Receptor guanylyl cyclase C (GC-C): regulation and signal transduction
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- Basu, N., Arshad, N. & Visweswariah, S.S. Mol Cell Biochem (2010) 334: 67. doi:10.1007/s11010-009-0324-x
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Receptor guanylyl cyclase C (GC-C) is the target for the gastrointestinal hormones, guanylin, and uroguanylin as well as the bacterial heat-stable enterotoxins. The major site of expression of GC-C is in the gastrointestinal tract, although this receptor and its ligands play a role in ion secretion in other tissues as well. GC-C shares the domain organization seen in other members of the family of receptor guanylyl cyclases, though subtle differences highlight some of the unique features of GC-C. Gene knock outs in mice for GC-C or its ligands do not lead to embryonic lethality, but modulate responses of these mice to stable toxin peptides, dietary intake of salts, and development and differentiation of intestinal cells. It is clear that there is much to learn in future about the role of this evolutionarily conserved receptor, and its properties in intestinal and extra-intestinal tissues.
KeywordsGuanylyl cyclase CGuanylinUroguanylinStable toxin peptidescGMP
This review focuses on receptor guanylyl cyclase C, initially identified as the target for the heat-stable enterotoxins (ST) produced by enterotoxigenic E. coli. E. coli-associated diarrheal diseases are important endemic problems confronting developing nations and also travellers to these countries [1, 2]. These pathogens cause disease through the production of either heat-labile or heat-stable enterotoxins . Symptoms such as increased fluid and ion secretion, decreased fluid absorption, and neuromuscular imbalance are similar to those elicited by the action of cholera toxin, but lead to a much milder form of the disease. The E. coli heat-labile toxin brings about an activation of adenylyl cyclases, resulting in an increase in intracellular cAMP, while the ST peptide elevates intracellular cGMP levels . ST binds to a receptor guanylyl cyclase, GC-C, which is localized primarily on the apical or brush border membrane of intestinal epithelial cells [5–7]. GC-C also serves as the receptor for the gastrointestinal peptides guanylin/uroguanylin [8, 9]. The role of GC-C in mediating ST-induced diarrhea suggested a vital function for this receptor in maintaining fluid-ion homeostasis in the intestine [10, 11]. In this review, we will discuss features of GC-C that include its structure, regulation, and signaling processes in intestinal cells. We provide historical perspectives on the discovery and characterization of a cGMP-signaling system in the gut, and also highlight areas of new research which may lead to a deeper understanding of GC-C in normal physiology and in disease. An earlier review on this receptor has appeared in the same journal , so we present a more detailed analysis of research findings from 2002 onwards in the current review.
Tissue specific expression and sub-cellular localization of GC-C
A study more than 30 years ago demonstrated that the bulk of guanylyl cyclase activity was present in the apical region of intestinal cells associated with microvillar enzymes such as sucrase isomaltase and alkaline phosphatase [5, 13]. This was followed by the first report that administration of ST to rat intestinal cells led to increases in intracellular cGMP . Receptor autoradiography and in situ hybridization showed that GC-C is primarily expressed in rat small intestinal and colonic epithelia . Indeed, targeted gene deletion showed that GC-C was responsible for most of the guanylyl cyclase and ST-binding activities in the rat brush border epithelia [10, 11]. Reduced receptor expression was seen in the mitotically active stem cells located within the intestinal glands. There was no gradation in the expression along the villus to crypt axis in the small intestine, whereas in the colon the majority of GC-C was found to be expressed in the crypts, with very little expression on the surface or in regions at the opening of the crypts . Some investigators have suggested that the almost exclusive intestinal expression of GC-C could serve as a marker for colon carcinoma metastasis in tissues other than the gut . Indeed, PCR primers for GC-C have been used to detect circulating tumor cells and occult micrometastases of colon tumors .
In rat and the North American opossum, specific binding sites for ST were found in extra-intestinal tissues such as the kidney, airway epithelium, peri-natal liver, stomach, brain, and adrenal glands [18, 19]. Additionally, immunohistochemical analyses demonstrated expression of GC-C in the tubular epithelial cells of rat epididymis suggesting that the GC-C signaling pathway could perhaps control fluid and ion balance for optimal sperm maturation and storage in this tissue .
Polarized sorting of membrane proteins and lipids to the apical or basolateral membranes of epithelial cells is a process necessary for directional ion and solute transport as well as cellular signaling. GC-C expression is limited to the apical or the luminal membrane of intestinal epithelia which is in contrast to atrial natriuretic factor receptor guanylyl cyclase (ANF-RGC) which localizes in part to the basolateral membrane with a lower amount found at the apical side . The apical sorting determinant for GC-C is a unique region of 11 highly conserved amino acids in the COOH terminus of the receptor. This sequence is also sufficient to selectively target an unpolarized reporter protein to the apical membrane . Additional work is needed to determine the identity of the proteins involved in this apical sorting which would also provide a more complete understanding of general protein targeting in colonic epithelia.
