An adenylyl cyclase pseudogene in Mycobacterium tuberculosis has a functional ortholog in Mycobacterium avium.

A number of genes similar to mammalian Class III nucleotide cyclases are found in mycobacteria, and biochemical characterization of some of these proteins has indicated that they code for adenylyl cyclases, with properties similar to the mammalian enzymes. Our earlier bioinformatic analysis had predicted that the Rv1120c gene in Mycobacterium tuberculosis is a pseudogene, while analysis of the genome of Mycobacterium avium indicated the presence of a functional ortholog. We therefore cloned and expressed Rv1120c and its ortholog from M. avium, Ma1120, in Escherichia coli, and find that while the protein from M. tuberculosis is misfolded and found in inclusion bodies, Ma1120 is expressed to high levels as a functional adenylyl cyclase. Sequence analysis of Ma1120 indicates interesting variations in critical amino acids that are known to be important for catalytic activity. Ma1120 is maximally active in the presence of MnATP as substrate ((app)Km approximately 400 microM), and is inhibited by P-site inhibitors (IC50 of 2',5'-dideoxy-3'-adenosine triphosphate approximately 730 nM) and tyrphostins (IC50 approximately 36 microM) in a manner similar to the mammalian enzymes. This therefore represents the first Class III cyclase biochemically characterized from M. avium, and the absence of a functional ortholog in M. tuberculosis suggests a unique role for this enzyme in M. avium.

Functional characterisation of mycobacterial DNA gyrase: an efficient decatenase.

A rapid single step immunoaffinity purification procedure is described for Mycobacterium smegmatis DNA gyrase. The mycobacterial enzyme is a 340 kDa heterotetrameric protein comprising two subunits each of GyrA and GyrB, exhibiting subtle differences and similarities to the well-characterised Escherichia coli gyrase. In contrast to E.coli gyrase, the M.smegmatis enzyme exhibits strong decatenase activity at physiological Mg2+ concentrations. Further, the enzymes exhibited marked differences in ATPase activity, DNA binding characteristics and susceptibility to fluoroquinolones. The holoenzyme showed very low intrinsic ATPase activity and was stimulated 20-fold in the presence of DNA. The DNA-stimulated ATPase kinetics revealed apparent K0.5 and kcat of 0.68 mM and 0.39 s(-1), respectively. The dissociation constant for DNA was found to be 9.2 nM, which is 20 times weaker than that of E.coli DNA gyrase. The differences between the enzymes were further substantiated as they exhibited varied sensitivity to moxifloxacin and ciprofloxacin. In spite of these differences, mycobacterial DNA gyrase is a functionally and mechanistically conserved enzyme and the variations in activity seem to reflect functional optimisation for its physiological role during mycobacterial genome replication.

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

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

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.

Protein kinase C regulates transcription of the human guanylate cyclase C gene.

Guanylate cyclase C is the receptor for the bacterial heat-stable enterotoxins and guanylin family of peptides, and mediates its action by elevating intracellular cGMP levels. Potentiation of ligand-stimulated activity of guanylate cyclase C in human colonic T84 cells is observed following activation of protein kinase C as a result of direct phosphorylation of guanylate cyclase C. Here, we show that prolonged exposure of cells to phorbol esters results in a decrease in guanylate cyclase C content in 4beta-phorbol 12-myristate 13-acetate-treated cells, as a consequence of a decrease in guanylate cyclase C mRNA levels. The reduction in guanylate cyclase C mRNA was inhibited when cells were treated with 4beta-phorbol 12-myristate 13-acetate (PMA) in the presence of staurosporine, indicating that a primary phosphorylation event by protein kinase C triggered the reduction in RNA levels. The reduction in guanylate cyclase C mRNA levels was not due to alterations in the half-life of guanylate cyclase C mRNA, but regulation occurred at the level of transcription of guanylate cyclase C mRNA. Expression in T84 cells of a guanylate cyclase C promoter-luciferase reporter plasmid, containing 1973 bp of promoter sequence of the guanylate cyclase C gene, indicated that luciferase activity was reduced markedly on PMA treatment of cells, and the protein kinase C-responsive element was present in a 129-bp region of the promoter, containing a HNF4 binding element. Electrophoretic mobility shift assays using an oligonucleotide corresponding to the HNF4 binding site, indicated a decrease in binding of the factor to its cognate sequence in nuclear extracts prepared from PMA-treated cells. We therefore show for the first time that regulation of guanylate cyclase C activity can be controlled at the transcriptional level by cross-talk with signaling pathways that modulate protein kinase C activity. We also suggest a novel regulation of the HNF4 transcription factor by protein kinase C.

