The Experts below are selected from a list of 159 Experts worldwide ranked by ideXlab platform
Mitchell B. Cohen - One of the best experts on this subject based on the ideXlab platform.
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Immunohistochemical Localization of Guanylin in the Rat Small Intestine and Colon
Biochemical and biophysical research communications, 1995Co-Authors: Mitchell B. Cohen, David P. Witte, J.a. Hawkins, M.g. CurrieAbstract:Guanylin is an endogenous mammalian ligand which binds to guanylate cyclase C (GC-C), the Escherichia coli heat-stable Enterotoxin Receptor. This interaction results in intestinal Cl- and fluid secretion, which is largely, if not exclusively, mediated through the cystic fibrosis transmembrane regulator (CFTR). Using in situ hybridization, we have previously localized guanylin mRNA to villus epithelial cells of the rat small intestine and to superficial epithelial cells of the rat colon. In the present study, we demonstrate immunoreactive guanylin in a subpopulation of goblet cells in the rat jejunum and ileum. In the colon, there was immunostaining of superficial epithelial cells and goblet cells. The immunohistochemical localization of guanylin parallels the observed distribution of guanylin mRNA. Localization of guanylin in goblet cells leads us to speculate that an in vivo function of guanylin regulated, CFTR-mediated Cl- secretion is to hydrate intestinal mucin.
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Upregulation of Escherichia coli heat-stable Enterotoxin Receptor in regenerating rat liver
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1994Co-Authors: D W Laney, J. A. Bezerra, J. L. Kosiba, Sandra J. Friezner Degen, Mitchell B. CohenAbstract:Guanylate cyclase C (GC-C) is a transmembrane protein that serves as a Receptor for the recently characterized endogenous ligand guanylin and for Escherichia coli heat-stable toxin (STa). Binding of either guanylin or STa to intestinal GC-C results in net chloride secretion. Although GC-C is expressed in the rat intestine throughout life, its expression in the rat liver has previously been shown to occur only during the perinatal period. As a step toward elucidating the role of this Receptor in the liver, we tested the hypothesis that GC-C mRNA expression could be induced in the adult rat liver following 1) partial hepatectomy, a stimulus for hepatocyte proliferation; 2) intraperitoneal carbon tetrachloride injection, a model of hepatocyte regeneration in the presence of inflammatory changes; and 3) subcutaneous turpentine injection, which generates an acute phase response without hepatocyte proliferation. We demonstrated expression of GC-C mRNA in the regenerating rat liver following either partial hepatectomy or CCl4-induced hepatic necrosis. We have also shown that GC-C mRNA expression occurred in association with an acute phase reaction. Coordinate with the expression of GC-C mRNA, there was upregulation of radiolabeled STa binding to liver plasma membranes prepared from turpentine-treated rats. Maximal expression of GC-C occurred in preparations enriched for the canalicular domain. Although the function of GC-C in the liver is unknown, localization to the canalicular domain would be consistent with a role for GC-C in hepatic chloride secretion, especially in the perinatal liver and during hepatocyte regeneration.
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guanylin mrna is expressed in villous enterocytes of the rat small intestine and superficial epithelia of the rat colon
Biochemical and Biophysical Research Communications, 1993Co-Authors: L G Lewis, M.g. Currie, David P. Witte, D W Laney, Mitchell B. CohenAbstract:Abstract Guanylin, an endogenous ligand for the Escherichia coli heat-stable Enterotoxin Receptor, is a recently characterized intestinal peptide. To understand the possible physiologic function of guanylin, we examined the cellular location of guanylin mRNA expression in the rat intestine. Intestinal cells were sequentially isolated from villous tip to crypt in rat jejunum and ileum. Northern blots of total RNA identified a single 0.65 kb guanylin transcript predominantly in the villous cell fractions. In situ hybridization studies demonstrated maximal signal intensity in villous cells in rat ileum and surface epithelial cells in the colon. In the ileum, the signal was nonuniform in distribution in the surface epithelial cells, with focal areas of intense signal in clusters of columnar absorptive cells. In both colon and ileum, signal intensity was near background level in deep crypt cells, lamina propria, and muscularis.