Domain structure and organization
The full length cDNA of human GC-C obtained from colonic epithelial cells is translated into a polypeptide of 1,073 amino acids [7, 23, 24]. There is an N-terminal signal sequence of 23 amino acid residues, which is responsible for targeting the protein to the endoplasmic reticulum. The signal sequence undergoes post-translational proteolysis to generate a mature polypeptide of 1,050 amino acids, which corresponds to a theoretical molecular mass of 120 kDa. GC-C is conserved evolutionarily with orthologs identified in birds and fish .
The ECD of receptor GCs exhibits the least sequence similarity to other members of the family, which is reflective of the diverse ligands to which they bind . There is only 15–20% sequence identity between the ECD of GC-C and ANF-RGC or retinal guanylyl cyclases (RetGCs), while the ECD of human GC-C shows a 70–75% sequence identity with GC-Cs from other species. The ECD of GC-C binds ST peptides with a high affinity (Kd ~0.1 nM) and the endogenous peptide ligands guanylin (Kd ~10 nM) and uroguanylin (Kd ~1 nM) with lower affinity [27, 28]. The region in the ECD of porcine GC-C that interacts with ST has been mapped by photoaffinity labelling to be very close to the transmembrane region in the sequence “SPTFIWK” (amino acids 409–416) [29, 30], with the tryptophan residue playing an important role in ligand interaction. Subsequently, it was shown that fragments of the ECD of porcine GC-C were capable of binding ST independently [30, 31].
In the absence of a crystal structure of the ECD of GC-C, comparative modeling has been performed using the ECD of ANF-RGC as a template [31, 32]. Analyses of the crystal structure of the ECD of ANF-RGC revealed that it existed as a dimer with each monomer consisting of N-terminal α-helical and C-terminal β-sheet domains. . Subsequently, the crystal structures of the ECD of NPR-C with and without bound CNP were determined . The ECD of GC-C is therefore predicted to contain a C-terminal region consisting of β-strands, which could contain the ligand-binding site, at a similar position to that seen in ANF-RGC [32, 34]. No information is available till date on the structural rearrangements that occur on ligand binding to GC-C. Heterologously expressed GC-C forms higher order complexes even in the absence of ligand, that are converted to monomers on exposure to reducing conditions [35, 36]. Since the ECDs of ANF-RGC and NPR-C crystallized as dimers in the absence of ligand, and no disulfide bonds appear to contribute to dimerization [33, 34], it is unclear why the presence of reducing agents alters the oligomeric status of GC-C. However, it is possible that unique disulfide bonds may be present in the ECD of GC-C, which will be known only when structural data becomes available.
GC-C is glycosylated in mammalian cells giving rise to two differentially glycosylated forms of approximately 130 and 145 kDa in size, as determined by mobility on SDS polyacrylamide gel electrophoresis . Both forms bound wheat germ agglutinin and protein N-glycosidase F treatment converted both forms to a single 120 kDa protein, the size predicted for the unmodified polypeptide chain encoding GC-C. Heterologous expression of GC-C in HEK293 cells in the presence of tunicamycin, an inhibitor of N-linked glycosylation, resulted in a protein of 120 kDa in size, indicating that GC-C is an N-linked glycoprotein . The 145 kDa form of GC-C contains sialic acid and galactose residues and is present on the plasma membrane of transfected HEK293 cells over-expressing GC-C, whereas the 130 kDa form contains high mannose structures and is primarily resident in the endoplasmic reticulum. The 130 kDa form serves as the precursor for the plasma membrane-associated 145 kDa form, and though both bind ST with equal affinity, cGMP production is increased only in the 145 kDa form in the presence of ligand .
Kinase homology domain
All receptor GCs possess a domain of approximately 250 residues between the juxtamembrane and the guanylyl cyclase domains, which shares a significant sequence similarity with protein tyrosine kinases, and is called the KHD. The position of the KHD in receptor guanylyl cyclases, as well as the apparent co-evolution of the KHD with their respective guanylyl cyclase domain  suggests that it transduces the signal of ligand binding from the ECD to the guanylyl cyclase domain. Thus, the role of the KHD appears to be analogous to the role of G proteins in mediating the signaling from the heptahelical receptors to the adenylyl cyclase . Though the KHDs of ANF-RGC and GC-C share a sequence identity of around 35%, they are not interchangeable. A chimeric receptor, in which the KHD of ANF-RGC was replaced with the KHD of GC-C, resulted in a protein that was unresponsive to natriuretic peptides. Deletion of the KHD of ANF-RGC led to constitutive, ligand independent production of cGMP . However, partial deletions in the KHD of ANF-RGC and GC-B resulted in inactive proteins. A similar effect of complete and partial KHD deletions was also observed in GC-C, indicating that the domain has a regulatory effect on receptor function [39, 40]. However, the exact mechanism of this regulation has remained elusive.
