Guanylyl Cyclase C
Guanylyl cyclase C (GC-C) is the target for a family of heat-stable enterotoxins (ST) produced by enterotoxigenic Escherichia coli (ETEC), Vibrio cholera non-01, and Yersinia enterocolitica. Stable toxin-mediated diarrheas are observed frequently in infants and contribute significantly to incidences of Travelers’ Diarrhea. Early studies demonstrated that the effects of ST were mediated by increases in intracellular cGMP levels in intestinal cells, and the receptor for ST was almost exclusively expressed in the apical microvillar compartment of the intestinal brush-border epithelia (De Jonge 1975; Schulz et al. 1990). Subsequently, the receptor for ST peptides was identified as a member of the membrane-associated guanylyl cyclase family, and binding of ST to GC-C led to activation of the receptor, resulting in the production and accumulation of cGMP in enterocytes (Schulz et al. 1990). Effectors of cGMP in intestinal cells include protein kinase G (PKG), cyclic nucleotide-gated ion channels (CNG), and the cystic fibrosis transmembrane conductance regulator (CFTR), which regulate net fluid and ion efflux into the intestinal lumen manifesting as watery diarrhea in humans and farm animals (Basu et al. 2010).
ST peptides are low molecular weight peptides that differ slightly in their amino acid composition, but have a cysteine-rich core with a conserved disulfide bonding pattern essential for the heat stability and biological activity of these peptides (Gariepy et al. 1987). Nearly a decade after the isolation of bacterial ST peptides, a 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 a related peptide, uroguanylin. These endogenous ligands as well as ST peptides are synthesized as precursor proteins which have little or no intrinsic biological activity until cleaved by converting enzymes that release the active peptides for interaction with GC-C (Forte 2004). Guanylin and uroguanylin are less potent in stimulating chloride secretion in comparison with ST peptides, since the affinity of guanylin and uroguanylin for GC-C is lower than that of ST. Uroguanylin is effective in stimulating cGMP production in cells at a pH of ∼5.5, whereas guanylin has a reduced efficacy at an acidic pH, and is active at an alkaline pH. This is because acidic pH markedly decreases the affinity of guanylin binding to GC-C but enhances that of uroguanylin. The presence of both guanylin and uroguanylin in the gastrointestinal tract allows for effective control of cGMP-mediated regulation of salt and water transport over a broad pH range that is encountered in the extracellular fluid of the intestinal tract (Hamra et al. 1997). Novel peptide agonists, linaclotide (Busby et al. 2010), and plecanatide (Shailubhai et al. 2013) have been developed for GC-C. Linaclotide is FDA approved and alleviates abdominal pain and constipation in constipation-predominant IBS (C-IBS).
Domain Organization of GC-C
The mRNA of GC-C is translated to a polypeptide of 1073 amino acids, which adopts the multidomain architecture similar to that seen in other receptor guanylyl cyclases. The N-terminal signal sequence of 23 amino acid residues undergoes proteolysis on transit to the endoplasmic reticulum, generating a mature polypeptide of 1050 amino acids with a theoretical molecular mass of 120 kDa. From the N to the C terminus of GC-C, an extracellular ligand-binding domain (ECD) is followed by a single transmembrane helix and an intracellular domain consisting of juxtamembrane, kinase-homology (KHD), linker, guanylyl cyclase (GCD), and the C-terminal tail (CTD) domains. The minimal catalytic unit required for the guanylyl cyclase activity of GC-C is a homodimer, although higher order oligomers have been reported and may be the physiologically relevant forms. As with all guanylyl cyclases, the active site is thought to lie at the interface of the two guanylyl cyclase domains of the homodimer. Other than the intestinal tract, GC-C is also expressed at lower levels in various extraintestinal tissues such as the kidney, seminiferous tubules of the testis, perinatal liver, brain, and epididymis (Basu et al. 2010).