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Comparison of Receptors for Escherichia coli heat-stable Enterotoxin : novel Receptor present in IEC-6 cells
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1993Co-Authors: Elizabeth A. Mann, Mitchell B. Cohen, Ralph A. GiannellaAbstract: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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Novel sites for expression of an Escherichia coli heat-stable Enterotoxin Receptor in the developing rat
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1992Co-Authors: D W Laney, Elizabeth A. Mann, Ralph A. Giannella, S. C. Dellon, D. R. Perkins, Mitchell B. CohenAbstract:Escherichia coli heat-stable Enterotoxin (STa) mediates diarrheal disease by binding to and activating an intestinal transmembrane Receptor, guanylate cyclase C (GC-C). To test the hypotheses that there was 1) increased perinatal expression of GC-C in rat intestine and 2) GC-C expression and STa binding in extraintestinal tissues of immature rat, we prepared whole cell membranes and total RNA from jejunum, ileum, colon, liver, kidney, heart, lung, brain, testis, and placenta of rats ranging in age from 12 days gestation to adult. Northern analysis demonstrated the presence of a unique 3.8-kb mRNA transcript at all ages in the jejunum, ileum, colon, and, to a lesser degree, in the testis. GC-C was also detected by Northern analysis in liver (from gestational age 18 days through 14 days postnatal) and in placenta. Steady-state mRNA encoding GC-C was not detected by Northern analysis in the other organs examined. GC-C-specific mRNA expression was greatest in the perinatal period in the jejunum, ileum, and liver. Specific binding of 125I-labeled STa was found in each of the tissue membranes in which GC-C mRNA was present; binding was not present in those tissues that had no detectable GC-C mRNA. The existence of GC-C in extraintestinal organs in the rat, and the development changes in GC-C expression support our hypothesis that GC-C, apart from its role as an STa Receptor in mediating diarrheal disease, also serves as a Receptor for an endogenous ligand.
Ralph A. Giannella - One of the best experts on this subject based on the ideXlab platform.
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Cell line-specific transcriptional activation of the promoter of the human guanylyl cyclase C/heat-stable Enterotoxin Receptor gene☆
Biochimica et biophysica acta, 1996Co-Authors: Elizabeth A. Mann, Mary Lynn Jump, Ralph A. GiannellaAbstract:Abstract The guanylyl cyclase C protein, expressed primarily in the intestine, is the Receptor for the heat-stable Enterotoxin of Escherichia coli . We have isolated and sequenced the promoter region and the first exon of human guanylyl cyclase C and determined the major site of transcription initiation. Transfection of a − 1973 + 124 promoter/luciferase gene fusion construct in the Caco-2 intestinal cell line resulted in a high level of expression; results with deletion constructs indicate the presence of multiple positive-acting sequence elements. These promoter elements were not active upon transfection into NIH/3T3 and LLC-PK 1 cell lines which do not express GC-C.
