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Enterotoxigenic Escherichia coli (ETEC) causes ∼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease., Methods: Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets., Results: Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens., Conclusions: We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Heat-stable toxins (STs) produced by enterotoxigenic bacteria cause endemic and traveler's diarrhea by binding to and activating the intestinal receptor guanylyl cyclase C (GC-C). Advances in understanding the biology of GC-C have extended ST from a diarrheagenic peptide to a novel therapeutic agent. Here, we summarize the physiological and pathophysiological role of GC-C in fluid-electrolyte regulation and intestinal crypt-villus homeostasis, as well as describe translational opportunities offered by STs, reflecting the unique characteristics of GC-C, in treating irritable bowel syndrome and chronic constipation, and in preventing and treating colorectal cancer.

A series of pyridopyrimidine derivatives were synthesized and evaluated for their ability to inhibit cyclic nucleotide synthesis in the presence of stable toxin a of Escherichia coli. The structure activity relationships around the basic core structure were examined and examples with better activity and potentially better pharmacological properties are presented.

The heat-stable enterotoxin of Escherichia coli (STa) is a potent stimulant of intestinal chloride and bicarbonate secretion. Guanylyl cyclase C (GC-C) has been shown to be the primary receptor involved in mediating this response. However, numerous studies have suggested the existence of an alternative STa-binding receptor. The aims of this study were to determine whether a non-GC-C receptor exists for STa and what is the functional relevance of this for intestinal bicarbonate secretion in mice. (125)I-STa-binding experiments were performed with intestinal mucosae from GC-C knockout (KO) and wild type (WT) mice. Subsequently, the functional relevance of an alternative STa-binding receptor was explored by examining STa-, uroguanylin-, and guanylin-stimulated duodenal bicarbonate secretion (DBS) in GC-C KO mice in vitro and in vivo. Significant (125)I-STa-binding occurred in the proximal small intestines of GC-C KO and WT mice. Analysis of binding coefficients and pH dependence showed that (125)I-STa-binding in GC-C KO mice involved a receptor distinct from that of WT mice. Functionally, STa, uroguanylin, and guanylin all stimulated a significant increase in DBS in GC-C KO mice. Uroguanylin- and guanylin-stimulated DBS were significantly inhibited by glibenclamide, but not by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). However, STa-stimulated DBS was unaffected by glibenclamide but inhibited by DIDS. Taken together, our results suggest that alternative, non-GC-C, receptors likely exist for STa, uroguanylin, and guanylin in the intestines of mice. While uroguanylin- and guanylin-stimulated DBS are cystic fibrosis transmembrane conductance regulator (CFTR) dependent, STa-stimulated DBS is CFTR independent. Further understanding of this alternative receptor and its signaling pathway may provide important insights into rectification of intestinal bicarbonate secretion in cystic fibrosis.

Guanylyl cyclase C (GC-C), universally overexpressed on primary and metastatic colorectal carcinoma cells, is activated by endogenous ligands, guanylin, and uroguanylin, and by exogenous 18-residue heat-stable enterotoxins (STa) produced by diarrheagenic bacteria. Two 12-residue STa analogs with alternate combinations of two interlocked disulfide bonds, peptides 3 and 6, were synthesized by orthogonal solid phase synthesis routes. Peptides 3 and 6 bound GC-C with a rank order potency of STa > peptide 3 > peptide 6. Peptides 3 and 6 behaved as agonists in stimulating cGMP production. The results reveal that the toxic domain of STa can be reduced to 12 amino acids.

The elaboration of heat stable enterotoxin (STa) is an important step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC), which causes severe diarrhea in newborn animals. In this study, the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from the anterior jejunum, posterior jejunum, ileum and colon of newborn kids was investigated. The density of STa-receptors on enterocytes and BBMVs was higher in the posterior jejunum than that in other segments of the kids' intestines. Additionally, the affinity of the posterior jejunum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. Our findings suggest that the posterior jejunum is a major target for STa within the intestinal tract of newborn kids.

Bacterial diarrheagenic heat-stable enterotoxins induce colon cancer cell cytostasis by targeting guanylyl cyclase C (GCC) signaling. Anticancer actions of these toxins are mediated by cyclic guanosine 3',5'-monophosphate (cGMP)-dependent influx of Ca2+ through cyclic nucleotide-gated channels. However, prolonged stimulation of GCC produces resistance in tumor cells to heat-stable enterotoxin-induced cytostasis. Resistance reflects rapid (tachyphylaxis) and slow (bradyphylaxis) mechanisms of desensitization induced by cGMP. Tachyphylaxis is mediated by cGMP-dependent protein kinase, which limits the conductance of cyclic nucleotide-gated channels, reducing the influx of Ca2+ propagating the antiproliferative signal from the membrane to the nucleus. In contrast, bradyphylaxis is mediated by cGMP-dependent allosteric activation of phosphodiesterase 5, which shapes the amplitude and duration of heat-stable enterotoxin-dependent cyclic nucleotide accumulation required for cytostasis. Importantly, interruption of tachyphylaxis and bradyphylaxis restores cancer cell cytostasis induced by heat-stable enterotoxins. Thus, regimens that incorporate cytostatic bacterial enterotoxins and inhibitors of cGMP-mediated desensitization offer a previously unrecognized therapeutic paradigm for treatment and prevention of colorectal cancer.

mRNA-based technologies and preclinical research in a variety of animal models have shown that guanylyl cyclase C (GCC) is a highly sensitive and specific molecular marker for the diagnosis of colorectal cancer (CRC). GCC is also a receptor for Escherichia coli (E. coli) heat-stable enterotoxin (STa) and can be used for STa-directed delivery of small-sized imaging agents to human CRC tumours. In this study, we have evaluated GCC as a new immunohistochemical (IHC) marker for CRC tissues and STa as a suitable vector for delivering high-sized protein molecules to CRC cells. Firstly, we have developed a highly sensitive EnVision(+)-based IHC staining method for detecting GCC in serial paraffin-embedded sections of primary and metastatic CRC (38 cases) or non-CRC (14 cases) adenocarcinomas. Carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) were chosen as controls. Our results indicate that GCC staining was positive in 100% of CRC tumours and was comparable to CEA (95%) or CK20 (92%). In contrast to CEA and CK20, GCC was negative in all of the extra-intestinal non-CRC tumours examined. GCC appears to display higher specificity than either CEA or CK20 while retaining high sensitivity, suggesting that it is a better CRC marker than CEA or CK20. Secondly, STa was genetically coupled to green fluorescent protein (GFP) and the resulting GFP-tagged STa was characterized for expression in E. coli and enterotoxicity in mouse. The binding characteristics of GFP-STa in CRC Caco-2 cells were followed by immunofluorescence microscopy. In this work we show that GFP-tagged STa is biologically active and has retained its ability to internalise into Caco-2 cells making it a potential vehicle for the delivery of anticancer therapeutic protein agents.

