Guanylyl cyclase C (GC-C) is a member of particulate guanylyl cyclases, discovered primarily as the target of a family of heat stable enterotoxins (ST), produced by enterotoxigenic Escherichia coli (ETEC). ST is acknowledged as a prime cause of traveller’s diarrhea and the leading cause of child mortality under the age of 5 years in developing nations. The bacterial expression of ST peptides represents molecular mimicry where the pathogen has exploited a gastrointestinal tract-signaling pathway to disperse and propagate. GC-C is primarily expressed on the apical or the brush border membranes of intestinal epithelial cells. GC-C agonists elaborated in the gastrointestinal tract are a family of guanylin peptides, which are responsible for maintaining fluid-ion homeostasis, essential for normal gut physiology. The signal of liigand binding to the extracellular domain of GC-C is transduced to the catalytic guanylyl cyclase domain, which results in production of intracellular cGMP. The elevated levels of cGMP influence multiple downstream targets, which finally regulate ion-flux through the transporters present on the membrane of an enterocyte. The ST peptide, a GC-C superagonist, produces physiologically abnormal levels of cGMP that manifest as secretory diarrhea. The purview of GC-C misregulation was confined to the notion of its hyperactivation caused by ETEC infection and the ensuing diarrhea. Recently, two seminal studies widened the scope of pathologies associated with GC-C. Studies described point mutations in GUCY2C, which were associated with human disease. One study identified a Norwegian family whose members demonstrated a dominantly inherited syndrome of frequent diarrhea associated with hyperactive GC-C. Following this study, inactivating mutations in GC-C in a small Bedouin population was reported. The current study reports the molecular phenotypes associated with the first germ line mutations in GC-C that result in a severe form of congenital sodium diarrhea. Our collaborators from Austria (Thomas Muller & Andreas Janecke, Department of Pediatrics Innsbruck Medical University) communicated to us their study of patients who had clinical diagnosis of congenital sodium diarrhea, with proportionally high fecal sodium loss, metabolic acidosis and dehydration. Exome sequencing in a cohort of 6 unrelated patients revealed four heterozygous missense mutations in GC-C (R792S, L775P, K507E, N850D). Novel GC-C mutations were de novo spontaneous mutations with the carrier being the only affected family member in contrast to the previous two reports with familial history. Biochemical characterization revealed that the mutants (GC-CR792S, GC-CL775P) were constitutively active with GC-CR792S, GC-CK507E, and GC-CN850D showing further stimulation upon treatment with ST and guanylin family of peptides. Interestingly, there was no change in the binding affinities of the ligands for the mutant receptors compared to wild type. However, a significant decrease (ranging from 10-100 fold) in ligand EC50 for the mutant GC-C receptors was prominent. The in vitro assays suggested that the mutations occupying different domains of GC-C might have resulted in distinct structural consequences reflected in the repertoire of phenotypes that were observed. The results presented in this thesis illustrate the molecular basis of the severe form of congenital diarrhea associated with the GC-C gain-of-function mutations. This study has also elaborated our understanding of the regulation of GC-C activity by its various domains.