The ECD of GC-C shows low sequence similarity to the ECDs of other receptor guanylyl cyclases, reflecting the differences in the ligands that bind each individual receptor. GC-C binds ST peptides with a high affinity (Kd ∼ 0.1 nM) and guanylin (Kd ∼ 10 nM) and uroguanylin (Kd ∼ 1 nM) with lower affinity. The ECD of GC-C shows N-linked glycosylation resulting in 130 kDa and 145 kDa forms of the receptor. The 145 kDa form contains sialic acid and galactose residues and is present on the plasma membrane, whereas the 130 kDa form contains high mannose structures and is primarily resident in the endoplasmic reticulum. The 130 kDa is the precursor for the 145 kDa form, and though both differentially glycosylated forms bind ST with equal affinity, cGMP production in the presence of ligands is seen only with the 145 kDa form (Ghanekar et al. 2004).
GC-C harbors a domain of approximately 250 residues between the juxtamembrane and the guanylyl cyclase domains, which shares a significant homology to protein tyrosine kinases, and is called the KHD. The crucial position of the KHD in receptor guanylyl cyclases suggests that it transduces the signal of ligand binding from the ECD to the guanylyl cyclase domain. This is supported by the fact that complete and partial deletions of the KHD in GC-C led to inactivation of the receptor. A sequence alignment of the KHD of GC-C with tyrosine and serine-threonine kinases reveals that the highly conserved HRD motif present in active protein kinases is replaced with a HGR motif in the KHD of GC-C. The absence of the catalytic aspartate residue predicts that the KHD of GC-C is a pseudokinase (Rudner et al. 1995).
A ∼70 amino acid region connecting the KHD and the guanylyl cyclase domain is referred to as the linker region. 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 adopts a coiled-coil structure. Mutational analysis of the linker region has demonstrated its importance in repressing the guanylyl cyclase activity of GC-C in the absence of ligand and permitting ligand-mediated activation of the cyclase domain (Saha et al. 2009).
The primary structure of the guanylyl cyclase domain is highly conserved in both receptor and soluble GCs and is similar to the catalytic domain of adenylyl cyclases. Guanylyl cyclases belong to the Class III nucleotide cyclase family. Homodimeric receptor GCs could have two active sites, and the allostery seen in biochemical assays suggests that there are indeed two substrate-binding sites per dimer of enzyme (Parkinson et al. 1994). Recent crystal structures of the guanylyl cyclases Cya2 from cyanobacterium Synechocystis PCC6803 and CYG12 from the green algae Chlamydomonasreinhardtii reveal the presence of a head-to-tail homodimer with the two monomers in a wreath-like arrangement.
GC-C and the retinal GCs (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 interaction of receptor GCs with the cytoskeleton. Deletion of the CTD in GC-C led to a loss of ligand-mediated activation of the receptor. The CTD contains a unique region of 11 highly conserved amino acids, which acts as an apical sorting signal (Hodson et al. 2006).
One of the hallmarks of any signaling system is the intricate and diverse modes of regulation that it is subjected to, and GC-C is no exception.
Transcriptional regulation. GC-C mRNA is encoded by the gene GUCY2C present at the locus 12p12 in Homo sapiens and contains 27 exons. The regulatory region lies ∼2 kb upstream of the gene and has binding sites for caudal type homeobox gene-2 (Cdx2), hepatocyte nuclear factor 4 (HNF4), GATA-4, glucocorticoid receptor, and nuclear factor-IL6 (NF-IL6). HNF4, Cdx2, and GATA-4 are expressed in the intestine, and Cdx2 and HNF4 are responsible for the predominant expression of GC-C in the intestine as well as in cell-lines derived from colorectal carcinoma tissue such as Caco2 and T84. Protein kinase C (PKC) has been shown to transcriptionally regulate expression of GC-C. Activation of PKC by phorbol esters such as phorbol 12-myristate 13-acetate (PMA) led to a downregulation of GC-C mRNA levels in T84 cells (Roy et al. 2001).