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Comparison of Receptors for Escherichia coli heat-stable Enterotoxin : novel Receptor present in IEC-6 cells
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1993Co-Authors: Elizabeth A. Mann, Mitchell B. Cohen, Ralph A. GiannellaAbstract: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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Novel sites for expression of an Escherichia coli heat-stable Enterotoxin Receptor in the developing rat
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1992Co-Authors: D W Laney, Elizabeth A. Mann, Ralph A. Giannella, S. C. Dellon, D. R. Perkins, Mitchell B. CohenAbstract:Escherichia coli heat-stable Enterotoxin (STa) mediates diarrheal disease by binding to and activating an intestinal transmembrane Receptor, guanylate cyclase C (GC-C). To test the hypotheses that there was 1) increased perinatal expression of GC-C in rat intestine and 2) GC-C expression and STa binding in extraintestinal tissues of immature rat, we prepared whole cell membranes and total RNA from jejunum, ileum, colon, liver, kidney, heart, lung, brain, testis, and placenta of rats ranging in age from 12 days gestation to adult. Northern analysis demonstrated the presence of a unique 3.8-kb mRNA transcript at all ages in the jejunum, ileum, colon, and, to a lesser degree, in the testis. GC-C was also detected by Northern analysis in liver (from gestational age 18 days through 14 days postnatal) and in placenta. Steady-state mRNA encoding GC-C was not detected by Northern analysis in the other organs examined. GC-C-specific mRNA expression was greatest in the perinatal period in the jejunum, ileum, and liver. Specific binding of 125I-labeled STa was found in each of the tissue membranes in which GC-C mRNA was present; binding was not present in those tissues that had no detectable GC-C mRNA. The existence of GC-C in extraintestinal organs in the rat, and the development changes in GC-C expression support our hypothesis that GC-C, apart from its role as an STa Receptor in mediating diarrheal disease, also serves as a Receptor for an endogenous ligand.
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A gradient in expression of the Escherichia coli heat-stable Enterotoxin Receptor exists along the villus-to-crypt axis of rat small intestine.
Biochemical and biophysical research communications, 1992Co-Authors: Mitchell B. Cohen, Elizabeth A. Mann, Chantal Lau, Susan J. Henning, Ralph A. GiannellaAbstract:Binding of Escherichia coli heat-stable Enterotoxin to its Receptor is critical to the initiation of toxin-induced secretion and diarrheal disease; it is also likely, however, that this Receptor binds an endogenous ligand. In order to characterize the expression of the heat-stable Enterotoxin Receptor in the small intestine, we isolated epithelial cells from villus tip to crypt in rat jejunum and ileum. Binding of radiolabeled toxin was maximal in the villus preparations and gradually decreased along the villus-to-crypt axis, paralleling the decline of sucrase activity. Northern blots of total RNA identified a single heat stable Enterotoxin Receptor transcript (3.8 kb), predominantly in the villus cell fractions. In situ hybridization demonstrated clear signal in the villus cells with no apparent signal in the crypt cells, lamina propria or muscularis. Expression of this Receptor was greatest after enterocytes leave the proliferative cycle and enter villi. This pattern of gene and protein expression may reflect a role of this Receptor in binding endogenous ligands which in turn may regulate intestinal ion flux along the villus-to-crypt axis.
Elizabeth A. Mann - One of the best experts on this subject based on the ideXlab platform.
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Cell line-specific transcriptional activation of the promoter of the human guanylyl cyclase C/heat-stable Enterotoxin Receptor gene☆
Biochimica et biophysica acta, 1996Co-Authors: Elizabeth A. Mann, Mary Lynn Jump, Ralph A. GiannellaAbstract:Abstract The guanylyl cyclase C protein, expressed primarily in the intestine, is the Receptor for the heat-stable Enterotoxin of Escherichia coli . We have isolated and sequenced the promoter region and the first exon of human guanylyl cyclase C and determined the major site of transcription initiation. Transfection of a − 1973 + 124 promoter/luciferase gene fusion construct in the Caco-2 intestinal cell line resulted in a high level of expression; results with deletion constructs indicate the presence of multiple positive-acting sequence elements. These promoter elements were not active upon transfection into NIH/3T3 and LLC-PK 1 cell lines which do not express GC-C.
