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STAT pathway in response to G-protein-coupled receptor stimulation
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Abstracts from the 11th InternationalSymposium on Regulatory Peptides
SCREENING FOR SIGNAL TRANSDUCTION THROUGH THE JAK/STAT PATHWAY IN RESPONSE TO G-PROTEIN-COUPLED RECEPTOR STIMULATION. P. Hildebrand, A. Pansky, M. Heim, C. Beglinger. Department of Research, University Hospital, CH-4031 Basel, Switzerland. The Jak/STAT (Janus kinase/signal transducers and activators of !ranscription) pathway is a newly discovered signal transduction pathway which directly conveys signals from the cell surface to the nucleus. Various cytokines and growth factors typically activate this pathway; as first seven-transmembrane domain, G protein-coupled receptor type, angiotensin II has recently been shown to induce rapid phosphorylation of Jak tyrosine kinases and subsequent activation of STAT proteins. The aim of the present study was therefore to identify peptides linked to G protein-coupled receptors that additionally activate the Jak/STAT pathway. Methods: After incubation of the cells with ligands, activation of the pathway (Statl to Stat6) was tested by electrophoretic mobility shift assays (STAT dimers + labeled DNA response elements) in nuclear extracts of rat cardiac fibroblasts, human lung fibroblasts, human melanoma cells (A375-6), rat pancreatic acinar tumor cells (AR4-2J), and rat pancreatic acini, respectively. Results: Interferon c~- and v-induced activation of Statl/Stat2 (positive controls) could be shown in cardiac and lung fibroblasts, A375-6 cells, AR4-2J cells, but not in pancreatic acini. IL-6 and EGF activated Stat3 in A375-6 cells, whereas IL-1 and bombesin showed no response despite the expression of functional receptors. Functional G protein-coupled receptors on AR4-2J cells have been demonstrated for bombesin, CCK, gastrin, secretin, VIP, CGRP, insulin, and carbachol; binding of specific ligands to these receptors did not activate the Jak/STAT pathway as tested with DNA response elements detecting Statl to Stat6. In pancreatic acini, STATs were neither detected in cytosolic nor in nuclear extracts, most likely due to the high content of digestive enzymes. Nuclear translocation of Statl (and other Stats) after stimulation of rat cardiac fibroblasts by angiotensin II could not be detected with all response elements tested. Summary: Activation of the Jak/STAT pathway by interferons and other positive controls could be demonstrated in all cell lines tested, which is in agreement with the effects of these ligands in other cell types. Bombesin and other G protein-coupled peptides that act as pancreatic secretagogues did not activate Jak/STAT-mediated DNA binding activity with the available DNA response elements. Angiotensin lI-induced activation of Statl could not be confirmed in this study.
STRUCTURE AND EXPRESSION PATTERN OF THE HUMAN GUANYLYL CYCLASE ACTIVATING PEPTIDE 11GENE (GCAP-II, UROGUANYLIN) O. Hill. A. Cieslak. M. Fuehs. W.G. Forssmann and H.J, Ma2ert Lower Saxony Institute for Peptide Research, 30625 Hannover, Germany Uroguanylin (Kita et al. 1994, Am J PhysioI 266, F342-F348). in its circulating molecular form (GCAP-II, guanylate f.yclaseoictivating 12eptide) is an endogenous ligand for intestinal guanylyl cyclase C receptors. Like guanylin, GCAP-II induces transepithelial CI- secretion by cGMP-mediated activation of the CFTR channel (Hess et at. 1995, FEBS Letters 374, 34-38). This peptide is a C-terminal fragment of a 112amino acid precursor molecule. A high level expression of the GCAP-II gene was detected in the human colon (Hill et at. 1995, Biochim. Biophys. Acta 1253, 146-149). The data so far known suggests that GCAPs (guanylins) are part of a system controlling the epithelial electrolyte balance at least in the intestine. Therefore, the molecular analysis of the GCAP-II gene and the documentation of its expression pattern, is a tool to better understand cellular control mechanisms of this new hormone. Northern blot and RT-PCR analysis revealed that GCAP-II mRNA is present in the stomach, duodenum, jejunum, ileum, cecum, and colon, as well as in lower concentrations in the gall bladder. For further investigations on structure and regulation, we have recently cloned the corresponding gene by a combination of genomic library screening and promoter finder walking. Sequence analysis revealed a structural homology of the GCAP-II gene with the guanylin (GCAP-I) gene. Like the guanylin (GCAP-I) gene (Hill et al. 1995, PNAS 92, 2046-2050) it is approximately 3 kbp in size and consists of three exons. Exon I codes for amino acids 1-30 of human GCAPII, Exon II, for amino acids 31-91 and exon llI for amino acids 92-112. The promoter region of the gene was determined by primer extension analysis. In addition to a weak TATA-box, it contains some potential regulatory elements such as, for example, a CAC-box, a progesterone response element, an AP 4 site and several putative cAMP response elements. The importance of these elements for promoter activity is currently under investigation.