A Mycobacterium smegmatis gyrase B specific monoclonal antibody reveals association of gyrase A and B subunits in the cell.

DNA gyrase is a unique topoisomerase, which plays important roles in macromolecular events like DNA replication, transcription and genetic recombination. In this study a high affinity monoclonal antibody to the gyrase B (GyrB) subunit of Mycobacterium smegmatis was characterized, which did not cross-react with either the Escherichia coli GyrB subunit or with GyrB subunits from other mycobacterial species. The antibody recognized an epitope in the N-terminus, novobiocin-binding domain of GyrB. Immunoprecipitation of gyrase from M. smegmatis cell lysate revealed an association, mediated by ionic interactions, of gyrase A and GyrB subunits in the cell. This antibody is a valuable tool for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer.

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn(2+), at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent cross-linking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

Tyrosine phosphorylation of the human guanylyl cyclase C receptor.

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

Expression of GC-C, a receptor-guanylate cyclase, and its endogenous ligands uroguanylin and guanylin along the rostrocaudal axis of the intestine.

Members of the receptor-guanylate cyclase (rGC) family possess an intracellular catalytic domain that is regulated by an extracellular receptor domain. GC-C, an intestinally expressed rGC, was initially cloned by homology as an orphan receptor. The search for its ligands has yielded three candidates: STa (a bacterial toxin that causes traveler's diarrhea) and the endogenous peptides uroguanylin and guanylin. Here, by performing Northern and Western blots, and by measuring [125I]STa binding and STa-dependent elevation of cGMP levels, we investigate whether the distribution of GC-C matches that of its endogenous ligands in the rat intestine. We establish that 1) uroguanylin is essentially restricted to small bowel; 2) guanylin is very low in proximal small bowel, increasing to prominent levels in distal small bowel and throughout colon; 3) GC-C messenger RNA and STa-binding sites are uniformly expressed throughout the intestine; and 4) GC-C-mediated cGMP synthesis peaks at the proximal and distal extremes of the intestine (duodenum and colon), but is nearly absent in the middle (ileum). These observations suggest that GC-C's activity may be posttranslationally regulated, demonstrate that the distribution of GC-C is appropriate to mediate the actions of both uroguanylin and guanylin, and help to refine current hypotheses about the physiological role(s) of these peptides.

Nature of linkage between the cationic headgroup and cholesteryl skeleton controls gene transfection efficiency.

Three novel cationic cholesterol derivatives with different modes of linkage between the cationic headgroup and the cholesteryl backbone have been synthesized and used as mixtures with 1, 2-dioleoyl-L-alpha-glycero-3-phosphatidyl ethanolamine (DOPE) for liposome-mediated gene transfection. A pronounced improvement in gene transfer efficiency was observed when the cationic center was appended to the cholesteryl backbone using an ether linkage as opposed to when the linkages were based on either ester or urethane groups. Amphiphiles with ether links such as cholest-5-en-3beta-oxyethane-N,N,N-trimethyl ammonium bromide (2) and cholest-5-en-3beta-oxyethane-N,N-dimethyl-N-2-hydroxyethyl ammonium bromide (3) showed transfection efficiencies considerably greater than commercially available gene transfer reagents. Notably, the transfection ability of 2 with DOPE in the presence of serum was significantly greater than Lipofectamine((R)) and Lipofectin((R)). Interestingly, 3 did not require the helper lipid DOPE for transfection. This suggests that these newly described cholesterol-based amphiphiles should be very promising in liposome-mediated gene transfection. The advantage that the ether linkage possesses would be important in the design of newer, more efficient cholesterol-based delivery reagents.

Expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) in human colonic epithelial cells: role in the induction of cellular refractoriness to the heat-stable enterotoxin peptide.

Stable toxin (ST) peptides are the causative agents for a severe form of watery diarrhea. These peptides bind to a membrane-associated form of guanylyl cyclase, guanylyl cyclase C. The result is an accumulation of cyclic guanosine monophosphate (cGMP) in the intestinal cell, regulating protein kinase activity and the phosphorylation of a number of proteins involved in ion transport across the intestine. Using the human T84 colonic cell line as a model system, we show that cGMP accumulation in these cells after ST application is regulated by the activity of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5). The presence of human PDE5 in this cell line was confirmed by Western blot analysis, using an antibody raised to the bovine enzyme, and by the observation that cGMP hydrolytic activity detected in T84 cell lysates was almost completely inhibited by low concentrations of zaprinast, a specific inhibitor of PDE5. An increase in activity of PDE5 was observed in T84 cell lysates on exposure to the ST peptide and prolonged exposure of T84 cells to the ST peptide led to the induction of cellular refractoriness in these cells, which was largely contributed in terms of an increased rate of degradation of cGMP in desensitized cells as a result of PDE5 activation. This activation was correlated with an increase in the affinity of the enzyme for the substrate cGMP, as well as an increased affinity for zaprinast. We provide evidence for the first time that cGMP levels in the human colonocyte are regulated by the cGMP-hydrolytic activity of PDE5 and suggest that the expression and regulation of PDE5 in the intestine could therefore be important in controlling cGMP-mediated signaling in this tissue.

Homologous desensitization of the human guanylate cyclase C receptor. Cell-specific regulation of catalytic activity.

Guanylate Cyclase C (GCC) serves as a receptor for the endogenous ligands, guanylin and uroguanylin, as well as the family of bacterial heat-stable enterotoxins (ST), which are one of the major causes of diarrhoea the world over. We had earlier provided evidence that GCC, present in the human colonic T84 cell line, is desensitized on prolonged exposure to ST, and this desensitization was reflected in a reduced ST-stimulated guanylate cyclase activity of GCC [Bakre, M.M. & Visweswariah, S.S. (1997) FEBS Lett. 408, 345-349]. In this study, we have investigated the mechanisms that underlie this cellular desensitization process. Desensitization of T84 cells was not a result of reduction in GCC present in membranes prepared from desensitized T84 cells, nor due to increased cGMP-phosphodiesterase activity associated with the membrane fraction. The decrease in ST-stimulatable guanylate cyclase activity of GCC was due to a dramatic reduction in the Vmax of the cyclase, which was also seen when MnGTP was used as the substrate. GCC undergoes ligand-induced inactivation in vitro, which is alleviated in the presence of ATP. In vivo desensitized GCC could be further inactivated in vitro when preincubated with ST, indicating that the two mechanisms of GCC inactivation are distinct. Cellular refractoriness as reflected by a reduced responsiveness to further ST-stimulation following prior exposure to IST, coupled with GCC desensitization was also observed in another colonic cell line, Caco2. However, HEK293 cells, stably transfected with GCC cDNA, when exposed to ST for prolonged periods, did not result in GCC desensitization, indicating that desensitization of GCC appeared to be a cell specific phenomenon. GCC expressed in HEK293-GCC cells, however, showed in vitro ligand induced inactivation, suggesting that there are two independent means of ligand-induced desensitization of GCC, perhaps distinct from the mechanisms that have been described earlier for other members of the guanylate cyclase receptor family.