Based on structure-function correlation in protein kinases, three motifs have been found to be essential for function: (i) the lysine residue in the VAIK motif which interacts with the α- and β-phosphate of ATP; (ii) the aspartate in the HRD motif which functions as a base to facilitate proton transfer; and (iii) the aspartate residue in the DFG motif which interacts with the metal ion, which in turn coordinates the β- and γ-phosphate of ATP . Approximately, 10% of the 518 kinases known in the human kinome lack one of the above mentioned three motifs in the kinase domain, and have therefore been predicted to be catalytically inactive [42, 43]. Thus, while the KHD of GC-C shares 23–25% sequence identity (45% sequence similarity) with the catalytic domain of Src family kinases (Src, Lck, and Hck), a sequence alignment of the KHD of GC-C with tyrosine and serine-threonine kinases reveals that the highly conserved HRD motif in active protein kinases  is replaced with a HGR motif in the KHD of GC-C. In addition, the glycine-rich loop seen in some protein kinases is substituted by a smaller loop containing large charged residues. Using homology modeling and mutational analysis, lysine residue 516 in the KHD was shown to be essential for ATP interaction , but the absence of the catalytic aspartate residue in the HRD motif in GC-C predicts this domain to be a pseudokinase domain. Since recent crystal structures have shown that many kinases predicted from sequence to be inactive can possess kinase activity [43, 45, 46], it is conceivable that the KHD of GC-C may adopt an unconventional mechanism to phosphorylate itself or other substrates.
A ~70 amino acid region between the KHD and the guanylyl cyclase domain in receptor GCs is referred to as the linker region. The linker region in ANF-RGC has been shown to mediate dimerization of the catalytic subunits . Truncated mutants of ANF-RGC possessing only the catalytic domain exist as monomers that are devoid of enzyme activity . The linker region of RetGC-1 has been shown to be critical for proper regulation by GCAP-1 and Ca2+ by mediating protein dimerization as judged by a phage λ repressor dimerization assay . A recent bioinformatic analysis has suggested that the linker region in receptor GCs serves as a signaling helix, which could regulate the activities of these receptors, and adopt a coiled-coil structure . However, the ability of the ECD in GC-C to dimerize suggests that the linker region may not be the main driving force for dimerization of the receptor. Recently, extensive mutational analysis of residues in the linker region suggests that the linker is important for repressing the guanylyl cyclase activity of the receptor in the absence of ligand, and permitting ligand-mediated activation of the cyclase domain .
Guanylyl cyclase domain
Guanylyl cyclases belong to the Class III nucleotide cyclase family . The primary structure of the guanylyl cyclase domain (approximately 250 amino acids) is highly conserved in both receptor and soluble GCs and is closely related to the catalytic domain of adenylyl cyclases (ACs) . Heterodimeric cyclases such as the ACs and soluble GCs have a single active site formed by two catalytic subunits and bind one substrate molecule per dimer . On the other hand, homodimeric receptor GCs could have two active sites, and may be capable of binding two substrate molecules per dimer. Recent crystal structures of the guanylyl cyclases Cya2 from cyanobacterium Syncheocystis PCC6803  and CYG12 from the green algae Chlamydomonas reinhardtii have shed light on the topology of the cyclase domain . The crystal structures reveal the presence of a head-to-tail homodimer with two monomers in a wreath-like arrangement. The central cleft formed at the dimer interface contains the two symmetric active sites which could mimic the homodimeric guanylyl cyclases .
The guanylyl cyclase fold contains a central seven-stranded β sheet surrounded by several α helices. This is a hallmark of the class III nucleotide cyclase fold and resembles the ‘palm’ fold in the active site of various DNA polymerases . Similarity in structure is also reinforced by the fact that both the polymerases and the adenylyl cyclases catalyze analogous reactions, which involve the nucleophilic attack of the 3′-hydroxyl group of a ribose unit on the α-phosphate of a nucleotide 5′-triphosphate . The active site of the GC domain is located at the dimer interface and contains two Asp residues that co-ordinate the metal co-factors ions and thereby catalyze the attack of the 3′-hydroxyl group. In addition, an Asn residue orients the ribose and an Arg residue serves to stabilize the transition-state. Our unpublished observations suggest that mutation of the transition stabilizing residues (Asn948 and Arg952) in GC-C to alanine completely abolishes the guanylyl cyclase activity of GC-C.
Purified soluble GCs exhibit Hill coefficients of 1.0 for GTP, consistent with a single class of substrate binding sites . However, receptor GCs, including GC-C, show Hill coefficients >1.0, suggestive of two substrate-binding sites that interact in a positively cooperative manner [59, 60].
Carboxyl terminal domain
GC-C and the receptor GCs present in sensory organs (GC-D, GC-E and GC-F) possess a short C-terminal domain (CTD) of approximately 60 residues . It has been suggested that the CTD may be involved in associating receptor GCs with the cytoskeleton. This is consistent with the finding that GC-C and RetGCs are resistant to non-ionic detergent extraction from membranes in comparison to ANF-RGC and CNP-RGC, which lack the CTD [28, 62] Deletion of the CTD in GC-C led to a loss of ligand-mediated activation of the receptor . The CTD also contains an apical sorting signal as stated above . The C-terminal domain interacts with IKEPP (intestinal and kidney-enriched PDZ protein). This protein is expressed in the apical region of intestinal cells and its association with GC-C inhibits ST-mediated cyclase activity, the mechanism of which is unknown .