Desensitization. Refractoriness to prolonged exposure of cells to ligands of GC-C, in terms of cGMP production, has been observed. In Caco2 cells, selective removal of the 145 kDa form from the surface of the receptor is seen on extended treatment with ST (Ghanekar et al. 2003). Increased activity of the cGMP-binding, cGMP-specific phosphodiesterase, PDE5, was found to contribute to cellular refractoriness in T84 cells, reducing the levels of cGMP in the cell (Bakre et al. 2000). Exposure of HEK293 cells stably expressing GC-C to ST for prolonged periods did not result in desensitization, indicating that cell-specific mechanisms contribute to cellular refractoriness.
Inactivation of GC-C occurs in vitro on ST incubation prior to addition of MgGTP as the substrate for the guanylyl cyclase assays. Inactivation of GC-C was alleviated in the presence of ATP, indicating a role for ATP (and perhaps the KHD) in regulation of GC-C activity (Gazzano et al. 1991).
Phosphorylation. PKC phosphorylates GC-C on Ser1029 in the CTD of GC-C and enhances ST-mediated GC-C activation in terms of increased cGMP production (Wada et al. 1996). This is in contrast to the role of PKC in transcriptional downregulation of GC-C. Phosphorylation at tyrosine residues 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). Recent data shows that GC-C is a substrate for inhibitory phosphorylation by c-src, resulting in reduced ligand-mediated cGMP production. The tyrosine at position 820 gets phosphorylated, and this generates a docking site for the c-src SH2 domain leading to interaction and further activation of c-src. Therefore, GC-C could be involved in cross-talk with tyrosine kinases especially in colon carcinoma cells where c-src kinase expression and activity is high (Basu et al. 2009).
Allosteric regulation. ATP is able to potentiate ligand-mediated activation of GC-C and consequent cGMP production in vitro, in the presence of Mg-GTP as substrate. In contrast, ATP inhibits the cyclase activity of GC-C when Mn-GTP is used as substrate. ATP mediates its effects by binding to the KHD of GC-C, and ATP hydrolysis is not required for inhibition of the guanylyl cyclase activity. Lysine 516 in the KHD of 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 regulation of GC-C activity by the adenine nucleotide (Jaleel et al. 2006). The KHD is also the site of action for some of the 2-substituted adenine nucleotides which were found to be potent allosteric inhibitors of GC-C (Parkinson et al. 1997). Tyrphostins, known to be inhibitors of tyrosine kinases, were found to noncompetitively inhibit the activity of GC-C, by binding to the catalytic domain of GC-C, and indeed nucleotide cyclases in general (Jaleel et al. 2004). Recently, piperidine derivatives were found to inhibit GC-C-mediated cGMP production in T84 cell lines and pig jejunal tissue. This inhibition was specific to cGMP pathway, and adenylyl cyclases or cAMP were not affected (Bijvelds et al. 2015).
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 soluble guanylyl cyclases.
Downstream Signaling and Disease Phenotypes
An increase in cGMP levels can regulate ion and fluid transport in the intestine in different ways. 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. Increase in levels of intracellular cGMP results in the activation of cGMP-dependent protein kinase II (PKGII) and protein kinase A, which phosphorylate CFTR thus activating the chloride channel. Sodium absorption in the intestine is partly governed by the Na+/H+ exchanger (NHE), which absorbs NaCl in combination with the chloride/anion exchanger. Activation of PKGII by cGMP results in phosphorylation of NHE3 at three residues and thereby inhibiting its activity, i.e., decreasing sodium absorption. PKGII also reduces the surface expression of NHE3 which also aggravates the decrease in sodium absorption (Chen et al. 2015). The reduced sodium absorption by the NHE and increased chloride secretion by the CFTR in response to cGMP are thought to be the chief mechanisms by which ST mediates its effects (Basu et al. 2010).
Preclinical studies with GC-C agonists suggested analgesic affects for visceral pain in C-IBS patients. The diminished pain perception is via GC-C-dependent cGMP production which acts on colonic nociceptors across the basolateral membrane (Castro et al. 2013).