Guanylyl cyclase C receptor: regulation of catalytic activity by ATP.

Guanylyl cyclase C (GCC), a member of the family of membrane bound guanylyl cyclases is the receptor for the heat-stable enterotoxin (ST) peptides and the guanylin family of endogenous peptides. GCC is activated upon ligand binding to increase intracellular cGMP levels, which in turn activates other downstream signalling events in the cell. GCC is also activated in vitro by nonionic detergents. We have used the T84 cell line as a model system to investigate the regulation of GCC activity by ATP. Ligand-stimulated GCC activity is potentiated in the presence of ATP, whereas detergent-stimulated activity is inhibited. The potentiation of GCC activity by ATP is dependent on the presence of Mg2+ ions, and is probably brought about by a direct binding of Mg-ATP to GCC. The protein kinase-like domain of GCC, which has earlier been shown to play a critical role in the regulation of GCC activity, may be a possible site for the binding of Mg-ATP to GCC.

Thermodynamic analyses reveal role of water release in epitope recognition by a monoclonal antibody against the human guanylyl cyclase C receptor.

The thermodynamics of a monoclonal antibody (mAb)-peptide interaction have been characterized by isothermal titration microcalorimetry. GCC:B10 mAb, generated against human guanylyl cyclase C, a membrane-associated receptor and a potential marker for metastatic colon cancer, recognizes the cognate peptide epitope HIPPENIFPLE and its two contiguous mimotopes, HIPPEN and ENIFPLE, specifically and reversibly. The exothermic binding reactions between 6.4 and 42 degrees C are driven by dominant favorable enthalpic contributions between 20 and 42 degrees C, with a large negative heat capacity (DeltaC(p)) of -421 +/- 27 cal mol(-1) K(-1). The unfavorable negative value of entropy (DeltaS(b)(0)) at 25 degrees C, an unusual feature among protein-protein interactions, becomes a positive one below an inversion temperature of 20.5 degrees C. Enthalpy-entropy compensation due to solvent reorganization accounts for an essentially unchanged free energy of interaction (DeltaDeltaG(b)(0) congruent with 0). The role of water molecules in the recognition process was tested by coupling an osmotic stress technique with isothermal titration microcalorimetry. The results provide direct and compelling evidence that GCC:B10 mAb recognizes the peptides HIPPENIFPLE, HIPPEN, and ENIFPLE differentially, with a concomitant release of variable and nonadditive numbers of water molecules (15, 7, and 3, respectively) from the vicinity of the binding site.

Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides.

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor.

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.

Identification and characterization of receptors for riboflavin carrier protein in the chicken oocyte. Role of the phosphopeptide in mediating receptor interaction.

Riboflavin carrier protein (RCP) is a phosphoglycoprotein found in the egg and the serum of laying birds and other animals. We have investigated the binding of chicken RCP (cRCP) to membranes prepared from the whole chicken oocytes. RCP binding had an absolute requirement for calcium, with an affinity (Kd 10(-8) M) high enough to be physiologically relevant. Ligand blotting experiments using labeled RCP and vitellogenin, with proteins solubilized from oocyte membranes, indicated that RCP and vitellogenin bound specifically to three proteins of Mr 380, 260 and 110 kDa. Vitellogenin also bound to proteins of Mr 515 kDa and 97 kDa, similar in size to those identified by receptor associated protein of RAP. Reduced and carboxyamidated RCP inhibited the binding of 125I-labeled RCP to chicken oocyte membranes, but recombinant RCP expressed in E. coli, and dephosphorylated RCP, failed to interact with the receptors, indicating that post-translational modifications were necessary for ligand-receptor interaction. The purified phosphopeptide, prepared from tryptic digests of egg white RCP, was able to inhibit the binding of RCP to the receptor proteins, with an affinity comparable to native RCP indicating that the phosphopeptide sequence present in RCP serves as the focal point for RCP-receptor interactions.