Peptide ligands of GC-C
The structure of an active analog of the 13-mer core sequence of E. coli STp (Cys5–Cys17 in STp, a variant of ST produced by an E. coli strain isolated from pigs; corresponds to Cys6–Cys18 in STh, a variant produced by E. coli isolated from humans), provides some insight into the mode of interaction of ST with GC-C. The peptide molecule forms a self-associated ring-shaped hexamer , which could have formed as an artifact of crystallization, since no other evidence exists for oligomerization of ST. All the monomer peptide molecules in the hexamer have identical conformation, which are fixed by three intramolecular disulfide linkages between Cys5 and Cys10, Cys6 and Cys14, and Cys9 and Cys17. Moreover, even though a minimum of two disulfide bridges is required for ST binding to GC-C, the bond between Cys6 and Cys14 is essential . Recently, a peptide of 12-amino acids containing only two disulfide bonds was synthesized that was a weak agonist of GC-C . Whether such a peptide could be modified to serve as an antagonist of GC-C or as a means of targeting the receptor in intestinal cells remains to be seen.
Almost a decade after the isolation of bacterial ST peptides, a 15-amino acid long peptide that increased cGMP levels in T84 human colon carcinoma cells was purified from the extracts of rat jejunum, and named guanylin . This was soon followed by the isolation of uroguanylin from opossum urine . Subsequently, uroguanylin was also isolated as biologically active peptides from human and rat urine, and also from opossum and rat intestinal mucosa [69–71]. Like ST, the active peptide domain in guanylin and uroguanylin is located in precursor proteins called pro-guanylin and pro-uroguanylin, respectively. These precursor proteins have little or no intrinsic biological activity until cleaved by converting enzymes that release the active peptides for interaction with target receptor GCs [71, 72]. However, these peptides are less potent in stimulating transepithelial chloride secretion in T84 cells compared to ST, since the affinity of guanylin and uroguanylin for GC-C is 100-fold and 10-fold lower than ST, respectively [9, 73]. A recent report has shown that uroguanylin can also activate GC-D which is primarily expressed in the necklace glomeruli of the olfactory bulb [74–76].
The biologically active forms of guanylin and uroguanylin have striking similarities in their structure, with four conserved cysteines (Fig. 2). Disulfide bonds are formed between the first and third, second and fourth cysteines in each peptide. These disulfides appear to be required for optimal peptide potencies in the stimulation of cGMP production in vitro [8, 9]. ST has an extra disulfide bond and this structural feature may account for the apparently higher potencies of ST peptides for activation of GC-C as compared to guanylin and uroguanylin .
Uroguanylin is highly potent and effective in stimulating cGMP production in cells at an acidic pH of ~5.5, whereas guanylin has a reduced efficacy at an acidic pH, and is active at an alkaline pH [77, 78]. This is because acidic pH markedly decreases the affinity of guanylin binding to GC-C and correspondingly enhances that of uroguanylin binding . Along the gastrointestinal tract, the extracellular fluid bathing the apical surfaces has a variable pH , which may influence the biological activities of guanylin and uroguanylin, through changes in peptide ionization states and/or molecular conformations. Thus, the presence of both guanylin and uroguanylin in the gastrointestinal tract broadens the spectrum of biological activity for cGMP-mediated control of salt and water transport.
Studies on the mRNA expression patterns for guanylin and uroguanylin in spleen and testis of North American opossum, Didelphis virginiana resulted in the identification of a unique uroguanylin-like mRNA . The unique feature of this peptide was the presence of three cysteine residues, which can form only one intramolecular disulfide bond. This peptide was named lymphoguanylin because the source of the first cDNA isolated was spleen and is expressed broadly in all lymphoid tissues . Synthetic lymphoguanylin activates GC-C in the T84 cell line, and OK-GC receptors in opossum kidney (OK) cells, but its potency is less than that of uroguanylin or guanylin . Till date, no lymphoguanylin ortholog has been identified in any species other than the opossum.
Regulation of guanylyl cyclase C
Any molecule in a signaling pathway must undergo modulation to ensure its proper function. The function of a protein can be regulated in several ways such as covalent modification of the protein, change in spatial or temporal expression of the protein, sub-cellular localization or half life. GC-C undergoes many of these types of regulation as described below.