The intestinal epithelium undergoes homeostatic cycles of proliferation, migration, differentiation, and apoptosis. Imbalance between cell proliferation and apoptosis leads to the formation of tumors within the intestinal tract. In colon cancer, the expression of mRNAs encoding uroguanylin and guanylin is markedly suppressed, whereas mRNA expression of GC-C is comparable in colon cancer and normal colonic mucosa. Oral administration of uroguanylin suppressed the formation and apparent progression of polyps in the APCMin/+ mouse model of colorectal cancer. Recent reports have suggested that ST or uroguanylin treatment of T84 and Caco2 cells inhibits proliferation by delaying progression through the cell cycle. 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+, prevented ST-mediated inhibition of proliferation. This suggested that the antiproliferative 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 (Pitari et al. 2007). GC-C-mediated cGMP production results in p53-independent transcriptional upregulation of p21 via activation of PKGII and p38 MAPK. Prolonged treatment of T84 colon carcinoma cells with ST leads to cell cytostasis and senescence (Basu et al. 2014).
Over a million people suffer from colon cancer in developed countries, but its incidence is relatively low in underdeveloped and developing countries. A common epidemiological characteristic of these developing nations is the prevalence of ETEC. Periodic infections with ST-producing bacteria in the intestine could be preventive for people living in the developing nations, since the action of the ST peptides induces cell cytostasis, thus providing resistance to intestinal neoplasia.
Mutations in GC-C and Its Functional Consequences
The first report of a mutation in the GUCY2C gene was a novel autosomal dominant “gain of function” mutation identified in a Norwegian family, members of which had a family history of chronic diarrhea and symptoms similar to inflammatory bowel disease. The mutation turned out to be a heterozygous missense mutation that converted a well-conserved Serine residue at position 840 to Isoleucine (p.S840I). In vitro analysis of the mutant receptor showed a very high increase in the production of cGMP in presence of its ligand (Fiskerstrand et al. 2012). De novo “gain of function” mutations were reported in GUCY2C gene recently. Two mutations (p.L775P and p.R792S) were mapped to the linker region of the receptor while other two (p.K507E and p.N850D) were in the kinase homology domain and the guanylyl cyclase domain, respectively. Patients harboring these mutations showed prenatal onset of secretory diarrhea and massive abdominal protrusion due to fluid-distended intestinal loops at birth. Other complications included painful obstruction of the ileum and also colitis. In vitro analysis of these mutant receptors revealed higher cGMP production in the absence of ligand, with the p.R792S mutant showing 100-fold higher intracellular cGMP as compared to a wild type receptor (Müller et al. 2015).
Inactivating autosomal recessive mutations of GC-C were reported in two unrelated Bedouin families. One such mutation was mapped to the extracellular ligand-binding domain of GC-C (p.D387G) which is a mutation in the conserved aspartic acid residue, resulting in a significant reduction of enzymatic activity accompanied by intestinal obstruction and meconium ileus. Another mutation found in a neonate was a duplication of a single nucleotide (c.2270dupA) resulting in a premature stop codon and a truncated receptor lacking the cyclase domain. The neonate having this mutation showed severe meconium ileus and required surgery (Romi et al. 2012). Two more autosomal recessive inactivating mutations were reported in a Lebanese family. Homozygous p.C928R and a compound heterozygous p.A670T/p.C928R were found. The p.C928R mutation, which resulted in the loss of a disulfide bond in the guanylyl cyclase domain, was associated with severe meconium ileus, requiring surgical intervention. The compound heterozygous state had a less severe phenotype, which indicates decreased penetrance and variable expressivity (Smith et al. 2015).
The severity of phenotypes related with gain or loss of function mutations in GC-C explains the importance of the receptor for normal gut functioning and its conservation across different species.
The GC-C/cGMP signaling axis is important in maintaining fluid-ion homeostasis in the intestinal tract. In addition, evidence for its antineoplastic role in the development of colon carcinoma is rapidly accumulating. It would be interesting in the future to look at the molecular players responsible for the cytostatic effect exerted by the ligands for GC-C. The function of GC-C expressed in extra-intestinal tissue is largely unknown, but some of the key components may be common to that operative in the intestine. The severe gastrointestinal effects of mutations in GC-C, including inflammation and symptoms that mimic Crohn’s disease and inflammatory bowel disease, warrants further investigations into the role of GC-C in modulating gut immunity.
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