHD is the site for ATP interaction. A monoclonal antibody, GCC:4D7, raised to the KHD of GC-C, fails to react with GC-C in the presence of ATP and ATP analogues that regulate GC-C catalytic activity, indicating that a conformational change occurs in the KHD on ATP binding. Mapping of the epitope of the antibody through the use of recombinant protein constructs and phage display showed that the epitope for GC-C:4D7 lies immediately C-terminal to a critical lysine residue (Lys516 in GC-C), required for ATP interaction in protein kinases. By employing a novel approach utilizing ATP-agarose affinity chromatography, we demonstrate that the intracellular domain of GC-C and the KHD bind ATP. Mutation of Lys516 to Ala abolishes ATP binding. Thus, this report is the first to show direct ATP binding to the pseudokinase domain of receptor guanylyl cyclase C, as well as to identify dramatic conformational changes that occur in this domain on ATP binding, akin to those seen in catalytically active protein kinases.     

Functional inactivation of the human guanylyl cyclase C receptor: modeling and mutation of the protein kinase-like domain

Receptor guanylyl cyclases possess an extracellular ligand-binding domain, a single transmembrane region, a region with sequence similar to that of protein kinases, and a C-terminal guanylyl cyclase domain. ATP regulates the activity of guanylyl cyclase C (GC-C), the receptor for the guanylin and stable toxin family of peptides, presumably as a result of binding to the kinase homology domain (KHD). Modeling of the KHD of GC-C indicated that it could adopt a structure similar to that of tyrosine kinases, and sequence comparison with other protein kinases suggested that lysine(516) was positioned in the KHD to interact with ATP. A monoclonal antibody GCC:4D7, raised to the KHD of GC-C, did not recognize ATP-bound GC-C, and its epitope mapped to a region in the KHD of residues 491--568 of GC-C. Mutation of lysine(516) to an alanine in full-length GC-C (GC-C(K516A)) dramatically reduced the ligand-stimulated activity of mutant GC-C, altered the ATP-mediated effects observed with wild-type GC-C, and failed to react with the GCC:4D7 monoclonal antibody. ATP interaction with wild-type GC-C converted a high-molecular weight oligomer of GC-C to a smaller sized oligomer. In contrast, GC-C(K516A) did not exhibit an alteration in its oligomeric status on incubation with ATP. We therefore suggest that the KHD in receptor guanylyl cyclases provides a critical structural link between the extracellular domain and the catalytic domain in regulation of activity in this family of receptors, and the presence of K(516) is critical for the possible proper orientation of ATP in this domain.     

Identification of a cell surface receptor common to germ and somatic cells.

The plasma membrane forms of guanylyl cyclase constitute a diverse family of cell surface receptors. An mRNA for the enzyme/receptor was first cloned from sea urchin testis after cross-linking studies suggested that guanylyl cyclase was a sperm receptor for egg peptides. The enzyme/receptor was shown to contain a single putative transmembrane domain, a large extracellular region that presumably binds peptide ligands, and an intracellular region that contains a protein kinase-like and a cyclase catalytic domain. The sea urchin cDNA was then used to isolate positive-hybridizing clones from mammalian tissues. At least two forms recognize natriuretic peptides and one form recognizes the heat-stable enterotoxins. In the case of the enterotoxin receptor, it remains to be shown whether or not an endogenous ligand exists that regulates enzyme activity. The discovery of this cell surface receptor family presents a new paradigm for second messenger signalling in that a low-molecular weight second messenger (cyclic GMP) is produced by the same protein that binds the extracellular ligand.     

Phosphorylation of the Kinase Homology Domain Is Essential for Activation of the A-Type Natriuretic Peptide Receptor

Natriuretic peptide receptor A (NPR-A) is the biological receptor for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyclase requires ANP binding to the extracellular domain and ATP binding to a putative site within its cytoplasmic region. The allosteric interaction of ATP with the intracellular kinase homology domain (KHD) is hypothesized to derepress the carboxyl-terminal guanylyl cyclase catalytic domain, resulting in the synthesis of the second messenger, cyclic GMP. Here, we show that phosphorylation of the KHD is essential for receptor activation. Using a combination of phosphopeptide mapping techniques, we have identified six residues within the ATP-binding domain (S497, T500, S502, S506, S510, and T513) which are phosphorylated when NPR-A is expressed in HEK 293 cells. Mutation of any one of these Ser or Thr residues to Ala caused reductions in the receptor phosphorylation state, the number and pattern of phosphopeptides observed in tryptic maps, and ANP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in NPR-A protein levels, as indicated by immunoblot analysis and determinations of cyclase activity in the presence of detergent. Conversion of Ser-497 to Ala resulted in the most dramatic decrease in cyclase activity (~20% of wild-type activity), but conversion to an acidic residue (Glu), which mimics the charge of the phosphoserine moiety, had no effect. Simultaneous mutation of five of the phosphorylation sites to Ala resulted in a dephosphorylated receptor which was unresponsive to hormone and had potent dominant negative inhibitory activity. We conclude that phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A.     