GC-C is encoded by the gene GUCY2C present at the locus 12p12 in the human . The full length GC-C mRNA is spliced from 27 exons and only a single splice variant of unknown function is known, which arises from an alternate 5′-splice acceptor in exon 1 forming a premature termination at codon 27. The specificity of expression of the alternatively spliced variant of GC-C parallels that of wild-type GC-C and is limited only to intestinally derived tissues . The regulatory region lies up to 2 kb upstream of the gene and has binding sites for several transcription factors such as caudal type homeobox gene-2 (Cdx2), hepatocyte nuclear factor 4 (HNF4), GATA-4, glucocorticoid receptor and nuclear factor-IL6 NF-IL6 . The factors HNF4, Cdx2, and GATA-4 are expressed in the intestine and of these, HNF4 and Cdx2 are responsible for expression of GC-C in the intestine as well as in cells derived from the intestine such as Caco2 and T84 [85, 86]. The role of the other factors has not yet been elucidated. Protein kinase C (PKC) has been shown to transcriptionally regulate expression of GC-C. Activation of PKC by phorbol esters such as 4β-phorbol 12-myristate 13-acetate (PMA) led to a down regulation of GC-C mRNA transcript levels in T84 cells . Active PKC prevents the binding of HNF4 to DNA, perhaps by direct phosphorylation of HNF4 or by reducing HNF4 expression.
Epithelial cells at the gastro-esophageal junction are frequently exposed to bile acids refluxed from the stomach [88, 89]. It has been observed that these bile salts can induce ectopic expression of GC-C in these cells which are undergoing neoplastic transformation . One of the bile salts, deoxycholate, increased nuclear translocation of NFkB thereby increasing cellular levels of the GC-C specific transcription factor Cdx2. This may represent a mechanism-based marker and target of transformation at the gastroesophageal junction .
Phosphorylation of GC-C
ANF-RGC is phosphorylated in the basal state and this phosphorylation is essential for its activation . There are no reports of basal phosphorylation of GC-C, and the serine and threonine residues that are phosphorylated in ANF-RGC, present in the juxtamembrane and kinase homology domain region, are not conserved in GC-C [92, 93]. Protein Kinase C phosphorylates GC-C on Ser1029 in the CTD of GC-C, and enhances ST-mediated GC-C activation in terms of increased cGMP production [94, 95]. This is in contrast to the role of PKC in transcriptional down-regulation of GC-C, which may serve as a mechanism of feedback inhibition of GC-C activity. Phosphorylation at tyrosine has not been reported in any receptor GC except GC-C . The GC-C intracellular domain was tyrosine phosphorylated when expressed in E. coli cells harboring the tyrosine kinase Elk (EphB1). The residues that were phosphorylated were not identified but results obtained with deletion constructs of GC-C showed that at least two distinct sites for tyrosine phosphorylation were present. Recent data shows that GC-C is a substrate for inhibitory phosphorylation by c-src, resulting in reduced ligand-mediated cGMP production . Therefore, GC-C could be involved in cross-talk with tyrosine kinases in colon carcinoma cells where src kinase activity is high.
Glycosylation of GC-C
GC-C has sites of glycosylation well conserved across species and the importance of these sites has been documented in porcine GC-C . Single mutations of the Asn residues, that could be potential sites for N-linked glycosylation, caused no change in the binding affinity of the mutants for ST, but a reduction in both total ST-binding activity and a concomitant activation of GC-C was seen. This suggests that mutation of these asparagines residues led to conformational instability of the protein. Mutation of a membrane proximal site of glycosylation abolished ligand binding, not due to loss of interaction of the carbohydrate moiety with the ligand, but because of a decrease in the stability of the functional form ECD which binds ST .
Enzymatic deglycosylation of the ECD of porcine GC-C resulted in a loss of ST-binding activity . However, the role of glycosylation in human GC-C seems more complex. The non-glycosylated form of the human ECD expressed in bacteria binds ST with the same affinity as both of the full length glycosylated forms of human GC-C . The 130 kDa form of human GC-C (130 kDa) has a high basal guanylyl cyclase activity in vitro, and is not stimulated further by ST. It therefore appears that further changes to the sugars which occur in the transit of GC-C from the endoplasmic reticulum to the plasma membrane 145 kDa form, render the receptor stimulatable by the ligand and inhibits its basal guanylyl cyclase activity . The glycosylation status of human GC-C thus mediates its response to ligand, and it would be interesting in future to identify sites for glycosylation in the extracellular domain of GC-C that modulate its ligand binding and further activation of the receptor.
Desensitization of GC-C
Once a signaling cascade is initiated, it is reset by a process known as desensitization to prevent indefinite perpetuation of the signal. While the desensitization of ANF-RGC is by dephosphorylation , this process does not appear to be operative in GC-C. The down regulation of GC-C signaling is different in various cell lines. In Caco2 cells, selective removal of only the ligand-stimulatable, higher glycosylated 145 kDa form from the surface of the receptor is seen on prolonged treatment with ST . Similar treatment in T84 cells rendered the receptor refractory to the ligand, although no removal of GC-C was seen from the surface. In this case, it was the increased activity of PDE5 that was found to accompany the process of desensitization which reduced the levels of cGMP in the cell . The decrease in ligand-mediated cyclase activity of GC-C was due to a dramatic reduction in the Vmax of the cyclase . However, exposure of HEK293 cells stably expressing GC-C to ST for prolonged periods did not result in desensitization, indicating that this process appeared to be a cell specific phenomenon .