Abstracts from the 11th InternationalSymposium on Regulatory Peptides
SCREENING FOR SIGNAL TRANSDUCTION THROUGH THE JAK/STAT PATHWAY IN RESPONSE TO G-PROTEIN-COUPLED RECEPTOR STIMULATION. P. Hildebrand, A. Pansky, M. Heim, C. Beglinger. Department of Research, University Hospital, CH-4031 Basel, Switzerland. The Jak/STAT (Janus kinase/signal transducers and activators of !ranscription) pathway is a newly discovered signal transduction pathway which directly conveys signals from the cell surface to the nucleus. Various cytokines and growth factors typically activate this pathway; as first seven-transmembrane domain, G protein-coupled receptor type, angiotensin II has recently been shown to induce rapid phosphorylation of Jak tyrosine kinases and subsequent activation of STAT proteins. The aim of the present study was therefore to identify peptides linked to G protein-coupled receptors that additionally activate the Jak/STAT pathway. Methods: After incubation of the cells with ligands, activation of the pathway (Statl to Stat6) was tested by electrophoretic mobility shift assays (STAT dimers + labeled DNA response elements) in nuclear extracts of rat cardiac fibroblasts, human lung fibroblasts, human melanoma cells (A375-6), rat pancreatic acinar tumor cells (AR4-2J), and rat pancreatic acini, respectively. Results: Interferon c~- and v-induced activation of Statl/Stat2 (positive controls) could be shown in cardiac and lung fibroblasts, A375-6 cells, AR4-2J cells, but not in pancreatic acini. IL-6 and EGF activated Stat3 in A375-6 cells, whereas IL-1 and bombesin showed no response despite the expression of functional receptors. Functional G protein-coupled receptors on AR4-2J cells have been demonstrated for bombesin, CCK, gastrin, secretin, VIP, CGRP, insulin, and carbachol; binding of specific ligands to these receptors did not activate the Jak/STAT pathway as tested with DNA response elements detecting Statl to Stat6. In pancreatic acini, STATs were neither detected in cytosolic nor in nuclear extracts, most likely due to the high content of digestive enzymes. Nuclear translocation of Statl (and other Stats) after stimulation of rat cardiac fibroblasts by angiotensin II could not be detected with all response elements tested. Summary: Activation of the Jak/STAT pathway by interferons and other positive controls could be demonstrated in all cell lines tested, which is in agreement with the effects of these ligands in other cell types. Bombesin and other G protein-coupled peptides that act as pancreatic secretagogues did not activate Jak/STAT-mediated DNA binding activity with the available DNA response elements. Angiotensin lI-induced activation of Statl could not be confirmed in this study.
STRUCTURE AND EXPRESSION PATTERN OF THE HUMAN GUANYLYL CYCLASE ACTIVATING PEPTIDE 11GENE (GCAP-II, UROGUANYLIN) O. Hill. A. Cieslak. M. Fuehs. W.G. Forssmann and H.J, Ma2ert Lower Saxony Institute for Peptide Research, 30625 Hannover, Germany Uroguanylin (Kita et al. 1994, Am J PhysioI 266, F342-F348). in its circulating molecular form (GCAP-II, guanylate f.yclaseoictivating 12eptide) is an endogenous ligand for intestinal guanylyl cyclase C receptors. Like guanylin, GCAP-II induces transepithelial CI- secretion by cGMP-mediated activation of the CFTR channel (Hess et at. 1995, FEBS Letters 374, 34-38). This peptide is a C-terminal fragment of a 112amino acid precursor molecule. A high level expression of the GCAP-II gene was detected in the human colon (Hill et at. 1995, Biochim. Biophys. Acta 1253, 146-149). The data so far known suggests that GCAPs (guanylins) are part of a system controlling the epithelial electrolyte balance at least in the intestine. Therefore, the molecular analysis of the GCAP-II gene and the documentation of its expression pattern, is a tool to better understand cellular control mechanisms of this new hormone. Northern blot and RT-PCR analysis revealed that GCAP-II mRNA is present in the stomach, duodenum, jejunum, ileum, cecum, and colon, as well as in lower concentrations in the gall bladder. For further investigations on structure and regulation, we have recently cloned the corresponding gene by a combination of genomic library screening and promoter finder walking. Sequence analysis revealed a structural homology of the GCAP-II gene with the guanylin (GCAP-I) gene. Like the guanylin (GCAP-I) gene (Hill et al. 1995, PNAS 92, 2046-2050) it is approximately 3 kbp in size and consists of three exons. Exon I codes for amino acids 1-30 of human GCAPII, Exon II, for amino acids 31-91 and exon llI for amino acids 92-112. The promoter region of the gene was determined by primer extension analysis. In addition to a weak TATA-box, it contains some potential regulatory elements such as, for example, a CAC-box, a progesterone response element, an AP 4 site and several putative cAMP response elements. The importance of these elements for promoter activity is currently under investigation.