Guanylate cyclase C (GC-C), a member of the membrane-bound GC family, consists of an extracellular domain (ECD) and an intracellular domain, which are connected by a single-transmembrane region. GC-C is a receptor protein, i.e. specifically stimulated by the endogenous peptides guanylin, uroguanylin, lymphoguanylin, and the exogenous peptide heat-stable enterotoxin (ST(a)), secreted by pathogenic Escherichia coli and acting on the intestinal brush border membranes. The binding of these peptide ligands to the ECD of GC-C results in the synthesis of cyclic GMP in cells, which, in turn, regulates a variety of intracellular physiologic processes. As the cloning of GC-C, its physiologic functions of each domain have been vigorously investigated. The structural characterization of the ligand-binding domain of the receptor promises to provide important clues for better understanding of the mechanisms of receptor recognition and activation. Recently, structural data for each domain of membrane-bound GCs and related proteins has become available. Coupling information obtained from such work and validation of structure-function relationships of GC-C and its ligands should allow for three-dimensional mapping of their interaction site in detail. Our approach to this issue involved designing photoaffinity-labeling ST(a) analogs, capable of binding covalently to the ligand-binding region of the ECD of GC-C. The photoaffinity-labeling ligand was used to covalently label a soluble form of the recombinant ECD protein. Mass spectrometric analyses of an endoproteinase digest of the ECD revealed that the ligand specifically bound to a narrow region contained in the membrane-proximal subdomain of the ECD of GC-C. These results will enable us to identify the possible binding motifs within the ligand-binding domain by computer modeling. In this review, we summarize the available data on the recognition mechanism between ST(a) and GC-C at the molecular level.

We examined the ability of blood group A-active glycoconjugates to act as receptors for Escherichia coli heat-labile type I enterotoxin (LT-I) in HT-29 cells. These cells contained ~4 times more specific binding sites for LT-I than for cholera toxin (CT). Binding of LT-I could not be blocked by the B subunit of CT (CT-B), indicating the existence of LT-I receptors in addition to the glycosphingolipid GM1. LT-I was able to increase levels of cyclic adenosine monophosphate (AMP), even in the presence of CT-B. Helix pomatia and anti-blood group A antibody caused a dose-dependent inhibition of binding of LT-I to cells and production of cyclic AMP. LT-I recognized several complex blood group A-active glycosphingolipids from cells, and this interaction was also interfered with by H. pomatia. Treatment of cells with D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol diminished surface expression of blood group A-active glycosphingolipids and binding of LT-I to non-GM1 receptors. These observations suggest that blood group A-active glycosphingolipids can function as alternative receptors for LT-I in HT-29 cells.

The heat-stable enterotoxin peptides (ST) produced by enterotoxigenic Escherichia coli are one of the major causes of transitory diarrhea in the developing world. Toxin binding to its receptor, guanylyl cyclase C (GC-C), results in receptor activation and the production of high intracellular levels of cGMP. GC-C is expressed in two differentially glycosylated forms in intestinal epithelial cells. Prolonged exposure of human colonic cell lines to ST peptides induces cellular refractoriness to the ST peptide, in terms of intracellular cGMP accumulation. We have investigated the mechanism of cellular desensitization in human colonic Caco2 cells, and observe that exposure of cells to ST leads to a time and dose-dependent inability of cells to respond to the peptide in terms of GC-C stimulation, both in whole cells and membranes prepared from desensitized cells. This is concomitant with a 50% reduction in ST-binding activity in desensitized cells. Desensitization was correlated with a loss of the plasma membrane-associated, hyperglycosylated 145 kDa form of GC-C, while the predominant 130 kDa form, localized both on the plasma membrane and the endoplasmic reticulum, continued to be present in ST-treated cells. Desensitized cells recovered ST-responsiveness on removal of the ST peptide, which was correlated with a reappearance of the 145 kDa form on the cell surface, following processing of the endoplasmic reticulum-associated pool of the 130 kDa form. Selective internalization of the 145 kDa form of the receptor was required for cellular desensitization, as ST-treatment of cells at 4 degrees C did not lead to refractoriness. We therefore show a novel means of regulation of cellular responsiveness to the ST peptide, whereby altering cellular levels of the differentially glycosylated forms of GC-C can lead to differential ligand-mediated activation of the receptor.

Receptor-binding by the B-subunit of Escherichia coli heat-labile enterotoxin (EtxB) induces apoptosis of peripheral CD8(+), but not CD4(+) T-cells. Given that peripheral CD8(+) and CD4(+) T cells arise from a common developmental pathway in the thymus, we investigated the effects of EtxB on different thymocyte populations. We show that the acquisition of sensitivity to EtxB-mediated cell death arises following transition of CD4(+)CD8(+) double positive cells into the CD4(-)CD8(+) pathway. Maturation of T cells into CD4(-)CD8(+) single positive cells is associated with upregulated expression of receptors for EtxB.