Desensitization occurs even in vitro in a manner which seems independent of cell line specificity. GC-C from either T84 or on overexpression in HEK293 cells was desensitized on incubation with ST prior to addition of the substrate Mg-GTP. This was alleviated in the presence of ATP, indicating a role for ATP (and perhaps the KHD) in regulation of GC-C activity [104–106].
Allosteric regulators of GC-C activity
ATP is able to potentiate ligand-activated cGMP production in vitro by GC-C in the presence of Mg-GTP as substrate (EC50 ~ 500 μM), while 2-substituted ATP derivatives (2-methyl thioadenosine 5′-triphosphate (2Me-SATP) and 2 chloroadenosine 5′-triphosphate (2Cl-ATP)) inhibit guanylyl cyclase activity at approximately similar concentrations . In contrast, ATP inhibits the cyclase activity of GC-C when Mn-GTP is used as substrate, or in the presence of detergents with magnesium as the metal co-factor . The intracellular domain of GC-C expressed in insect cells has constitutive guanylyl cyclase activity which is also inhibited by ATP . ATP mediates its effects by binding to the KHD of GC-C and ATP hydrolysis is not required. Lys516 in GC-C corresponds to the conserved lysine in the VAIK motif present in tyrosine kinases that is involved in coordinating the α and β phosphates of ATP. Mutation of Lys516 to an Alanine abolished ATP binding and the subsequent inhibition in the presence of detergents .
The use of inhibitors has also contributed to the understanding of GC-C regulation. Previously documented inhibitors include 2-substituted adenine nucleotides which were found to be potent allosteric inhibitors of GC-C [110, 111]. Since the KHD binds ATP, it is likely that these adenine nucleotides bind in the same region. Another group of inhibitors, the tyrosine kinase inhibitors or tyrphostins, were found to noncompetitively inhibit the activity of GC-C [44, 112]. The inhibition was seen both in the presence and absence of the KHD region of the receptor implying that the site of action is in the guanylyl cyclase domain, although the possibility of the tyrphostins influencing the KHD region is not ruled out. A recent study revealed that a pyridopyrimidine derivative, [5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2,1:5,6] pyrido [2,3-d]pyrimidine-2,4,6-trione; BPIPP] inhibited both soluble as well as receptor guanylyl cyclases and adenyl cyclases . However, the mechanism of BPIP-dependent inhibition appears to be complex and indirect. Till date, there are no specific inhibitors of any receptor guanylyl cyclase, but it is conceivable that inhibitors directed to the individual KHDs could be more specific, since this domain is unique to receptor guanylyl cyclases, and is not found in other guanylyl cyclases.
Signal transduction mediated by GC-C
Regulation of salt and water homeostasis
The endogenous peptides, guanylin, and uroguanylin are involved in the regulation of salt and water transport across the intestinal epithelia . ST peptides serve as super-agonists of the receptor resulting ST-induced watery diarrhea. GC-C knockout (KO) mice are refractory to the actions of ST that are seen in WT mice [10, 11]. Indeed, ST peptides represent molecular mimicry wherein enterotoxigenic bacteria exploit normal intestinal physiology for their dissemination and propagation.
An increase in cGMP levels can regulate ion and fluid transport in the intestine in different ways . Sodium absorption in the intestine is partly governed by Na+/H+ exchanger (NHE) which absorbs NaCl in combination with the chloride/anion exchanger . Cyclic GMP is known to inhibit the NHE, thereby decreasing sodium and chloride absorption [120, 121]. Intestinal chloride efflux is mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) which is located in the apical membrane of Cl− secreting epithelial cells . It belongs to the ATP binding cassette (ABC) transporter family  and functions as a Cl− channel, passively transporting Cl− in either direction across a membrane, depending on the electrochemical gradient . Guanylyl cyclase-C induced increase in levels of intracellular cGMP results in the activation of cGMP-dependent protein kinase II (PKGII) which phosphorylates CFTR thus activating  the chloride channel. . PKGII co-localizes with CFTR on the apical brush border intestinal membrane and is able to phosphorylate CFTR in vivo [125, 126]. The reduced chloride absorption by the NHE in response to cGMP and increased chloride secretion by the CFTR are thought to be the chief mechanisms by which ST mediates its effects.
Cyclic GMP in intestinal cells produced by activation of GC-C could also act by inhibiting PDE3 (a cGMP-regulated, cAMP-hydrolyzing phosphodiesterase), which results in decreased cAMP hydrolysis, local cAMP accumulation, and subsequent activation of PKA [127, 128]. Interestingly, very high levels of cGMP can also directly cross-activate PKA . The PKGII-independent, PKA-dependent cGMP regulatory pathway is probably predominant in the colon, where the expression of PKGII is relatively low as compared to the small intestine . However, one study has shown that cGMP analogs can activate the CFTR in primary cultures of human distal colonocytes and T84 cells through a PKGII-dependent pathway .
