Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.2 (guanylate cyclase)
8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.
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PMID:Guanylyl cyclase is a heat-stable enterotoxin receptor. 170 94

Heat stable enterotoxins (STs) are low molecular-weight peptides secreted by enterotoxigenic bacteria. One type of these enterotoxins (STa) induces intestinal secretion leading to acute diarrhea by binding to a membrane form of guanylate cyclase. We have isolated a cDNA from a human colonic cell line, T84, encoding for a guanylate cyclase-coupled enterotoxin receptor (STaR). The predicted amino acid sequence of the human STa receptor is 81% identical with the previously cloned enterotoxin receptor (GC-C) from rat intestine. COS-7 cells transiently transfected with the cloned cDNA expressed specific concentration-dependent response to STa as measured by cyclic GMP accumulation and is about 20 times more sensitive to the stimulation by STa than has been shown for GC-C.
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PMID:Isolation and expression of a guanylate cyclase-coupled heat stable enterotoxin receptor cDNA from a human colonic cell line. 171 70

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.
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PMID:Regulation of cell signaling by the cytoplasmic domains of the heat-stable enterotoxin receptor: identification of autoinhibitory and activating motifs. 753 43

The systematic isolation of circulating regulatory peptides which generate cGMP as second messenger resulted in the identification of a novel member of the guanylin family. In the present study we describe the purification and amino acid sequence of a new guanylate cyclase C activating peptide (GCAP-II). GCAP-II contains 24 amino acids in the following sequence: FKTLRTIANDDCELCVNVACTGCL. Its molecular mass is 2597.7 Da. The 16 C-terminal amino acids are identical to uroguanylin from human urine. native and synthetic GCAP-II activate GC-C, the specific guanylate cyclase receptor, of cultured human colon carcinoma (T84) cells. GCAP-II stimulates chloride secretion in isolated human intestinal mucosa mediated by intracellular cGMP increase. GCAP-II specific antibodies were used to localize the peptide by immunohistochemistry in entero-endocrine cells of the colonic mucosa.
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PMID:GCAP-II: isolation and characterization of the circulating form of human uroguanylin. 758 7

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.
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PMID:Rat guanylyl cyclase C expressed in COS-7 cells exhibits multiple affinities for Escherichia coli heat-stable enterotoxin. 761 7

The quantification and distinction of particulate guanylyl cyclases in the human intestine were considered by an enzymatic approach, which comprised the signal transduction from receptor binding to cGMP formation, and, in addition, by showing the expression of an intracellular portion of these transmembrane proteins. Basal guanylyl cyclase (GC) activities were 50 to 80 pmol cGMP formation/min/mg protein and were stimulated up to twofold by heat stable enterotoxin, but were not significantly influenced by atrial natriuretic factor. Enzymatic analysis of colonoscopic specimens pointed to the prevalence of guanylyl cyclase C in the terminal ileum and in the large bowel including colon ascendens, colon descendens, sigmoid, and rectum. The availability of sequence information on human guanylyl cyclases permitted the development of a polymerase chain reaction approach for distinguishing the expression of GC-A and GC-C in human tissue samples. The expression levels of particulate guanylyl cyclases found by polymerase chain reaction in surgical biopsy specimens confirmed the enzymatic data, in that substantial expression of GC-C was found not only in the small intestine but also in the large bowel. According to the restriction mapping of amplificates, GC-C prevailed over GC-A throughout the human intestine, particularly in the mucosal layers.
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PMID:Distribution of membrane bound guanylyl cyclases in human intestine. 795 32

We studied the activation and inactivation of recombinant guanylyl cyclase (GC) C stably expressed in human kidney 293 cells. Activation of GC-C by heat-stable enterotoxin (STa), Cd2+, hemin, or the detergent Triton X-100 was followed by a rapid inactivation of the enzyme. Adenine nucleotides were found to prevent the inactivation process in native membranes, as well as following enzyme solubilization and immunopurification. Inactivation of GC-C was not associated with dephosphorylation. However, the phosphorylation of GC-C was promoted by phorbol esters, known activators of protein kinase C. The activation of purified GC-C by STa required the presence of a nonspecific factor as exemplified by bovine serum albumin. When immunopurified GC-C, stabilized by ATP and bovine serum albumin, was analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, proteins with almost twice the molecular mass (220 and 245 kDa) of the monomer were observed. The mobility of these high M(r) GC-C forms was reduced by STa, suggesting that STa induces a conformation change in a preexisting GC-C dimer. These results indicate that ATP interacts directly with GC-C, stabilizing its active conformation and that the activation of GC-C may occur in the absence of other specific regulatory factors.
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PMID:Heat-stable enterotoxin activation of immunopurified guanylyl cyclase C. Modulation by adenine nucleotides. 810 20

Enterotoxigenic Escherichia coli elaborate a heat-stable enterotoxin that causes diarrhea in humans and animals. The primary event in the diarrheal cascade is the binding of this enterotoxin to specific receptors on enterocytes and activation of guanylyl cyclase. Two intestinal cell lines, Caco-2 and IEC-6, were tested for the presence of these receptors. Although both cell lines exhibited specific binding, only the Caco-2 cell line responded to heat-stable enterotoxin with increased guanylyl cyclase activity. Cloning and expression studies confirmed that the receptor present in Caco-2 cells is a homologue of guanylyl cyclase C, a known transmembrane heat-stable enterotoxin receptor. Expression of the receptor in differentiating Caco-2 cells increases with cell maturation, indicating that these cells are a suitable model for future studies. However, Northern and polymerase chain reaction analyses demonstrated that guanylyl cyclase C is not expressed in IEC-6 cells, strongly suggesting the presence of a novel heat-stable enterotoxin receptor that is not coupled to guanylyl cyclase activity.
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PMID:Comparison of receptors for Escherichia coli heat-stable enterotoxin: novel receptor present in IEC-6 cells. 838 96

Guanylin (GCAP-I, guanylate cyclase activating peptide I) and uroguanylin (GCAP-II, guanylate cyclase activating peptide II) are regulatory peptides involved in the regulation of the intestinal chloride / water balance. They share significant structural homology to the E. coli enterotoxin STa, which binds to the particulate guanylyl cyclase C causing diarrhea in mammals. In this study we report the functional analysis of the guanylin / GCAP-I gene promoter region. By means of the luciferase reporter gene assay, we demonstrate a strong promoter activity in T84 cells. Especially the first 160 bp of the 5'-flanking region of the gene seem to be essential for gene induction. Our findings are the basis for further identification of important regulatory elements of the corresponding gene.
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PMID:Functional analysis of the human guanylin gene promoter. 871 1

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.
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PMID:Biogenesis, cellular localization, and functional activation of the heat-stable enterotoxin receptor (guanylyl cyclase C). 871 57


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