One half million patients suffer from colorectal cancer in industrialized nations, yet this disease exhibits a low incidence in under-developed countries. This geographic imbalance suggests an environmental contribution to the resistance of endemic populations to intestinal neoplasia. A common epidemiological characteristic of these colon cancer-spared regions is the prevalence of enterotoxigenic bacteria associated with diarrheal disease. Here, a bacterial heat-stable enterotoxin was demonstrated to suppress colon cancer cell proliferation by a guanylyl cyclase C-mediated signaling cascade. The heat-stable enterotoxin suppressed proliferation by increasing intracellular cGMP, an effect mimicked by the cell-permeant analog 8-br-cGMP. The antiproliferative effects of the enterotoxin and 8-br-cGMP were reversed by L-cis-diltiazem, a cyclic nucleotide-gated channel inhibitor, as well as by removal of extracellular Ca(2+), or chelation of intracellular Ca(2+). In fact, both the enterotoxin and 8-br-cGMP induced an L-cis-diltiazem-sensitive conductance, promoting Ca(2+) influx and inhibition of DNA synthesis in colon cancer cells. Induction of this previously unrecognized antiproliferative signaling pathway by bacterial enterotoxin could contribute to the resistance of endemic populations to intestinal neoplasia, and offers a paradigm for targeted prevention and therapy of primary and metastatic colorectal cancer.

Some E. coli cause diarrhea by elaborating heat-labile and heat-stable (ST) enterotoxins which stimulate intestinal secretion. E. coli ST's are small peptides which bind to intestinal luminal epithelial cell receptors. The ST receptor, one of a family of receptor-cyclases called guanylyl cyclase C (GC-C), is a membrane spanning protein containing an extracellular binding domain and intracellular protein kinase and catalytic domains. The intestine synthesizes and secretes homologous peptides, guanylin and uroguanylin. The kidney also synthesizes uroguanylin. ST, guanylin or uroguanylin binding to GC-C results in increased cGMP, phosphorylation of the CFTR Cl- channel and secretion. Proguanylin and prouroguanylin circulate in blood and bind to receptors in intestine, kidney, liver, brain etc. In the kidney, they stimulate the excretion of Na+ and K+. Study of GC-C "knock-out" mice reveal that GC-C is important to intestinal salt and water secretion, duodenal bicarbonate secretion, recovery from CCl4-induced liver injury, and to intestinal polyp formation in Min mice lacking GC-C.

Membrane guanylate cyclase C (GC-C) is the receptor for guanylin, uroguanylin, and heat-stable enterotoxin (STa) in the intestine. GC-C-deficient mice show resistance to STa in intestine but saluretic and diuretic effects of uroguanylin and STa are not disturbed. Here we describe the cellular effects of these peptides using immortalized human kidney epithelial (IHKE-1) cells with properties of the proximal tubule, analyzed with the slow-whole-cell patch clamp technique. Uroguanylin (10 or 100 nm) either hyperpolarized or depolarized membrane voltages (V(m)). Guanylin and STa (both 10 or 100 nm), as well as 8-Br-cGMP (100 microm), depolarized V(m). All peptide effects were absent in the presence of 1 mm Ba(2+). Uroguanylin and guanylin changed V(m) pH dependently. Pertussis toxin (1 microg/ml, 24 h) inhibited hyperpolarizations caused by uroguanylin. Depolarizations caused by guanylin and uroguanylin were blocked by the tyrosine kinase inhibitor, genistein (10 microm). All three peptides increased cellular cGMP. mRNA for GC-C was detected in IHKE-1 cells and in isolated human proximal tubules. In IHKE-1 cells GC-C was also detected by immunostaining. These findings suggest that GC-C is probably the receptor for guanylin and STa. For uroguanylin two distinct signaling pathways exist in IHKE-1 cells, one involves GC-C and cGMP as second messenger, the other is cGMP-independent and connected to a pertussis toxin-sensitive G protein.

Binding of the 48 amino acid polypeptide of the mature heat-stable Escherichia coli enterotoxin b (STb) to the functional receptor sulfatide (SFT) constitutes the first step in inducing secretory diarrhoea in the intestinal lumen of animals. The NMR structure of this toxin dictated the choice of amino acids for site-directed mutagenesis to delineate the binding site of STb to SFT. Amino acids facing the solvent either in the loop or the hydrophobic alpha-helix were selected. Seventeen site-specific mutants of STb toxin were produced and purified by high-pressure liquid chromatography. Enterotoxicity of the 17 mutants was determined using a rat loop assay and binding was evaluated using a microtitre plate binding assay. Both hydrophobic and electrostatic interactions are important for STb attachment. When mutations (F37K, I41S and M42S) were introduced into the hydrophobic alpha-helix to lessen hydrophobicity, binding activity and enterotoxicity decreased by more than sixfold. The loop defined by C21 and C36 also made specific contributions. Mutants generated at basic residues (K22, K23 and R29) within this region exhibited both reduced binding activities and reduced toxic activities. For all STb mutants constructed and analysed, when binding to SFT was reduced, a reduction in toxicity equivalent or greater was noted, indicating that binding to SFT is a step that precedes the toxic effect observed for STb toxin. Significantly, when the negatively charged D30 was substituted for either alanine or valine, the binding to SFT was about twice that of native STb, whereas the enterotoxicity was reduced by half.

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [(3)H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [(3)H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.

Heat-stable enterotoxin (ST(a)) elaborated by E. coli is a major cause of diarrhea. The transmembrane protein guanylyl cyclase C (GC-C) is the acknowledged receptor for ST(a) and for the mammalian peptides guanylin and uroguanylin. Binding to GC-C results in generation of cGMP, activation of type II cGMP-dependent protein kinase, phosphorylation of CFTR and increased chloride and bicarbonate secretion. We had previously shown that ST(a) receptors (GC-C) are found on the brush border membranes of small intestinal enterocytes and of colonocytes. However, since it has subsequently been shown that the endogenous ligands for these receptors, guanylin and uroguanylin, circulate in blood, we proposed the existence of ST(a) binding sites on the basolateral membranes (BLM) of colonocytes. Specific binding of 125I-ST(a) to rat colonocyte BLM was seen. The kinetics of binding to the BLM were similar to binding to BBM. The nature of the BLM receptor is unknown. This suggests that circulating guanylin and uroguanylin, analogues of ST(a), may also function via the basolateral surface., (Copyright 2001 Academic Press.)

Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique. The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules. Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin. These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed. Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay. The results demonstrated that LT-A-CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A. Specifically, LT-A-CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-gamma) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A-LT-B and native CT both produced lower levels of antigen-specific IFN-gamma. Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.