CFTR relocates from sub-apical vesicles to the apical membrane following increase in intracellular cAMP . Recently, cGMP produced by GC-C in response to ST has also been shown to induce a greater than four-fold increase in surface CFTR in enterocytes, that was prevented by PKG inhibitors . This re-distribution of CFTR is dependent on an intact microtubular network as well as PKA and PKG. This suggests that the cytoskeletal and cyclic nucleotide-dependent recruitment of CFTR to the apical membrane plays a role in chloride transport in colonic epithelial cells .
Recently, there has been some debate on the relative importance of fluid secretion versus fluid absorption in causing the watery diarrhea associated with the ST peptides. It has been argued that there is a lack of direct evidence for increased chloride secretion following ST application to cell lines and intestinal loops, and that fluid secretion occurs not because epithelial cells actively pump water out, but instead inhibit fluid absorption, increase conductivity through tight junctions, and increase hydrostatic driving force through elevated capillary pressure . In this context it is worthwhile to note that till date no drug is available that can inhibit diarrhea purely by using inhibitors of chloride secretion through CFTR.
Regulation of intestinal cell proliferation by GC-C and its ligands
Cyclic GMP is known to inhibit cell proliferation in cells such as cardiac fibroblast [134, 135] and cGMP-dependent proteins kinases are mediators of both proliferative and anti-proliferative effects in diverse cell types . Therefore, one would predict that cGMP produced in the intestine by GC-C could regulate intestinal cell proliferation. The intestinal epithelium undergoes homeostatic cycles of proliferation, migration, differentiation, and apoptosis driven by multipotent stem cells . An imbalance between cell proliferation and apoptosis may lead to the formation of tumors within the intestinal tract . In colon cancer, the expression of mRNAs encoding uroguanylin and guanylin are markedly suppressed, whereas mRNA expression of GC-C is comparable in colon cancer and normal colon mucosa [139, 140].
Activation of GC-C by uroguanylin treatment has been shown to induce apoptosis in human T84 and Caco2 colon carcinoma cell lines by a cGMP-dependent mechanism . Furthermore, oral administration of uroguanylin suppressed the formation and apparent progression of polyps in the ApcMin/+ mouse model (a strain containing a dominant mutation in the adenomatous polyposis coli, Apc gene) of colorectal cancer . Activation of GC-C opposes pro-proliferative Wnt/β-catenin/Tcf-4 signaling and induces apoptosis by promoting PKG1β-dependent degradation of β-catenin, inhibiting its nuclear translocation .
Recent reports have suggested that ST or uroguanylin treatment of T84 and Caco2 cells inhibit proliferation by delaying progression of the cell cycle and not by inducing apoptosis [142, 143]. The cytostatic effects of GC-C agonists could be mimicked by 8-Br-cGMP, a cell-permeable cGMP analog, but could not be prevented by inhibitors of the known downstream effectors of elevated cGMP, such as PKG, PKA or PDE3. However, L-cis-diltiazem, a CNG channel inhibitor, as well as removal of extracellular Ca2+ or chelation of intracellular Ca2+ prevented ST-mediated inhibition of proliferation . This suggested that the anti-proliferative action of GC-C agonists was mediated by a cGMP signaling mechanism, which regulates Ca2+ influx through CNG channels. Thus, Ca2+ serves as the third messenger in the signaling cascade linking GC-C at the cell surface to regulation of proliferation in the nucleus.
It has been suggested that ST-induced GC-C signaling leads to trafficking of calcium sensing receptors (CaRs) to the surface of human colonocytes/enterocytes. Elimination of GC-C signaling in mice abolished surface expression of CaR in enterocytes highlighting the importance of GC-C in Ca2+ mediated cytostasis . Prolonged GC-C stimulation leads to resistance in tumor cells to ST-induced cytostasis . GC-C activates a cGMP-regulated, cGMP-specific phosphodiesterase (PDE5), which lowers intracellular cGMP levels , which in turn can effect Ca2+ influx through CNG channels . Thus, these negative feedback mechanisms in cGMP signaling help in shaping the duration and amplitude of agonist-induced cytostasis.
Approximately, 1.5 million individuals suffer from colon cancer in developed countries, but its incidence is relatively low in underdeveloped and developing countries . A common epidemiological characteristic of these colon cancer-spared regions is the prevalence of enterotoxigenic E. coli. Periodic infections with ST producing bacteria in the intestine could elicit beneficial therapeutic actions for people living in the developing nations, since the action of the ST peptides could prevent cell proliferation at relatively early stages of tumor formation, thus providing resistance to intestinal neoplasia. It is conceivable of course that there are additional signaling pathways that regulate and are regulated by GC-C in the colonic cell, that could either alleviate or enhance tumor formation, and could be controlled by actions of GC-C ligands .