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Comparison of Receptors for Escherichia coli heat-stable Enterotoxin : novel Receptor present in IEC-6 cells
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1993Co-Authors: Elizabeth A. Mann, Mitchell B. Cohen, Ralph A. GiannellaAbstract: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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Novel sites for expression of an Escherichia coli heat-stable Enterotoxin Receptor in the developing rat
American Journal of Physiology-Gastrointestinal and Liver Physiology, 1992Co-Authors: D W Laney, Elizabeth A. Mann, Ralph A. Giannella, S. C. Dellon, D. R. Perkins, Mitchell B. CohenAbstract:Escherichia coli heat-stable Enterotoxin (STa) mediates diarrheal disease by binding to and activating an intestinal transmembrane Receptor, guanylate cyclase C (GC-C). To test the hypotheses that there was 1) increased perinatal expression of GC-C in rat intestine and 2) GC-C expression and STa binding in extraintestinal tissues of immature rat, we prepared whole cell membranes and total RNA from jejunum, ileum, colon, liver, kidney, heart, lung, brain, testis, and placenta of rats ranging in age from 12 days gestation to adult. Northern analysis demonstrated the presence of a unique 3.8-kb mRNA transcript at all ages in the jejunum, ileum, colon, and, to a lesser degree, in the testis. GC-C was also detected by Northern analysis in liver (from gestational age 18 days through 14 days postnatal) and in placenta. Steady-state mRNA encoding GC-C was not detected by Northern analysis in the other organs examined. GC-C-specific mRNA expression was greatest in the perinatal period in the jejunum, ileum, and liver. Specific binding of 125I-labeled STa was found in each of the tissue membranes in which GC-C mRNA was present; binding was not present in those tissues that had no detectable GC-C mRNA. The existence of GC-C in extraintestinal organs in the rat, and the development changes in GC-C expression support our hypothesis that GC-C, apart from its role as an STa Receptor in mediating diarrheal disease, also serves as a Receptor for an endogenous ligand.
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A gradient in expression of the Escherichia coli heat-stable Enterotoxin Receptor exists along the villus-to-crypt axis of rat small intestine.
Biochemical and biophysical research communications, 1992Co-Authors: Mitchell B. Cohen, Elizabeth A. Mann, Chantal Lau, Susan J. Henning, Ralph A. GiannellaAbstract:Binding of Escherichia coli heat-stable Enterotoxin to its Receptor is critical to the initiation of toxin-induced secretion and diarrheal disease; it is also likely, however, that this Receptor binds an endogenous ligand. In order to characterize the expression of the heat-stable Enterotoxin Receptor in the small intestine, we isolated epithelial cells from villus tip to crypt in rat jejunum and ileum. Binding of radiolabeled toxin was maximal in the villus preparations and gradually decreased along the villus-to-crypt axis, paralleling the decline of sucrase activity. Northern blots of total RNA identified a single heat stable Enterotoxin Receptor transcript (3.8 kb), predominantly in the villus cell fractions. In situ hybridization demonstrated clear signal in the villus cells with no apparent signal in the crypt cells, lamina propria or muscularis. Expression of this Receptor was greatest after enterocytes leave the proliferative cycle and enter villi. This pattern of gene and protein expression may reflect a role of this Receptor in binding endogenous ligands which in turn may regulate intestinal ion flux along the villus-to-crypt axis.
Arie B. Vaandrager - One of the best experts on this subject based on the ideXlab platform.
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Structure and function of the heat-stable Enterotoxin Receptor/guanylyl cyclase C
Molecular and Cellular Biochemistry, 2002Co-Authors: Arie B. VaandragerAbstract:Guanylyl cyclase C (GC-C) was found to function as the principal Receptor for heat-stable Enterotoxins (STa), major causative factors in E. coli -induced secretory diarrhea. GC-C is enriched in intestinal epithelium, but was also detected in other epithelial tissues. The enzyme belongs to the family of Receptor guanylyl cyclases, and consists of an extracellular Receptor domain, a single transmembrane domain, a kinase homology domain, and a catalytic domain. GC-C is modified by N-linked glycosylation and, at least in the small intestine, by proteolysis, resulting in a STa Receptor that is coupled non-covalently to the intracellular domain. So far two endogenous ligands of mammalian GC-C have been identified i.e. the small cysteine-rich peptides guanylin and uroguanylin. The guanylins are released in an auto- or paracrine fashion into the intestinal lumen but may also function as endocrine hormones in gut-kidney communication and as regulators of ion transport in extra-intestinal epithelia. They are thought to activate GC-C by inducing a conformational change in the extracellular portion of the homotrimeric GC-C complex, which allows two of the three intracellular catalytic domains to dimerize and form two active catalytic clefts. In the intestine, activation of GC-C results in a dual action: stimulation of Cl and HCO_3 secretion, through the opening of apical CFTR Cl channels; and inhibition of Na absorption, through blockade of an apical Na/H exchanger. The principal effector of the GC-C effect on ion transport is cGMP dependent protein kinase type II, which together with GC-C and the ion transporters, may form a supramolecular complex at the apical border of epithelial cells.