It has previously been demonstrated that sulfatide (3'-sulfogalactosyl-ceramide), present at the surface of epithelial cells of the small intestine of pigs, interacts with the thermostable enterotoxin b (STb) produced by ETEC, and that this molecule is implicated in the mechanism of action of the toxin. However, few things are known about the affinity and physical characteristics of the interaction between these two macromolecules. In this study, using a microtiter plate binding assay (MPBA), we showed that STb toxin has a strong specificity for sulfatide and that this binding is dose-dependent and saturable. A very weak binding occurred with galactosyl-ceramide whereas attachment to 3'-sulfolactosyl-ceramide corresponded to 76% of the binding to sulfatide. STb toxin was shown to possess a lectin-like property; a significative binding was observed when a terminal beta-galactose was present in the glycosphingolipids tested and an increased binding was observed in presence of a sulfate group in position 3 on the galactose. These findings suggest that a sulfated galactosyl residue seems to represent the epitope recognized by the toxin. The reaction between sulfatide and STb toxin is also time and temperature dependent and is not affected by pH. The interaction was not inhibited by free sugars, sulfated polymers, glycolipids or free ions, but was partly inhibited by high concentrations of charged sugars. STb-sulfatide binding process is a low affinity interaction, as demonstrated by the determined Kd of 2-6 +/- 1.5 microM.

Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains.

Guanylyl cyclase C, one of the family of membrane-bound guanylyl cyclases, consists of an extracellular domain and an intracellular domain, which are connected by a single transmembrane polypeptide. The extracellular domain binds unique small polypeptides with high specificity, which include the endogenous peptide hormones, guanylin and uroguanylin, as well as an exogenous enterotoxigenic peptide, heat-stable enterotoxin, secreted by pathogenic Escherichia coli. Information on this specific binding is propagated into the intracellular domain, followed by the synthesis of cGMP, a second messenger that regulates a variety of intracellular physiological processes. This study reports the design of a photoaffinity labeled analog of heat-stable enterotoxin (biotinyl-(AC(5))(2)-[Gly(4), Pap(11)]STp(4-17)), which incorporates a Pap residue (p-azidophenylalanine) at position 11 and a biotin moiety at the N terminus, and the use of this analog to determine the ligand-binding region of the extracellular domain of guanylyl cyclase C. The endoproteinase Lys-C digestion of the extracellular domain, which was covalently labeled by this ligand, and mass spectrometric analyses of the digest revealed that the ligand specifically binds to the region (residue 387 to residue 393) of guanylyl cyclase C. This region is localized close to the transmembrane portion of guanylyl cyclase C on the external cellular surface. This result was further confirmed by characterization of site-directed mutants of guanylyl cyclase C in which each amino acid residue was substituted by an Ala residue instead of residues normally located in the region. This experiment provides the first direct demonstration of the ligand-binding site of guanylyl cyclase C and will contribute toward an understanding of the receptor recognition of a ligand and the modeling of the interaction of the receptor and its ligand at the molecular level.

Background and Purpose: Enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) is a major cause of diarrhea in young animals. Age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa was investigated., Methods: Four age groups (2-day-, 1- and 2-week-, and 2-month-old) of Swiss Webster mice were studied (8 to 10 mice/group). Flow cytometry and radiolabeled STa (125I-STa) assays were used as reliable quantitative measures for characterization of STa-enterocyte receptor interaction., Results and Conclusions: Interaction of STa with its putative receptor was stronger for enterocytes of 2-day-old mice. Scatchard analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at higher numbers on enterocytes from 2-day-old (7.2 nmol/mg) than older (0.30, 0.36, and 0.40 nmol/mg for 1-week-, 2-week-, and 2-month-old mice, respectively). Additionally, receptors from 2-day-old mice had greater affinity for STa (Kd = 75 nM) than did receptors from older mice (Kd = 125, 1,430, and 1,111 nM for 1-week-, 2-week-, and 2-month-old mice, respectively). Density of STa receptors on enterocytes and their affinity to STa may determine extent of binding and severity of the secretory response, and may explain the high susceptibility of newborn animals and human infants to STa-mediated diarrhea.

The study examines the age-related differences in the density of Escherichia coli heat-stable enterotoxin (STa) receptors in the small intestine of rabbits. The number of STa receptors was found to be 1.7 x 10(12) in 14-day old rabbits compared to 2.4 x 10(9) in 14-week old rabbits per milligram brush-border membrane protein. The STa-induced guanylyl cyclase activity in the intestinal brush-border membranes was found to be stimulated by 6.2 folds over the basal enzyme activity in 14-day old rabbits, whereas in the 14-week old rabbits, it was 4 folds over the basal activity. Moreover, the enzyme activity remained lower in the adult rabbits compared to the younger ones. Autoradiographic analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis showed two STa-binding proteins of apparent molecular weights of 140 and 38 kDa in the intestinal brush-border membranes of rabbits.

Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied. Four groups (8-10 in each group) of two day old Swiss Webster suckling mice were used. Five, 10, 25, and 50 micrograms of insulin were given orally to half the mice in each group respectively. The rest of the mice in each group were given normal saline as intra-litter controls. After 7 days, the suckling mouse assay for STa was performed on three mice from each insulin-treated and control groups. Enterocyte suspensions were prepared from mice in all groups. Intestinal tissue samples were taken for electron microscopy. Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry. The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin treated groups compared to control mice (p < 0.05). Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an upregulatory effect on the STa receptor level. Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa. Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa. This study may provide further insights into the mechanism of STa/receptor interaction in diarrhea in newborn animals and human infants.

Binding of Escherichia coli heat-stable enterotoxin (STa) to its putative receptor on the brush border membrane of enterocytes is a prerequisite for the induction of diarrhea in infected humans and animals. Humans and animals of different ages vary in their susceptibility to the effect STa, perhaps due to the difference in STa interaction with its intestinal receptor. Flow cytometry was compared to indirect immunofluorescence and 125I-STa binding assays to measure the STa-enterocytes receptor interaction in different age groups of Swiss Webster mice (2-, 7-, 14-day-old). Flow cytometry indicated stronger interaction between STa and its putative receptor on enterocytes from the 2-day-old mice than enterocytes from older mice. 125I-STa-binding assay suggested that the stronger fluorescence intensity on enterocytes from younger mice is due to higher STa receptor density and higher receptor affinity to STa. Flow cytometry is more sensitive quantitative assay to measure the interaction between STa and its intestinal receptor than indirect immunofluorescence microscopy.