The GC-C knockout mouse
Given the evolutionary conservation of GC-C, from its first appearance in the class Pisces to Mammalia , its function may be of critical importance to intestinal physiology. Two groups have generated GC-C knockout mice (KO) to investigate the effects of this gene deletion in the mammalian system [10, 11]. The disruption of such a highly conserved gene did not result in a drastic phenotypic difference wherein the mice were viable, fertile, and developed normally. As expected, the KO mice were not susceptible to ST-induced diarrhea. Ironically, fluid-ion homeostasis in the intestine was also maintained in the KO mice upon oral administration of fluids containing high concentrations of salt [10, 11]. However, the preliminary analysis of the effects of GC-C deletion was on mice grown in disease free conditions in the laboratory which are not exposed to any stresses that a mammal outside this bubble environment would face. Therefore, the appropriate stressors, and the requirement for GC-C in facing such stresses, if any, are yet to be identified.
Contrary to what was hypothesized, the lack of GC-C in the KO mice did not completely abolish ST binding to the intestine. Low affinity receptors which were reported earlier in normal mice [146, 147] are still found in the KO mice . The binding in the KO mice was about 11% of the wild-type mice (WT) and a 40-fold difference in affinity was estimated between GC-C and this alternate putative receptor. ST binding to this receptor brought about significant duodenal bicarbonate secretion, although it was considerably lower than the secretion seen in WT mice. The KO mice show normal fluid-ion balance, suggesting that guanylin and uroguanylin can maintain homeostasis via receptors other than GC-C. For example, a receptor was found in rat intestinal cell line IEC-6 cells (which lack GC-C) which was not coupled to guanylyl cyclase activity, but bound ST .
Orally administered sodium chloride can be excreted more rapidly than equivalent amounts administered intravenously [149, 150] giving rise to the concept that NaCl intake is monitored by the intestine and the cue is subsequently given to the kidney to alter ion secretion and extracellular fluid volume. Intravenous administration of guanylin or uroguanylin in GC-C null mice also resulted in a rapid, time and dose dependent sodium excretion in a manner similar to that seen in normal mice [151, 152]. There was an increase in cGMP levels in both mice (2.5-fold over the basal level of cGMP in urine) indicating that this is a GC-C-independent but cGMP-dependent mechanism of fluid-ion secretion. Transcriptional upregulation of Na+/K+ ATPase γ-subunit or ClC-K2 mRNA was seen and may be regarded as a long term effect of peptide application . Clearly, evidence suggests that there are alternate receptors for ligand hitherto considered exclusive activators of GC-C.
Apart from the role of fluid-ion homeostasis in the intestine and kidney, uroguanylin and guanylin are also to regulate proliferation of the intestinal epithelium. Inactivation of the guanylin gene in mice led to lower levels of cGMP in intestinal cells than that seen in normal mice and the rate of proliferation was greatly increased . In the GC-C KO mouse, the reduced cGMP levels promote cell division, leading to the increase in the fraction of proliferating cells in the crypts [154, 155]. ApcMin/+ mice when crossed with the GC-C KO mouse resulted in increased tumor incidence and multiplicity, as was the case when GC-C KO were exposed to carcinogens such as azoxymethane . In contrast, another study showed that the ApcMin/+; Gucy2c−/− mice have a reduced polyp number, increased levels of apoptosis and an increased caspase-3 and caspase-7 gene expression in the intestines as compared to the ApcMin/+ mice . It has been suggested that absence of normal GC-C expression during development and the corresponding reduction in GC-C signaling may have caused alterations in other cell growth regulatory pathway(s) that partially bypass the neoplastic progression in the ApcMin/+; Gucy2c−/− mice.
We have discussed here much of the available information on GC-C and its role in normal intestinal physiology as well as in regulating intestinal cell proliferation. It is clear that despite its early identification as a target for the ST peptides, its function in the intestine is still somewhat enigmatic. The fact that the GC-C KO mouse is viable and shows no obvious abnormalities indicates that the role for this receptor is more subtle. Perhaps a critical requirement for signaling via GC-C and its ligands is evident under certain stresses that could lead to increased cell proliferation and/or abnormal development during formation of the intestinal villus. In addition, since GC-C continues to be expressed on metastases of tumors of colon origin to extra-intestinal tissues such as the liver, cGMP produced following activation of GC-C by circulating guanylin or uroguanylin may regulate cellular responses in these tissues as well. We anticipate a number of new findings on GC-C in terms of features of its regulation at the structural level, since the receptor serves as a good model to understand intramolecular and multidomain regulation of a protein. In addition, since no signaling pathway operates in isolation, we also expect the emergence of new pathways that either affect GC-C directly, or modulate cGMP production on activation by its ligands.
The work in the authors’ laboratory has been supported by the Departments of Science and Technology, and Biotechnology, Government of India. NB is a Junior Research Fellow of the Council of Scientific and Industrial Research, and NA is supported by a fellowship from the Indian Institute of Science. We thank all the current and past members of the laboratory for their stimulating discussions and their outstanding enthusiasm in pursuing aspects of GC-C regulation, its ligands and signaling mechanisms.