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structure and function of the heat stable Enterotoxin Receptor guanylyl cyclase c
Molecular and Cellular Biochemistry, 2002Co-Authors: Arie B. VaandragerAbstract:Guanylyl cyclase C (GC-C) was found to function as the principal Receptor for heat-stable Enterotoxins (STa), major causative factors in E. coli-induced secretory diarrhea. GC-C is enriched in intestinal epithelium, but was also detected in other epithelial tissues. The enzyme belongs to the family of Receptor guanylyl cyclases, and consists of an extracellular Receptor domain, a single transmembrane domain, a kinase homology domain, and a catalytic domain. GC-C is modified by N-linked glycosylation and, at least in the small intestine, by proteolysis, resulting in a STa Receptor that is coupled non-covalently to the intracellular domain. So far two endogenous ligands of mammalian GC-C have been identified i.e. the small cysteine-rich peptides guanylin and uroguanylin. The guanylins are released in an auto- or paracrine fashion into the intestinal lumen but may also function as endocrine hormones in gut-kidney communication and as regulators of ion transport in extra-intestinal epithelia. They are thought to activate GC-C by inducing a conformational change in the extracellular portion of the homotrimeric GC-C complex, which allows two of the three intracellular catalytic domains to dimerize and form two active catalytic clefts. In the intestine, activation of GC-C results in a dual action: stimulation of Cl and HCO3 secretion, through the opening of apical CFTR Cl channels; and inhibition of Na absorption, through blockade of an apical Na/H exchanger. The principal effector of the GC-C effect on ion transport is cGMP dependent protein kinase type II, which together with GC-C and the ion transporters, may form a supramolecular complex at the apical border of epithelial cells.
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Heat-stable Enterotoxin Receptor/guanylyl cyclase C is an oligomer consisting of functionally distinct subunits, which are non-covalently linked in the intestine.
The Journal of biological chemistry, 1994Co-Authors: Arie B. Vaandrager, E Van Der Wiel, M L Hom, L H Luthjens, H. R. De JongeAbstract: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.
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heat stable Enterotoxin Receptor guanylyl cyclase c is an oligomer consisting of functionally distinct subunits which are non covalently linked in the intestine
Journal of Biological Chemistry, 1994Co-Authors: Arie B. Vaandrager, E Van Der Wiel, M L Hom, L H Luthjens, H. R. De JongeAbstract: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.
David V. Goeddel - One of the best experts on this subject based on the ideXlab platform.
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Processing and characterization of human proguanylin expressed in Escherichia coli.
The Journal of biological chemistry, 1993Co-Authors: K C Garcia, F. De Sauvage, M Struble, William J. Henzel, D Reilly, David V. GoeddelAbstract:Abstract Guanylin is a 15-amino acid peptide hormone that was originally isolated from the jejunum of the rat small intestine and shown to be an endogenous activator of the intestinal heat-stable Enterotoxin Receptor-guanylyl cyclase. Guanylin is synthesized as a 115-amino acid prohormone, proguanylin, which is processed at a site yet to be determined, into a C-terminal bioactive fragment(s). In order to examine the processing of proguanylin in vitro, we have generated large quantities of the properly folded prohormone by constructing an expression vector that directs its secretion into the periplasmic space of Escherichia coli. The bacterially expressed human proguanylin was then processed to smaller C-terminal fragments by protease digestion. Digestion with trypsin or lysine-C generated C-terminal peptides of different length, which have been purified and characterized. Guanylin-22 and guanylin-32 have binding affinities and biological activities similar to guanylin-15, while guanylin-63 and the entire proguanylin have only minimal bioactivity. Circular dichroism spectroscopy reveals that proguanylin is a stably folded protein containing mostly beta-sheet and beta-turn structure.