Using a quantitative dot blot overlay assay of polyvinylidene difluoride membranes, we investigated the ability of Escherichia coli heat-stable enterotoxin b (STb) to bind to various glycolipids of defined structure. STb bound strongly to acidic glycosphingolipids, including sulfatide (or 3'-sulfogalactosylceramide) and several gangliosides, but not significantly to their derivatives, galactosylceramide and asialogangliosides, respectively. STb exhibited the highest binding affinity for sulfatide. STb bound to pure sulfatide in a dose-dependent and saturable manner, with a detection level of a few nanograms. The binding was not inhibited by tetramethylurea, which is a strong disrupter of hydrophobic interactions, or by the anionic sulfated polymer of glucose, dextran sulfate, indicating that the binding is not due solely to either hydrophobic or ionic interactions via the sulfate group of the sulfatide. The specificity of the binding was confirmed by the finding that a 500-fold molar excess of sulfatide inhibited STb binding by approximately 45%, whereas no competition was obtained with galactosylceramide under the same conditions. Taken together, our data indicated that a galactose residue linked to a sulfate group is required for the binding specificity of STb. Then, total lipids extracted either from the mucous layer or from the epithelial cells of the pig jejunum brush border, the natural target of STb, were analyzed by thin-layer chromatography (TLC). Both extracts contained a lipidic molecule with a relative mobility on a TLC plate similar to that of the sulfatide standard. The migrated lipid extracted directly from a preparative TLC plate was confirmed to be sulfatide, as it was recognized by laminin, a sulfated glycolipid binding protein, and by a monoclonal antibody directed against sulfatide. In an overlay assay on PVDF membranes, STb bound to the sulfatide prepared from porcine jejunum as well as to the sulfatide standard. Thus, these findings suggest that the terminal oligosaccharide sequence Gal(3SO4)beta1- on sulfatide could mediate binding of STb to its target cells and, in support of a recent report (E. Rousset, J. Harel, and J. D. Dubreuil, Microb. Pathog. 24:277-288, 1998), probably terminal sialic acid residue on another glycosphingolipid. Moreover, pretreatment in the ligated intestinal loop assay with laminin or sulfatase altered the biological activity of STb. In summary, we present data indicating that sulfatide represents a functional receptor for the STb toxin.

Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied. Four groups (8-10 in each group) of 2-day-old Swiss Webster suckling mice were used. For this study, 5, 10, 25 and 50 micrograms of insulin was given orally to half the mice in each group for 7 days. The rest of the mice in each group were given normal saline as intra-litter controls. After 7 days, a suckling mouse assay in which 1 microgram of STa was given to all mice in insulin-treated and control groups was performed. Enterocyte suspensions were prepared from mice in all groups. Intestinal tissue samples were taken for electron microscopy. Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry. The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin-treated groups compared to control mice (P < 0.05). Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an up-regulatory effect on the STa-receptor level. Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa. Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa. This study may provide further insights into the mechanism of STa/receptor interaction, which is a major cause of diarrhea in newborn animals and human infants.

Escherichia coli heat-stable enterotoxin (STa) was found to bind on the surface of human colonic (COLO 205) cells. The binding of [125I]STa to cell membranes was found to be specific, reversible and saturable. Scatchard analysis of the equilibrium binding demonstrated a single class of binding sites with a Kd of 0.5 x 10(-10) M. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed the specific incorporation of [125I]STa into a single STa binding protein with a molecular mass of 95 kDa. Following incubation of COLO 205 cells with STa, a rise of intracellular cGMP was also evident.

Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), exhibits multiple binding affinities, including high (RH) and low (RL) affinity sites and a ligand-induced conversion of low-affinity sites from a higher (RL1) to a lower (RL2) affinity state. Occupancy of the lowest affinity state of low-affinity sites is coupled to ligand-induced catalytic activation. In the present studies, ligand binding and catalytic activation properties of a series of intracellular deletion mutants of GCC were examined to identify the structural domains underlying expression of high- and low-affinity sites and the ligand-induced shift in low-affinity sites. These studies demonstrated that the cytoplasmic domains of GCC are not required, but extracellular and transmembrane domains are sufficient, for expression of high-affinity binding sites. In addition, the cytoplasmic juxtamembrane and kinase homology domains are required for expression of the ligand-induced affinity shift in low-affinity sites. Of significance, this shift in affinity was insensitive to adenine nucleotides, in contrast to other members of the receptor guanylyl cyclase family, such as guanylyl cyclase A (GCA). Also, the juxtamembrane and kinase homology domains are critical for coupling ST-receptor binding and guanylyl cyclase catalytic activation. Indeed, deletion of those domains from GCC results in a constitutively inhibited enzyme, suggesting that they function as positive effectors of ligand activation, in contrast to GCA and GCB, in which the kinase homology domain represses basal catalytic activity. These data suggest that the mechanisms regulating different members of the receptor guanylyl cyclase family are overlapping but not identical.

Heat-stable enterotoxin (STa) is an important causative agent of diarrheal disease throughout the world. STa is known to bind specifically to receptors in the intestine, provoking intense intestinal secretion. Binding of STa, or of the mammalian endogenous ligands guanylin and uroguanylin, activates the guanylyl cyclase C receptor (GC-C); the resulting elevation of cGMP levels stimulates chloride secretion via CFTR. We have generated knockout mice which completely lack the GC-C receptor. These mice are viable and show no obvious alteration in intestinal fluidity. However, GC-C null mice are refractory to the secretory action of STa, proving that the GC-C receptor is necessary for the diarrheal response induced by STa., (Copyright 1997 Academic Press.)