All cells are equipped to sense changes in their environment and make adaptive responses according to the stimuli. Signal recognition usually occurs at the cell membrane (with the exception of steroid signalling) where the ligand, which can be a small molecule, a peptide or a protein, binds its cognate receptor. This results in a change in the conformation of the receptor which in turn can regulate the production of second messengers. Second messengers can now modulate specific pathways which control gene expression and modify various aspects of cell behaviour. The signalling cascade is terminated by the removal of second messenger and/or by desensitisation of the receptor to the extracellular signal. Cyclic guanosine monophosphate (cGMP) was first identified in the rat urine and since then has emerged as an important second messenger regulating diverse cell processes. Subsequent to its discovery in mammalian cells, enzymes responsible for its synthesis (guanylyl cyclases), hydrolysis (phosphodiesterases) and its most common effectors (cGMP-dependent protein kinases) were identified. Guanylyl cyclases exist in two forms, cytosolic and membrane bound. Both have a conserved guanylyl cyclase domain, but differ in their choice of ligands, overall structure and tissue localization. It is now known that cytosolic and the membrane-bound forms are involved in eliciting distinct cellular responses. Receptor guanylyl cyclase C (GC-C) was identified as the target for a family of heat-stable enterotoxin toxins (ST) produced by enterotoxigenic E.coli. Stable toxin-mediated diarrhoeas are observed frequently in infants and contribute significantly to the incidence of Travellers’ Diarrhea. Early studies demonstrated that the effects of ST were mediated by an increase in intracellular cGMP levels in intestinal cells, and the receptor for ST was almost exclusively expressed in the apical microvilli of the intestinal brush-border epithelia. Effectors of cGMP in intestinal cells include protein kinase G (PKG), cyclic nucleotide gated ion channel 3 (CNG), and the cystic fibrosis transmembrane conductance regulator (CFTR). ST is an exogenous ligand which serves as a hyperagonist for GC-C, in comparison with the endogenous ligands guanylin and uroguanylin, which maintain fluid-ion homeostasis in the intestinal epithelia. The GC-C/cGMP signal transduction pathway also modulates intestinal cell proliferation along the crypt-villus axis by exerting a cytostatic effect on the epithelial cells, thereby regulating their turnover and neoplastic transformation. The current study describes in molecular detail two signalling pathways, one impinging on and one emerging from GC-C, which regulate colonic cell proliferation. The first part identifies the cross-talk and cross-regulation of GC-C and c-src. The second part delves into the molecular basis of GC-C/cGMP-mediated cytostasis and its effect on colonic tumorigenesis. Cross-talk between signalling pathways is believed to play a key role in regulating cell physiology. Phosphorylation of signalling molecules by protein kinases is frequently used as a means of achieving this cross-regulation. Aberrant hyperactivation of the c-src tyrosine kinase is an early event in the progression of colorectal cancer, and activated c-src specifically phosphorylates a number of proteins in the cell. It was found that c-src can phosphorylate GC-C in T84 colorectal carcinoma cells, as well as in the rat intestinal epithelia. Tyrosine phosphorylation of GC-C resulted in attenuation of ligand-mediated cGMP production; an effect which was reversed by chemical or transcriptional knockdown of c-src. These effects were found to be cell line-independent and relied only on the extent of c-src expression and activation in the cell. Mutational analysis revealed GC-C to be phosphorylated on a conserved tyrosine residue (Y820) in the guanylyl cyclase domain. The sequence of GC-C around Y820 allowed for efficient phosphorylation by c-src, and indeed, kinase assays indicated that the affinity of c-src for the GC-C Y820 peptide was one of the highest reported till date. A phospho-mimetic mutation at this site, which mimics a constitutively phosphorylated receptor, resulted in a sharp reduction of guanylyl cyclase activity of the receptor, reiterating the inhibitory role of Y820 phosphorylation on GC-C activity. Phosphorylation of GC-C at Y820 generated a docking site for the SH2 domain of c-src which could interact and thereby co-localize with GC-C on the cell membrane. Intriguingly, this interaction resulted in activation of c-src, setting-up a feed-forward loop of inhibitory GC-C phosphorylation and c-src activation. Treatment of colorectal carcinoma cells with ligands for GC-C reduces cell proliferation and inhibits tumorigenesis. It was observed that this cytostatic effect can be modulated by the status of c-src activation, and consequently, the fraction of tyrosine phosphorylated GC-C in these cells. Since activation of c-src is a frequent event in intestinal neoplasia, phosphorylation of GC-C by active c-src may be one of the means by which the cytostatic effects of GC-C agonists (guanylin and uroguanylin) in the intestine are bypassed, thereby leading to cancer progression. Colonisation of the gut with enteropathogenic microorganisms induces secretion of IFNγ from the host mucosal immune system, which subsequently activates c-src in intestinal epithelial cells. Ligand-stimulated activity of GC-C was found to be reduced in IFNγ treated cells. This could be one of the host defence mechanisms initiated in response to enterotoxigenic E. coli infection. These results provide the first evidence of cross-talk between a receptor guanylyl cyclase and a tyrosine kinase that results in heterologous desensitisation of the receptor. Populations with a higher incidence of enterotoxigenic E.coli infections appear to be protected from intestinal neoplasia. It was found that mice lacking GC-C, and therefore unable to respond to ST, displayed an increased cell proliferation in colonic crypts and enhanced carcinogen-induced aberrant crypt foci formation, which is a surrogate marker for colorectal carcinogenesis. However, pharmacological elevation of cGMP was able to efficiently induce cytostasis even in GC-C knockout mice, indicating a key role for cGMP in regulating colonic cell proliferation. Through microarray analyses, genes regulated by ST-induced GC-C activation in T84 colorectal carcinoma cells were identified. Genes involved in a number of cellular pathways were differentially expressed, including those involved in signal transduction, protein and solute secretion, transcriptional regulation and extracellular matrix formation. One of the genes found to be significantly up-regulated was the cell-cycle inhibitor, p21. The increase in p21 expression was validated at both the transcript and protein level. This p53-independent up-regulation of p21 was coupled to the activation of the cGMP-responsive kinase, PKGII, since knockdown of PKGII using specific siRNAs abolished ST-induced p21 induction. Activation of PKGII led to phosphorylation and activation of the stress responsive p38 MAPK. Similar to what was seen following knockdown of PKGII, inhibition of p38 MAPK activity attenuated the up-regulation of p21 in response to cGMP, indicating that PKGII and p38 MAPK could be a part of a pathway regulating p21 expression. It was found that active p38 MAPK phosphorylated the ubiquitous transcription factor SP1, enhancing its occupancy at the proximal p21 promoter. Therefore, SP1 could be one of the factors linking cGMP to transcription of the p21 mRNA. Chronic activation of GC-C led to nuclear accumulation of p21 in colonic cells, which entered a quiescent state. These cells arrested in the G1 phase of the cell cycle, consequent to p21-dependent inhibition of the G1 cyclin-CDK complexes. A fraction of these quiescent cells stochastically initiated a cGMP-dependent senescence programme and displayed all the hallmarks of senescent cells, including flattened cell morphology, expression of SA- galactosidase and formation of senescence-associated heterochromatic foci. Activation of senescence and loss of tumorigenicity in these cells was crucially dependent on the up-regulation of p21. This irreversible exit from the cell cycle due to cGMP-mediated activation of the PKGII/p38/p21 axis was well correlated with reduced colonic polyp formation in mice exposed to ST. In summary, these observations may provide a possible explanation for the low incidence of colorectal carcinoma seen in countries with a high incidence of ST-mediated diarrhoea. Interestingly, c-src mediated tyrosine phosphorylation of GC-C prevented p21 accumulation following ligand application. The findings described in this thesis may have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.

The survival of the any living organism depends on its availability to communicate, and a breakdown of cellular signaling can have dire consequences such as uncontrolled cellular proliferations or even cell death. Environmental cues or ligands are perceived by cognate receptors, expressed primarily on the cell surface, and transmitted to the interior of the cell to elicit a response. This universal phenomenon is termed as signal transduction. During this process, second messengers such as cyclic nucleotides, cAMP and cGMP, are produced which serve to amplify the signal. Cyclic GMP is emerging as a universal second messenger, and is found in both prokaryotes and eukaryotes. It is synthesized from GTP by the action of guanylyl cyclases. Vertebrate guanylyl cyclases are of two forms, soluble and membrane-associated. Soluble guanylyl cyclases are heterodimeric enzymes which are activated by nitic oxide. Membrane-associated guanylyl cyclases on the other hand are homodimeric enzymes that act as receptors for divers polypeptide ligands. In mammals, there are seven isoforms of receptors guanylyl cyclase, GC-A through GC-G. These recptors have a highly conseved modular domin organization with an N-terminal extracellular domain, a single transmembrane domain and a C- terminal intra cellular regions. The intracellular region contains a juxtamembrane domain followed by a protein-kinase domain, a linker region and a catalytic