Tyrphostins are inhibitors of guanylyl and adenylyl cyclases

Guanylyl cyclase C (GC-C), the receptor for guanylin, uroguanylin, and the heat-stable enterotoxin, regulates fluid balance in the intestine and extraintestinal tissues. The receptor has an extracellular domain, a single transmembrane spanning domain, and an intracellular domain that harbors a region homologous to protein kinases, followed by the C-terminal guanylyl cyclase domain. Adenine nucleotides can regulate the guanylyl cyclase activity of GC-C by binding to the intracellular kinase homology domain (KHD). In this study, we have tested the effect of several protein kinase inhibitors on GC-C activity and find that the tyrphostins, known to be tyrosine kinase inhibitors, could inhibit GC-C activity in vitro. Tyrphostin A25 (AG82) was the most potent inhibitor with an IC(50) of approximately 15 microM. The mechanism of inhibition was found to be noncompetitive with respect to both the substrate MnGTP and the metal cofactor. Interestingly, the activity of the catalytic domain of GC-C (lacking the KHD) expressed in insect cells was also inhibited by tyrphostin A25 with an IC(50) of approximately 5 microM. As with the full-length receptor, inhibition was found to be noncompetitive with respect to MnGTP. Inhibition was reversible, ruling out a covalent modification of the receptor. Structurally similar proteins such as the soluble guanylyl cyclase and the adenylyl cyclases were also inhibited by tyrphostin A25. Evaluation of a number of tyrphostins allowed us to identify the requirement of two vicinal hydroxyl groups in the tyrphostin for effective inhibition of cyclase activity. Therefore, our studies are the first to report that nucleotide cyclases are inhibited by tyrphostins and suggest that novel inhibitors based on the tyrphostin scaffold can be developed, which could aid in a greater understanding of nucleotide cyclase structure and function.     

Interaction of guanylyl cyclase C with SH3 domain of Src tyrosine kinase. Yet another mechanism for desensitization

Protein-protein interactions mediated by the Src homology 3 (SH3) domain have been implicated in the regulation of receptor functions for subcellular localization of proteins and the reorganization of cytoskeleton. The experiments described in this article begin to identify the interaction of the SH3 domain of Src tyrosine kinase with the guanylyl cyclase C receptor after activation with Escherichia coli heat-stable enterotoxin (ST). Only one of two post-translationally modified forms of guanylyl cyclase C from T84 colonic carcinoma cells bind to GST-SH3 fusion protein of Src and Hck tyrosine kinases. Interestingly, the GST-Src-SH3 fusion protein showed 2-fold more affinity to native guanylyl cyclase C in solution than the GST-Hck-SH3 fusion protein. The affinity of the GST-Src-SH3 fusion protein to guanylyl cyclase C increased on desensitization of receptor in vivo. An in vitro cyclase assay in the presence of GST-Src-SH3 fusion protein indicated inhibition of the catalytic activity of guanylyl cyclase C. The catalytic domain recombinant protein (GST-GCD) of guanylyl cyclase C could pull-down a 60-kDa protein that reacted with Src tyrosine antibody and also showed autophosphorylation. These data suggest that SH3 domain-mediated protein-protein interaction with the catalytic domain of guanylyl cyclase C inhibited the cyclase activity and that such an interaction, possibly mediated by Src tyrosine kinase or additional proteins, might be pivotal for the desensitization phenomenon of the guanylyl cyclase C receptor.     