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Conservation of the kinaselike regulatory domain is essential for activation of the natriuretic peptide Receptor guanylyl cyclases.
Molecular and cellular biology, 1992Co-Authors: Kerry J. Koller, David G. Lowe, F. De Sauvage, David V. GoeddelAbstract:The natriuretic peptide Receptors, NPR-A and NPR-B, are two members of the newly described class of Receptor guanylyl cyclases. The kinaselike domain of these proteins is an important regulator of the guanylyl cyclase activity. To begin to understand the molecular nature of this type of regulation, we made complete and partial deletions of the kinase domain in NPR-A and NPR-B. We also made chimeric proteins in which the kinase domains of NPR-A and NPR-B were exchanged or replaced with kinase domains from structurally similar proteins. Complete deletion of the kinase homology domain in NPR-A and NPR-B resulted in constitutive activation of the guanylyl cyclase. Various partial deletions of this region produced proteins that had no ability to activate the enzyme with or without hormone stimulation. The kinase homology domain can be exchanged between the two subtypes with no effect on regulation. However, structurally similar kinaselike domains, such as from the epidermal growth factor Receptor or from the heat-stable Enterotoxin Receptor, another member of the Receptor guanylyl cyclase family, were not able to regulate the guanylyl cyclase activity correctly. These findings suggest that the kinaselike domain of NPR-A and NPR-B requires strict sequence conservation to maintain proper regulation of their guanylyl cyclase activity.
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Characterization of the recombinant human Receptor for Escherichia coli heat-stable Enterotoxin.
The Journal of biological chemistry, 1992Co-Authors: F. De Sauvage, Richard Horuk, Gregory L. Bennett, Clifford Quan, John Burnier, David V. GoeddelAbstract:We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable Enterotoxin Receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM for the cloned Receptor expressed in 293 cells and 166 pM for the Receptor present in T84 cells. Kinetic measurements of STa binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2, of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was 67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only Receptor for STa present in T84 cells.
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Conservation oftheKinaselike Regulatory Domain IsEssential forActivation oftheNatriuretic Peptide Receptor Guanylyl Cyclases
1992Co-Authors: Kerry J. Koller, Frederic J. De Sauvage, David G. Lowe, David V. GoeddelAbstract:Thenatriuretic peptide Receptors, NPR-AandNPR-B,aretwomembersofthenewlydescribed class of Receptor guanylyl cyclases. Thekinaselike domainofthese proteins isan important regulator oftheguanylyl cyclase activity. Tobegin tounderstand themolecular nature ofthis typeofregulation, we madecomplete and partial deletions ofthekinase domaininNPR-AandNPR-B.We alsomadechimeric proteins inwhichthe kinase domains ofNPR-AandNPR-Bwere exchanged or replaced withkinase domains fromstructurally similar proteins. Complete deletion ofthekinase homology domaininNPR-AandNPR-Bresulted in constitutive activation oftheguanylyl cyclase. Various partial deletions ofthisregion produced proteins that hadno ability toactivate theenzyme withorwithout hormonestimulation. Thekinase homology domain can beexchanged between thetwosubtypes withno effect on regulation. However, structurally similar kinaselike domains, suchasfromtheepidermal growthfactor Receptor or fromtheheat-stable Enterotoxin Receptor, another memberoftheReceptor guanylyl cyclase family, were notabletoregulate theguanylyl cyclase activity correctly. Thesefindings suggest thatthekinaselike domainofNPR-AandNPR-Brequires strict sequence conservation tomaintain properregulation oftheir guanylyl cyclase activity.