Refolding of native and recombinant chicken riboflavin carrier (or binding) protein: evidence for the formation of non-native intermediates during the generation of active protein.

Riboflavin carrier (or binding) protein (RCP) is a phosphoglycoprotein originally purified from the egg white, yolk and serum of laying hens. The 18 cysteine residues present in RCP form nine disulfide bridges, allowing the protein to form a compact structure to generate a hydrophobic pocket in which riboflavin sits. We studied the refolding of totally reduced and denatured egg white RCP and found that the protein initially folded to generate a molecule that did not possess riboflavin-binding activity, despite near-complete oxidation of the cysteine residues. Riboflavin-binding activity was then slowly regained, but the final refolded form of the protein was less compact in structure than the native molecule, due to incomplete oxidation of all the cysteine residues. Denatured and reduced dephosphorylated RCP refolded as efficiently as the native protein, with similar rates of disulfide-bond oxidation and generation of riboflavin binding, showing that the phosphoserine stretch of RCP has little role to play during refolding. In order to study the role of glycosylation in the refolding process, the cDNA for full-length RCP was expressed in Escherichia coli and purified. Recombinant RCP refolded only in the presence of redox buffers, demonstrating that glycosylation of RCP could allow the formation of high yields of productive intermediates in the folding pathway. Using a panel of conformation-specific monoclonal antibodies to RCP, it appeared that the folding intermediates of RCP possessed a structure distinctly different to the native protein, indicating that the correct folding pathway of RCP passed through conformation(s) generated by non-native disulfide bridges.

Epitope conservation and immunohistochemical localization of the guanylin/stable toxin peptide receptor, guanylyl cyclase C.

The heat-stable enterotoxins (ST) are a family of cysteine-rich low-molecular weight peptides produced by pathogenic bacteria, and are one of the major causes of watery diarrhea all over the world. These toxins mediate their action by binding to an intestinal cell surface receptor that is a membrane-associated guanylyl cyclase (GCC). This receptor also serves as the receptor for the recently characterised endogenous ligand, guanylin. We have expressed various domains of the receptor in Escherichia coli and used purified proteins for the generation of both polyclonal and monoclonal antibodies. While polyclonal antibodies were able to partially inhibit ST binding to the native receptor present in the T84 human colonic cell line, GCC:B10 monoclonal antibody did not interfere with ligand binding. Western blot analysis, using membranes prepared from human colonic T84 cells, detected two bands of size 160 and 140 kDa, representing alternately glycosylated forms of the receptor. Using the recombinant proteins, we could map the epitope of GCC:B10 monoclonal antibody to the intracellular domain of the receptor. We used the antibody to localize the receptor throughout the rat intestine, and in the porcine and bonnet monkey colon. We could detect receptor expression in the villus and the crypts of the duodenum, jejunum, ileum, and caecum, and in the crypts of the colon. Receptor expression was observed in cells that had earlier been shown to express cGMP-dependent kinase, but not the cystic fibrosis transmembrane regulator, a known downstream target of cGMP/G-kinase, which suggests that GCC/ cGMP could regulate additional cellular signal transduction machinery.

Dual regulation of heat-stable enterotoxin-mediated cGMP accumulation in T84 cells by receptor desensitization and increased phosphodiesterase activity.

We report the regulation of cGMP accumulation induced by the heat-stable enterotoxin, STh, in the T84 human colonic cell line. STh binding to its receptor, guanylyl cyclase C (GCC), leads to elevated intracellular levels of cGMP. Prolonged exposure of T84 cells to STh induced refractoriness to further cGMP accumulation, without significant receptor internalization, but with reduced STh-induced cGMP synthesis by the receptor. Significantly, increased degradation of cGMP by a cGMP-specific phosphodiesterase was observed in desensitized cells. This is the first report on the desensitization of GCC, as well as the role of the Type V phosphodiesterase in inducing cellular refractoriness.