Structural insights into the ligand binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors

Membrane bound guanylyl cyclases are single chain transmembrane receptors that produce the second messenger cGMP by either intra- or extracellular stimuli. This class of type I receptors contain an intracellular catalytic guanylyl cyclase domain, an adjacent kinase-like domain and an extracellular ligand binding domain though some receptors have their ligands yet to be identified. The most studied member is the atrial natriuretic peptide (ANP) receptor, which is involved in blood pressure regulation. Extracellular ANP binding induces a conformational change thereby activating the pre-oligomerized receptor leading to the production of cGMP. The recent crystal structure of the dimerized hormone binding domain of the ANP receptor provides a first three-dimensional view of this domain and can serve as a basis to structurally analyze mutagenesis, cross-linking, and genetic studies of this class of receptors as well as a non-catalytic homolog, the clearance receptor. The fold of the ligand binding domain is that of a bilobal periplasmic binding protein (PBP) very similar to that of the Leu/Ile/Val binding protein, AmiC, multi-domain transmembrane metabotropic glutamate receptors, and several DNA binding proteins such as the lactose repressor. Unlike these structural homologs, the guanylyl cyclase receptors bind much larger molecules at a site seemingly remote from the usual small molecule binding site in periplasmic binding protein folds. Detailed comparisons with these structural homologs offer insights into mechanisms of signal transduction and allosteric regulation, and into the remarkable usage of the periplasmic binding protein fold in multi-domain receptors/proteins.     

Guanylyl cyclases, a growing family of signal-transducing enzymes.

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified. These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.     

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

Mutational analysis of the Mycobacterium tuberculosis Rv1625c adenylyl cyclase: residues that confer nucleotide specificity contribute to dimerization

The mycobacterial Rv1625c gene product is an adenylyl cyclase with sequence similarity to the mammalian enzymes. The catalytic domain of the enzyme forms a homodimer and residues specifying adenosine triphosphate (ATP) specificity lie at the dimer interface. Mutation of these residues to those present in guanylyl cyclases failed to convert the enzyme to a guanylyl cyclase, but dramatically reduced its adenylyl cyclase activity and altered its oligomeric state. Computational modeling revealed subtle differences in the dimer interface that could explain the biochemical data, suggesting that the structural and catalytic features of this homodimeric adenylyl cyclase are in contrast to those of the heterodimeric mammalian enzymes.     

A disulfide-bridged mutant of natriuretic peptide receptor-A displays constitutive activity. Role of receptor dimerization in signal transduction

Natriuretic peptide receptor-A (NPR-A), a particulate guanylyl cyclase receptor, is composed of an extracellular domain (ECD) with a ligand binding site, a transmembrane spanning, a kinase homology domain (KHD), and a guanylyl cyclase domain. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), the natural agonists, bind and activate the receptor leading to cyclic GMP production. This receptor has been reported to be spontaneously dimeric or oligomeric. In response to agonists, the KHD-mediated guanylate cyclase repression is removed, and it is assumed that ATP binds to the KHD. Since NPR-A displays a pair of juxtamembrane cysteines separated by 8 residues, we hypothesized that the removal of one of those cysteines would leave the other unpaired and reactive, thus susceptible to form an interchain disulfide bridge and to favor the dimeric interactions. Here we show that NPR-AC423S mutant, expressed mainly as a covalent dimer, increases the affinity of pBNP for this receptor by enhancing a high affinity binding component. Dimerization primarily depends on ECD since a secreted NPR-A C423S soluble ectodomain (ECDC423S) also documents a covalent dimer. ANP binding to the unmutated ECD yields up to 80-fold affinity loss as compared with the membrane receptor. However, the ECD C423S mutation restores a high binding affinity. Furthermore, C423S mutation leads to cellular constitutive activation (20-40-fold) of basal catalytic production of cyclic GMP by the full-length mutant. In vitro particulate guanylyl cyclase assays demonstrate that NPR-AC423S displays an increased sensitivity to ATP treatment alone and that the effect of ANP + ATP joint treatment is cumulative instead of synergistic. Finally, the cellular and particulate guanylyl cyclase assays indicate that the receptor is desensitized to agonist stimulation. We conclude the following: 1) dimers are functional units of NPR-A guanylyl cyclase activation; and 2) agonists are inducing dimeric contact of the juxtamembranous region leading to the removal of the KHD-mediated guanylyl cyclase repression, hence allowing catalytic activation.     

Agonistic induction of a covalent dimer in a mutant of natriuretic peptide receptor-A documents a juxtamembrane interaction that accompanies receptor activation

The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-A(D435C) mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-A(D435C) dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-A(D435C) has been truncated (DeltaKC(D435C)) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type DeltaKC and DeltaKC(D435C) is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the juxtamembrane domain of NPR-A. In addition, this process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain.     