The B subunits of cholera toxin (CtxB) and Escherichia coli heat-labile enterotoxin (EtxB) are non-toxic lectins that bind and cross-link a ubiquitous cell glycolipid receptor, ganglioside GM1, and are recognized as potent mucosal and systemic immunogens. Here we examine the role of EtxB receptor occupancy in modulating the activation of B cells, in vitro, in primary lymphocyte cultures containing B and T cells. When 48-hr spleen cell cultures containing EtxB were compared with those in the presence of a non-receptor binding mutant, EtxB(G33D), a marked shift in the ratio of CD4+ T cells: B cells was noted. Evidence suggested that this was the result of either enhanced survival or proliferation of B cells associated with receptor occupancy by EtxB. Investigation revealed that EtxB induced only a minimal increase in proliferation above that of EtxB(G33D), in mixed cell cultures, and failed to induce any cell division of purified B cells or T cells. In contrast, receptor-binding by EtxB markedly up-regulated the expression of major histocompatability complex (MHC) class II, B7, intracellular adhesion molecule-1 (ICAM-1), CD40 and CD25 on the B-cell surface. These results indicate that the polyclonal effects of EtxB on B cells are not associated with wide-scale proliferation, but more likely with maintenance of B-cell survival by activation of molecules essential for B-cell differentiation. The findings also highlight the essential role of GM1-interaction with EtxB in the regulation of lymphocyte responses.

Guanylin and uroguanylin are peptides that activate receptor guanylate cyclases (GCs) and elicit increased intestinal secretion. Bacteria that cause traveler's diarrhea produce heat-stable toxins (STs) that mimic this action. Investigation of the distribution and identity of receptor GCs in the gastrointestinal tract of rats revealed that receptors were localized to epithelial cells in stomach and intestine. Clusters of cells in gastric mucosa and enterocytes lining the intestine exhibited specific binding of 125I-labeled ST. Ligated loops of stomach and intestine treated with intraluminal ST had significant increases in guanosine 3',5'-cyclic monophosphate (cGMP), with duodenum exhibiting the greatest response. Expression of guanylate cyclase C (GCC) mRNA and a truncated, GCC-like mRNA was found in both stomach and intestine. Both mRNAs were isolated as cDNAs encoding the GC catalytic domain. The 0.9-kilobase (kb) cDNA is 99.8% identical to GCC, whereas the truncated, 0.75-kb GCC-like cDNA has a 159-nucleotide deletion and is 96.6% identical to GCC at the protein level. Uroguanylin and guanylin mRNAs were detected in stomach and intestine. Uroguanylin mRNA was most abundant in small intestine, whereas guanylin mRNA was highest in large intestine. Thus the stomach and intestine are targets for regulation of transport by guanylin and uroguanylin via cGMP.

The heat-stable enterotoxins are a family of low molecular weight, cysteine rich peptide toxins which are one of the major causes of watery diarrhea in children and adults. These toxins bind to a cell surface receptor in intestinal cells and mediate their action through elevation of intracellular cyclic GMP. We have generated a monoclonal antibody to these peptide toxins which is able to neutralise the activity of the peptides in a human colonic cell line, the T84 cell line. The monoclonal antibody, ST:G8, appears to be directed to an epitope distinct from antibodies previously generated, and prior incubation of this antibody, or Fab generated from this antibody, with full length STh and STp peptides prevents cGMP accumulation in T84 cells. This inhibition is a direct result of the antibody preventing binding of the peptides to the receptor. ST:G8 Mab does not recognize a 13-mer biologically active analog of STp, comprising the core sequence of STp peptide, suggesting that it is directed to a region in the N-terminus of the peptides, which may modulate receptor interaction/activation. The antibody recognizes a conformational epitope in the ST peptides, since reduction and carboxyamidation of ST abolishes antibody cross-reactivity. Differential cross-reactivity of the Mab with STh and STp peptides which differ markedly only in their N-termini, suggests that this antibody recognizes a distinct conformation in the two peptides, which is essential for receptor interaction.

Enterotoxigenic Escherichia coli elaborate a peptide called heat-stable enterotoxin (ST), which binds to and activates the intestinal ST receptor (STaR). STaR, also known as guanylyl cyclase C (GC-C), is a member of the transmembrane guanylyl cyclase receptor family. The mRNA for STaR encodes an approximately 120 kDa protein with the N-terminal ligand binding domain on the cell surface. Ligand affinity cross-linking studies have previously demonstrated several species of potential ST binding proteins, ranging in size from approximately 50 to 160 kDa. Although these smaller forms of STaR (50-80 kDa) have been proposed to act in vivo as toxin binding proteins, their biogenesis and localization have not previously been examined. Using pulse labeling in vivo and synchronized translation in vitro, we demonstrate that these smaller forms represent incomplete translational products and are not formed through limited proteolysis of the full-length receptor, as had previously been believed. We determined, using fluorescence confocal microscopy and surface labeling, that only approximately 25% of cellular receptors are expressed at the surface, while the remaining population is retained within the endoplasmic reticulum. Only full-length receptor is found at the surface of the cell, indicating this to be the biologically active form of STaR responsible for interacting with the heat-stable enterotoxin and other luminal intestinal peptides. The large intracellular receptor population, and potential for function before translocation to the cell surface, may impact on how pharmacologic modulators of this clinically important receptor are designed.

Background & Aims: Clostridium difficile toxin A causes secretion and intestinal inflammation in rodents by binding to a specific trisaccharide Gal alpha 1-3Gal beta 1-4 GlcNAc on enterocyte receptors. The purpose of this study was to explore the ability of Synsorb 90 (Synsorb Biotech Inc., Calgary, Alberta, Canada), and inert support carrying this trisaccharide, to bind toxin A in vitro and to inhibit its enterotoxic effects in vivo., Methods: Binding of [3H]toxin A to Synsorb 90, Synsorb 83 (beta-mannose attached), and Chromosorb P (inert support with no sugar attached) (Synsorb Biotech Inc.) was measured. The inhibitory effects of these compounds on toxin A-mediated fluid secretion, mannitol permeability, and histological damage were measured in ileal loops in vivo., Results: Toxin A showed specific binding to Synsorb 90, bearing the specific trisaccharide that binds toxin A, but not to Synsorb 83 or to Chromosorb P. Pretreatment of rats with Synsorb 90 by gavage (200 mg/kg body wt), but no Synsorb 83 or Chromosorb P at the same doses, dramatically reduced toxin A-associated fluid secretion and permeability., Conclusions: An immobilized toxin A receptor sequesters toxin A in the intestinal lumen and inhibits its effects of ileal mucosa. These results suggest a potential use for this agent in treating patients with C. difficile colitis.