guanylyl cyclase domain. The coordinated actions of these domains ensure fine tuned-regulations of the catalytic domain. Guanylyl cyclase-C (GC-C) is a member of the membrane-bound guanylyl cyclases. GC-C is predominantely present in the intestine, on the apical surface of epithelial cells, but has also been detected in the rat epididymis. In the intestine it serves as the guanylin, uroguanylin and lymphoguanylin which are the endogenous peptide ligands, while heat- stable entrotoxins (ST) peptides secreted by enterotoxigenic E.coil, are exogenic ligands. Activation of GC-C by these ligands results in an increase in intracellular cGMP levels, which then activates cGMP-dependent protein kinase and cross-activates protein kinase A. In turn, these activated kinases phosphorylate and active the cystic fibrosis transmembrane conductance regulator (CFTR), resulting in chloride and water secretion into the intestine lumen, thus regulating salt and water homeostasis in the intestine. ST peptide has a greater affinity for GC-C than the endogenous ligands and activation of GC-C by ST results in masiive fluid and ion efflux from the intestine cells from which manifests as Travelers’ Diarrhea. The GC-C mediated cGMP signal transduction pathway also maintains intestinal crypt-villus axis homeostatis by exerting a cytostatic effect on the epithelial cells, there by regulating their turn over. Over the years multiple mechanisms of regulation of GC-C activity has been identified including allosteric controlled by various domains in the receptor and phosphorylation-mediated regulation of guanylyl cyclase activity. The current study describes aspects of the regulation of GC-C by gycosylation, and also reports the molecular phenotypes of a naturally occurring mutation in GC-C causes sever diarrhea in affected individuals. GC-C is expressed as a differentially glycosylated protein (130k Da and 145kDa). While both forms bind with equal affinity, only one the 145 kDa form is activated by its ligands. It is this higher glycosylated form which is selectively downregulated in the process of decensitization of GC-C in colomn carcinoma cells (Caco2). Give the critical role gycosylation plays in protein folding, trafficking, receptor activity and mediating protein inter actions, its influence on GC-C was analysed.

No description available.

Signal transduction is a crucial event that enables cells to sense and respond to cues from their immediate environment. Guanylyl cyclase C (GC-C) is a member of the family of receptor guanylyl cyclases. GC-C is a single transmembrane protein that responds to its ligands by the production of the second messenger cGMP. The guanylin family of peptides, (including the bacterially produced heat-stable enterotoxin ST) is the ligand for GC-C, elevates intracellular cGMP levels and activates downstream pathways. GC-C regulates the cystic fibrosis transmembrane conductance regulator (CFTR) by inducing phosphorylation by protein kinase G, resulting in chloride ion and fluid efflux. GC-C also regulates cell cycle progression through cGMP-gated Ca2+ channels. These functions are seen in the intestinal epithelium, the primary site for GC-C expression. GC-C as a molecule has been studied in detail, but its functioning in the context of other signaling pathways remains unknown. The aim of the present investigation was to understand the regulation of signal transduction by GC-C and its cross-talk with other signaling pathways operating in the cell. Molecular events that commonly connect components in a signaling pathway are protein phosphorylation and protein-protein interaction. These two aspects are explored in this thesis. The possibility of tyrosine phosphorylation of GC-C has been explored earlier in our laboratory. In vitro studies indicated that the residue Tyr820 was a site for phosphorylation by the Src family of non-receptor tyrosine kinases and those studies also suggested that phosphorylated Tyr820 could bind to the SH2 domain of Src. We generated a nonphosphorylatable mutant of GC-C, GC-CY820F, and a phosphomimetic mutant GC-CY820E to study the effect of phosphorylation of Tyr820, on the functioning of GC-C. A stable cell line of HEK293:GC-CY820F cells was generated and compared with HEK293:GC-CWT. Dose response to ST in the two cell lines showed that cGMP accumulation by GC-CY820F was greater than that of GC-CWT, although the EC50 remained unchanged. The phosphomimetic GC-CY820E mutant receptor was non-responsive to ST. Further in HEK293 cells, phosphorylation of GC-CWT by constitutively active v-Src resulted in decreased ST stimulation and this effect of v-Src was reduced with GC-CY820F. Inhibition of ST stimulation brought about by v-Src required catalytically active Src, as the kinase inactive v-SrcK295R did not inhibit ST stimulation. These results were corroborated by in vitro studies by using the recombinant catalytic domain of GC-C expressed in insect cells and by phosphorylation using a purified kinase, Hck. Observations suggested that phosphorylation of Tyr820 in the catalytic domain of GC-C compromises the guanylyl cyclase activity of GC-C. T84 and Caco-2 