Reduced activity of the NPR-A kinase triggers dephosphorylation and homologous desensitization of the receptor

NPR-A, the receptor for the atrial natriuretic peptide (ANP), is a 130-kDa protein presenting an extracellular ANP-binding domain, a single transmembrane domain, an intracellular regulatory kinase homology domain (KHD), and a guanylyl cyclase catalytic domain. Upon stimulation, NPR-A receptors are activated to produce cyclic guanosine monophosphate (cGMP) and are subsequently desensitized through dephosphorylation of residues at their KHD. We used wild-type rat (r) NPR-A (WT) and a disulfide-bridged mutant (C423S) expressed in human embryonic kidney (HEK) 293 cells to study receptor phosphorylation. We have previously characterized the C423S receptor as constitutively active and desensitized. At basal state, 32P incorporation in the rNPR-A(C423S) covalent dimer is about 24 times less efficient than incorporation in the WT rNPR-A. When membranes from WT and rNPR-A(C423S) are incubated with [35S]ATPgammaS, the mutant dimer receptor displays 3.5% of the thiophosphate incorporation found for WT rNPR-A. Since the rNPR-A(C423S) dimer is already extensively dephosphorylated, we then used the WT rNPR-A to study dephosphorylation. As previously documented, adding ANP globally induces time-dependent dephosphorylation of the receptor. However, in pulse-chase experiments with the WT rNPR-A, adding ANP during the chase does not lead to a significant effect on receptor dephosphorylation. On the other hand, thiophosphorylation of the WT rNPR-A previously desensitized with ANP is reduced to 8.3% of the incorporation for untreated receptor, similar to results found with the rNPR-A(C423S) at basal state. These results demonstrate that ANP-induced rNPR-A desensitization is modulated by a significant reduction in the activity or affinity of the rNPR-A kinase that contributes to the low phosphorylation level after induction. Moreover, we further document a close relationship between tight dimerization, dephosphorylation, and desensitization.     

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module: MUTATIONAL ANALYSIS OF GUANYLYL CYCLASE C*

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of ∼70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.In transmembrane receptors a series of conformational changes are required to transmit the information of ligand binding (an extracellular signal) to the interior of the cell, resulting in either altered interaction with signaling intermediates or in the regulation of a catalytic activity present in the receptor. In these multidomain receptors, where the ligand binding and effector domains are present in the same polypeptide chain, the relay of conformational changes is under the exquisite control of post-translational modifications or precise structural alterations.Receptor guanylyl cyclases (GCs)⁴ have an N-terminal extracellular ligand binding domain, a single transmembrane domain, and a C-terminal intracellular domain (1). Binding of ligands to the extracellular domain elicits a conformational change that increases the guanylyl cyclase activity of the receptor, resulting in increased cGMP production. The intracellular domain of receptor GCs contains a region that shares considerable sequence similarity to protein kinases and is referred to as the kinase homology domain (KHD). Binding of ATP to the KHD induces a conformational change that regulates cGMP production by the guanylyl cyclase domain (2). Thus, receptor GCs exemplify the intricate interactions between domains in transducing the signal from an extracellular ligand to the interior of the cell.The amino acid sequences of the extracellular domain of mammalian receptor GCs vary (less than ∼15% similarity), as would be expected given the diversity in the ligands that bind to and activate these receptors. The KHD shows ∼25–30% conservation in amino acid sequence across receptor GCs, and computational modeling has not only suggested that this region could adopt the overall structure of a protein kinase but also identified specific residues that could interact with ATP (2, 3). The catalytic domains of mammalian receptor GCs are more conserved (∼80% sequence similarity). The gradual increase in sequence similarity across the various domains, with the extracellular domain being the most diverse and the cyclase domains sharing the maximum sequence similarity, is a reflection of the ability of these receptor GCs to converge diverse extracellular signals to a unified output of cGMP production. The guanylyl cyclase domains of receptor GCs can be classified as members of the Class III family of nucleotide cyclases (4). The recent crystal structures of a bacterial guanylyl cyclase (5) and a eukaryotic soluble guanylyl cyclase (6) show similarities in the overall three-dimensional structure of adenylyl and guanylyl cyclases and also highlight the critical residues that determine substrate utilization (either ATP or GTP) in these enzymes.Guanylyl cyclase C (GC-C) serves as the receptor for the guanylin family of endogenous peptides as well as for the exogenous heat-stable enterotoxin (ST) peptides secreted by enterotoxigenic bacteria (7, 8). GC-C is predominantly expressed on the apical surface of epithelial cells in the intestine, although robust extra-intestinal expression is observed in the kidney and reproductive tissues of the rat (9–12). The extracellular domain of GC-C is glycosylated, and we have shown the importance of glycosylation in regulating receptor desensitization in colonic cells. We have also identified a critical residue (Lys-516) in the KHD of GC-C as being important for KHD-mediated modulation of the guanylyl cyclase activity (2, 3).A sequence of ∼70 amino acids is found between the KHD and the guanylyl cyclase domain of receptor GCs, which we refer to here as the linker region (13). This region is predicted to form an amphipathic α-helix and could also adopt a coiled coil conformation (14, 15). The linker region is also present in soluble (cytosolic) guanylyl cyclases where it connects the N-terminal heme binding regulatory domain to the C-terminal catalytic cyclase domain. The linker region is suggested to act as a dimerization module in receptor GCs (16–18) and has also been implicated in heterodimerization of the α and β subunits of soluble guanylyl cyclases (19, 20). However, there are several reports to the contrary that indicate that the linker does not affect the dimerization of receptor GCs (14, 15). Nevertheless, the critical importance of the linker in regulating the activity of receptor GCs is shown by the fact that mutations in this region of the retinal guanylyl cyclase (RetGC-1) are associated with autosomal dominant cone-rod dystrophy in humans (16, 21). We show here through extensive mutational and biochemical analysis that the linker regions in two receptor GCs, GC-C and guanylyl cyclase A (GC-A), play an important role in repressing the catalytic activity of the receptors in the absence of their ligands. In addition, our results provide for the first time a molecular explanation for detergent-enhanced guanylyl cyclase activity in this family of receptors and suggest a mechanism for this activation that could involve a hydrophobic interaction between the linker region and the guanylyl cyclase domain.     