Background: Cholera toxin from Vibrio cholerae and the type I heat-labile enterotoxins (LT-Is) from Escherichia coli are oligomeric proteins with AB5 structures. The type II heat-labile enterotoxins (LT-IIs) from E. coli are structurally similar to, but antigenically distinct from, the type I enterotoxins. The A subunits of type I and type II enterotoxins are homologous and activate adenylate cyclase by ADP-ribosylation of a G protein subunit, G8 alpha. However, the B subunits of type I and type II enterotoxins differ dramatically in amino acid sequence and ganglioside-binding specificity. The structure of LT-IIb was determined both as a prototype for other LT-IIs and to provide additional insights into structure/function relationships among members of the heat-labile enterotoxin family and the superfamily of ADP-ribosylating protein toxins., Results: The 2.25 A crystal structure of the LT-IIb holotoxin has been determined. The structure reveals striking similarities with LT-I in both the catalytic A subunit and the ganglioside-binding B subunits. The latter form a pentamer which has a central pore with a diameter of 10-18 A. Despite their similarities, the relative orientation between the A polypeptide and the B pentamer differs by 24 degrees in LT-I and LT-IIb. A common hydrophobic ring was observed at the A-B5 interface which may be important in the cholera toxin family for assembly of the AB5 heterohexamer. A cluster of arginine residues at the surface of the A subunit of LT-I and cholera toxin, possibly involved in assembly, is also present in LT-IIb. The ganglioside receptor binding sites are localized, as suggested by mutagenesis, and are in a position roughly similar to the sites where LT-I binds its receptor., Conclusions: The structure of LT-IIb provides insight into the sequence diversity and structural similarity of the AB5 toxin family. New knowledge has been gained regarding the assembly of AB5 toxins and their active-site architecture.

Internalization of Escherichia coli heat-stable enterotoxin (ST) mediated by guanylyl cyclase C was examined in T84 human colon carcinoma cells. Surface-associated, receptor-bound ST was quantitatively separated from intracellular ligand employing acidic guanidine-HCl. ST was internalized in a time-, temperature-, and ligand concentration-dependent fashion only by cells specifically expressing guanylyl cyclase C. Only receptors which bound reversibly to ST appeared to mediate endocytosis. The rate of internalization of ST empirically determined in these studies was 0.23 min-1. The density of surface receptors for ST was similar at 4 degrees C and 37 degrees C, suggesting that these receptors recycle back to the cell surface following internalization of ligand. Similarly, internalized ST was rapidly cleared from the intracellular compartment following endocytosis. These studies demonstrate that ST undergoes ligand-dependent receptor-mediated endocytosis in human colon carcinoma cells.

Intestinal cells exhibit binding sites with different affinities for Escherichia coli heat-stable enterotoxin (ST) and guanylin, suggesting the existence of different receptors for these peptides. Guanylyl cyclase C from intestinal cells has been identified as one receptor for these peptides. Equilibrium and kinetic binding characteristics of rat guanylyl cyclase C expressed in COS-7 cells were examined, employing ST, to determine if this receptor exhibited multiple affinities. Scatchard analysis of equilibrium binding yielded curvilinear isotherms consistent with the presence of high (pM) and low (nM) affinity sites. Kinetic analysis of binding demonstrated that these sites exhibited similar dissociation but different association kinetics. In addition, two distinct affinity states of low affinity sites were identified with dissociation constants of 0.15 and 5.85 nM. Association of ST and low affinity sites was biphasic, while dissociation from these sites was unimodal. Close agreement of equilibrium and kinetic dissociation constants suggested that low affinity sites were in the lowest affinity state at equilibrium. Comparison of the ligand dependence of guanylyl cyclase activity (EC50 = 110 nM) with receptor occupancy revealed that binding of ST to the lowest affinity state of low affinity sites (EC50 = 80 nM) is directly coupled to catalytic activation. These studies suggest that binding sites with different affinities for ST exhibited by intestinal cells reflect the expression of a single gene product, guanylyl cyclase C, rather than different receptors for the ligand. The shift in affinity state of low affinity sites and its correlation with catalytic activation suggest a central role for this phenomenon in mechanisms mediating receptor-effector coupling of membrane guanylyl cyclases.

Infection with enterotoxigenic Escherichia coli is a leading cause of traveler's diarrhea. Many enterotoxigenic E. coli strains produce heat-stable enterotoxin (ST), a peptide that binds to the intestinal receptor guanylyl cyclase C known as STaR. The toxin-receptor interaction elevates intracellular cGMP, which then activates apical chloride secretion, resulting in secretory diarrhea. In this report, we examine how the intracellular domains of STaR participate in the propagation and regulation of signaling. We show that STaR exists as an oligomer in both the presence and the absence of toxin. We also demonstrate that deletion of the intracellular kinase-homology domain produces a constitutively active mutant, suggesting that this domain subserves an autoinhibitory function. Finally, we constructed a point mutant within a highly conserved region of the cyclase domain that completely inactivates the catalytic activity of guanylyl cyclase. Cotransfection of this point mutant with wild-type receptor causes a dominant-negative effect on receptor activation. This suggests that interaction of receptor subunits is required for toxin-induced activation and that the cyclase domain is involved in this essential interaction. We propose that the binding of ST to STaR promotes a conformational change across the cell membrane. This removes the inhibitory effects of the kinase-homology domain and promotes an interaction between cyclase domains that leads to receptor activation. The data suggest a paradigm of signal transduction that may also be relevant to other members of the guanylyl cyclase receptor family.

The heat-stabile enterotoxin produced by Escherichia coli (ST) induces diarrhea by binding to receptors on intestinal cells, activating guanylyl cyclase, and increasing cyclic GMP. High- and low-affinity receptors for this toxin have been identified previously. ST induces intestinal secretion in suckling mice in picomole doses, suggesting a role for high-affinity receptors in this process. The present studies examine the relative roles of high- and low-affinity receptors in this process. The time course of changes in free ST concentrations in suckling mouse intestine was determined after intragastric inoculation. Also, binding characteristics of high- and low-affinity receptors and their coupling to guanylyl cyclase were defined in intestinal membranes from suckling mice. Intestinal concentrations of toxin and receptor binding characteristics empirically determined were used in a dynamic model correlating fractional occupancy of high- and low-affinity receptors with intestinal secretion to estimate their relative contributions to ST-induced diarrhea.