colon carcinoma cells endogenously express GC-C. The effect of tyrosine phosphorylation of GC-C was studied by using HgCl2, a known activator of Src kinases, and by the inhibition of protein tyrosine phosphatases using pervanadate, an irreversible inhibitor. Both these ways of achieving increased tyrosine phosphorylation resulted in decreased ST-stimulated cGMP production by GC-C, as suggested from v-Src transfection studies. This decrease was reversed by using a Src kinase specific inhibitor PP2, confirming the role of Src kinases in the inhibition of GC-C activity. Interestingly, in Caco-2 cells that differentiate in culture, the effect of pervanadate on the inhibition of ST-stimulated GC-C activation was dependent on the differentiation stage. Crypt-like cells showed higher inhibition with pervanadate. As they matured into villus-like cells, the effect of pervanadate on GC-C activation was gradually lost. This effect also correlated with a decrease in the expression of Lck, suggesting that in the context of the intestine there could be differential regulation of tyrosine phosphorylation of GC-C along the crypt-villus axis. Intestinal ligated loop assays in rats demonstrated that ST-induced fluid accumulation in the intestine was abrogated on pervanadate treatment. Reduction in this fluid accumulation by pervanadate was not observed with 8-Br-cGMP, a cell permeable analogue of cGMP. This indicated that tyrosine phosphorylation of proteins is important for ST-induced fluid accumulation, and perhaps pervanadate modulates this by phosphorylation of GC-C, thereby causing a reduction in fluid accumulation. Earlier in vitro studies on Src-SH2 binding from the laboratory had suggested the possibility of activation of Src family kinases by GC-C. The activation status of Src kinases was monitored by using phosphorylation-state specific antibody, pSFK416. ST stimulation in T84 cells increased Tyr416 phosphorylation of Src kinases in a time dependent manner, indicating that Src kinases are activated downstream of GC-C. This activation of Src kinases was also seen with the endogenous ligand of GC-C, uroguanylin. Interestingly, 8-Br-cGMP a cell permeable analogue of cGMP that is known to mimic other cellular effects of GC-C, namely Cl-secretion and cell cycle progression, did not activate Src kinases, suggesting that the mechanism of Src kinase activation by GC-C could be independent of cGMP. Binding affinities of Src, Lck, Fyn and Yes SH2 domains to Tyr820 phosphorylated GCC peptide were in the nM range, indicating a high affinity of interaction. In vitro GST-SH2 pull down experiments suggested that phosphorylation of Tyr820 in full length GC-C allows interaction of GC-C to the SH2 domain of Src. These studies suggest a dual cross-talk between Src kinases and GC-C; Src phosphorylation inhibits GC-C signaling and stimulation of GC-C by its ligands activates Src kinases. Interaction of proteins containing SH2 and SH3 domains are commonly found in signaling molecules. In accordance with the observation that there are three PXXP motifs in GCC, many SH3 domains could interact with GC-C. GC-C appears to show a preference to bind the SH3 domains of Fyn, Hck, Abl tyrosine kinases, Grb2 and Crk adaptor proteins, the α-subunit of P85 PI3 kinase, PLC-γ and cortactin to various extents. The SH3 domains of spectrin and Nck did not show any detectable interaction with GC-C. In SH3 pull-down assays, the N-terminal SH3 domain of Crk, CrkSH3 (N), bound GC-C maximally, suggesting that Crk is a good candidate for interaction with GC-C. By overlay analysis, the region of GC-C that binds CrkSH3 (N) was narrowed down to the catalytic domain of GC-C containing a ‘PGLP’ motif. Mutations were generated in GC-C at this site to generate GC-CP916Q and GC-CW918R. These mutations compromised the binding of full length receptor to CrkSH3 (N). In cells, CrkII and GC-C co-transfection inhibited the ST stimulation of GC-C. A CrkII mutant, that has compromised binding through its SH3 domain, did not inhibit the activity of GC-C. CrkII from T84 cells co-immunoprecipitated with GC-C and interestingly, the phosphorylated form of CrkII did not, indicating that GC-C - Crk interaction could be regulated by the phosphorylation of Crk. In summary, this study places GC-C, in the context of tyrosine kinase signaling pathway and interaction with the adaptor protein Crk. These studies suggest that GC-C signal transduction can be altered by cross-talk with other signaling events in the cell. Reversible phosphorylation of tyrosine residues inhibits the activity of GC-C, and this is mediated by Src family kinases. Src kinases themselves are activated on stimulation of GC-C by its ligands, possibly because of SH2 domain interaction with GC-C. Association of Crk by its SH3 domain regulates GC-C functioning primarily by inhibiting ST-stimulated cGMP production. This opens up the possibility of GC-C signaling through a multimeric complex involving other binding partners of Crk, and these cross-talks involving GC-C with the two proto-oncogenes, Src and Crk, might have far reaching consequences in the regulation of cellular functions.