Atrial natriuretic peptide-dependent photolabeling of a regulatory ATP-binding site on the natriuretic peptide receptor-A

The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular ligand-binding domain, a transmembrane-spanning domain, a kinase homology domain (KHD) and a guanylyl cyclase domain. Because the presence of ATP or adenylylimidodiphosphate reduces atrial natriuretic peptide (ANP) binding and is required for maximal guanylyl cyclase activity, a direct interaction of ATP with the receptor KHD domain is plausible. Therefore, we investigated whether ATP interacts directly with a binding site on the receptor by analyzing the binding of a photoaffinity analog of ATP to membranes from human embryonic kidney 293 cells expressing the NPR-A receptor lacking the guanylyl cyclase moiety (DeltaGC). We demonstrate that this receptor (NPR-A-DeltaGC) can be directly labeled by 8-azido-3'-biotinyl-ATP and that labeling is highly increased following ANP treatment. The mutant receptor DeltaKC, which does not contain the KHD, is not labeled. Photoaffinity labeling of the NPR-A-DeltaGC is reduced by 50% in the presence of 550 microm ATP, and competition curve fitting studies indicate a Hill slope of 2.2, suggestive of cooperative binding. This approach demonstrates directly that the interaction of ANP with its receptor modulates the binding of ATP to the KHD, probably through a conformational change in the KHD. In turn, this conformational change is essential for maximal activity. In addition, the ATP analog, 8-azido-adenylylimidodiphosphate, inhibits guanylyl cyclase activity but increases ANP binding to the extracellular domain. These results suggest that the KHD regulates ANP binding and guanylyl cyclase activity independently.     

Tyrosine phosphorylation of the human guanylyl cyclase C receptor

Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) inEscherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.     

A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor

Secretory diarrhea is the leading cause of infectious diarrhea in humans. Secretory diarrhea may be caused by binding of heat-stable enterotoxins to the intestinal receptor guanylyl cyclase C (GCC). Activation of GCC catalyzes the formation of cGMP, initiating a signaling cascade that opens the cystic fibrosis transmembrane conductance regulator chloride channel at the apical cell surface. To identify proteins that regulate the trafficking or function of GCC, we used the unique COOH terminus of GCC as the "bait" to screen a human intestinal yeast two-hybrid library. We identified a novel protein, IKEPP (intestinal and kidney-enriched PDZ protein) that associates with the COOH terminus of GCC in biochemical assays and by co-immunoprecipitation. IKEPP is expressed in the intestinal epithelium, where it is preferentially accumulated at the apical surface. The GCC-IKEPP interaction is not required for the efficient targeting of GCC to the apical cell surface. Rather, the association with IKEPP significantly inhibits heat-stable enterotoxin-mediated activation of GCC. Our findings are the first to identify a regulatory protein that associates with GCC to modulate the catalytic activity of the enzyme and provides new insights in mechanisms that regulate GCC activity in response to bacterial toxin.     

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.