Background/aims: Escherichia coli heat-stable enterotoxins (ST) are small peptides of 18 or 19 amino acids that bind to specific cell surface receptors located on the intestinal brush border and activate guanylate cyclase, resulting in an increase in the intracellular cyclic guanosine 3',5'-monophosphate content of the cell. The present study examined whether receptors for ST are expressed by primary and metastatic human colonic tumors in vivo., Methods: Plasma membranes prepared from surgical tissue samples from normal colon, liver and lung, primary colonic adenocarcinomas, and colon carcinomas metastatic to lung and liver were analyzed for the structural and functional characteristics of constituent ST receptors., Results: All primary and metastatic colonic tumors examined bound ST, showing receptors of high (pmol/L) and low (nmol/L) affinity with densities that were similar to those in normal colon. Also, affinity cross-linking of labeled ST to membranes showed similar binding proteins in primary and metastatic tumors and normal colon. ST binding and affinity-labeled proteins were not detected in normal extraintestinal tissues. Guanylate cyclase was activated by ST in membranes from all colonic tumors studied, with efficacies and potencies that were similar to those in normal colon. ST did not activate this enzyme in normal extraintestinal tissues., Conclusions: Receptors for ST are expressed by primary and metastatic human colonic tumors in vivo, with structural and functional characteristics that are similar to those in normal human colon.

The E. coli type I heat-labile enterotoxin (LT-I) shares considerable functional, structural, and immunological homology with cholera toxin (CT). Although the ganglioside GM1 is the sole receptor for CT, LT-I also appears to utilize additional, unique receptors on intestinal cells not recognized by CT. We characterized this second class of LT-I receptors using the human intestinal epithelial cell line, CaCo-2. CaCo-2 cells bound 8-fold more LT-I than CT, and some of these additional LT-I receptors appeared to be functional, as CT-B only partially inhibited LT-I activity at concentrations that completely inhibited CT activity. Membranes from unlabeled or [3H]galactose-labeled cells were incubated with toxin B subunits and extracted with Triton X-100, and the solubilized toxin B-receptor complexes were immunoabsorbed with anti-B bound to protein A-Sepharose. When organic extracts of the complexes were separated by thin-layer chromatography and overlayed with [125I]toxin, both toxins were found to bind only GM1. Separation of the complexes from [3H]galactose-labeled membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a series of galactoproteins specifically recognized by LT-I but not by CT. Similar proteins were detected on Western blots probed with [125I]toxin. LT-I activity on intact cells and binding to membranes and the above galactoproteins were enhanced by neuraminidase treatment even in the presence of CT-B. beta-1,4-Galactosidase and endo-beta-1,4-galactosidase, but not beta-1,3-galactosidase, significantly reduced LT-I binding. LT-I binding to fetuin and transferrin exhibited a similar glycosidase sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

The protein binding Escherichia coli heat-stable enterotoxin II (STII) was isolated from cell membranes of mouse intestine. The binding of 125I-labeled STII to the proteins was inhibited by unlabeled STII, showing that it is specific. Proteins cross-linked with 125I-STII were purified by column chromatography on hydroxyapatite and TSK gel. Analyses of the purified protein by SDS-polyacrylamide gel electrophoresis and gel filtration showed that the molecular mass was 25 kDa.

The heat-stable enterotoxins (ST) elaborated by enterotoxigenic Escherichia coli are a family of small cysteine-rich peptides that bind to specific epithelial receptors in the mammalian intestine, causing a secretory diarrhea. The expression of ST receptors is tightly regulated; they are found primarily in intestine, and their expression is developmentally modulated. One receptor for ST has been cloned, and its cDNA encodes a approximately 120-kDa particulate guanylyl cyclase (guanylyl cyclase-C). Recent studies suggest that there are additional ST receptors that are not homologous to guanylyl cyclase-C. We used an expression cloning strategy to identify intestinal mRNAs that lead to expression of ST receptor activity in transfected cells. Using an ST-specific affinity panning system, we identified a novel 1891-base pair cDNA that does not encode a receptor protein, but instead, consists primarily of untranslated sequence. This cDNA induced receptor activity in both COS and 293 embryonic kidney cells. Northern analysis of the T84 human intestinal cell line, from which this cDNA was cloned, suggests that it is part of a 7.8-kilobase mRNA transcript. This transcript was also identified in human small intestine and colon, as well as in several extra-intestinal tissues. Functional analysis of subcloned fragments reveals that ST binding activity is induced by a 457-base pair human Alu repetitive sequence within the cDNA and that the phenotype is independent of orientation. These findings suggest that a human Alu element induces expression of a unique ST receptor by a transacting mechanism. An unrelated Alu-rich genomic clone did not confer ST binding, suggesting that there may be structural and functional specificity within individual Alu sequences.

Guanylyl cyclase (GC) C is a heat-stable enterotoxin (STa) receptor with a monomeric M(r) of approximately 140,000. We calculated from its hydrodynamic parameters that an active GC-C complex has a M(r) of 393,000, suggesting that GC-C is a trimer under native conditions. Both trimeric and dimeric GC-C complexes were detected by 125I-STa binding and SDS-polyacrylamide gel electrophoresis under non-reducing conditions. The GC activity and STa binding from intestinal brush border membranes comigrated in gel filtration and velocity sedimentation with recombinant GC-C. However, 125I-STa cross-linking demonstrated that STa receptors with molecular masses of 52 and 74 kDa are non-covalently attached to GC in the intestine. Radiation inactivation revealed different functional sizes for basal GC activity, STa-stimulated GC activity, and STa binding (59, 210-240, and 32-52 kDa, respectively). At low radiation doses, basal GC activity was stimulated, suggesting that GC-C is inhibited by a relatively large, probably internal structure. These results suggest that STa may activate GC-C by promoting monomer-monomer interaction (internal "dimerization") within a homotrimeric GC-C complex, and that GC-C is proteolytically modified in the brush border membrane but retains its function.