<p>The duodenal epithelium secretes bicarbonate ions and this is regarded as the primary defence mechanism against the acid discharged from the stomach. For an efficient protection, the duodenum must also function as a sensory organ identifying luminal factors. Enteroendocrine cells are well-established intestinal “taste” cells that express signaling peptides such as orexins and guanylins. Luminal factors affect the release of these peptides, which may modulate the activity of nearby epithelial and neural cells.</p><p>The present thesis considers the effects of orexins and guanylins on duodenal bicarbonate secretion. The duodenal secretory response to the peptides was examined in anaesthetised rats <i>in situ</i> and the effects of orexin-A on intracellular calcium signaling by human as well as rat duodenal enterocytes were studied <i>in vitro</i>.</p><p>Orexin-A, guanylin and uroguanylin were all stimulants of bicarbonate secretion. The stimulatory effect of orexin-A was inhibited by the OX₁-receptor selective antagonist SB-334867. The muscarinic antagonist atropine on the other hand, did not affect the orexin-A-induced secretion, excluding involvement of muscarinic receptors. Orexin-A induced calcium signaling in isolated duodenocytes suggesting a direct effect at these cells. Interestingly, orexin-induced secretion and calcium signaling as well as mucosal orexin-receptor mRNA and OX₁-receptor protein levels were all substantially downregulated in overnight fasted rats compared with animals with continuous access to food. Further, secretion induced by Orexin-A was shown to be dependent on an extended period of glucose priming.</p><p>The uroguanylin-induced bicarbonate secretion was reduced by atropine suggesting involvement of muscarinic receptors. The melatonin receptor antagonist luzindole attenuated the secretory response to intra-arterially administered guanylins but had no effect on secretion when the guanylins were given luminally. </p><p>In conclusion, the results suggest that orexin-A as well as guanylins may participate in the regulation of duodenal bicarbonate secretion. Further, the duodenal orexin system is dependent on the feeding status of the animals.</p>

The duodenal epithelium secretes bicarbonate ions and this is regarded as the primary defence mechanism against the acid discharged from the stomach. For an efficient protection, the duodenum must also function as a sensory organ identifying luminal factors. Enteroendocrine cells are well-established intestinal “taste” cells that express signaling peptides such as orexins and guanylins. Luminal factors affect the release of these peptides, which may modulate the activity of nearby epithelial and neural cells. The present thesis considers the effects of orexins and guanylins on duodenal bicarbonate secretion. The duodenal secretory response to the peptides was examined in anaesthetised rats in situ and the effects of orexin-A on intracellular calcium signaling by human as well as rat duodenal enterocytes were studied in vitro. Orexin-A, guanylin and uroguanylin were all stimulants of bicarbonate secretion. The stimulatory effect of orexin-A was inhibited by the OX1-receptor selective antagonist SB-334867. The muscarinic antagonist atropine on the other hand, did not affect the orexin-A-induced secretion, excluding involvement of muscarinic receptors. Orexin-A induced calcium signaling in isolated duodenocytes suggesting a direct effect at these cells. Interestingly, orexin-induced secretion and calcium signaling as well as mucosal orexin-receptor mRNA and OX1-receptor protein levels were all substantially downregulated in overnight fasted rats compared with animals with continuous access to food. Further, secretion induced by Orexin-A was shown to be dependent on an extended period of glucose priming. The uroguanylin-induced bicarbonate secretion was reduced by atropine suggesting involvement of muscarinic receptors. The melatonin receptor antagonist luzindole attenuated the secretory response to intra-arterially administered guanylins but had no effect on secretion when the guanylins were given luminally. In conclusion, the results suggest that orexin-A as well as guanylins may participate in the regulation of duodenal bicarbonate secretion. Further, the duodenal orexin system is dependent on the feeding status of the animals.