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CCK-8S induced increase in protein phosphorylation in rat glioma C6 cells
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3
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post-transcriptional level. In the case of the cerebellum, only a completely unspliced mRNA form was detected, in agreement with previous studies in which the presence of CCK, ligand binding sites were not observed. In contrast, a truncated CCK,-R mRNA, lacking 250 bp, was detected in all the studied brain regions except for the cerebellum. This mRNA, for which a cellular function has not been assigned, potentially encodes a protein consisting of 168 amino acids. P1/2 NKD: Neuropeptide Receptor in Neurogenesis P. Rosay, J.-F. Colas and L. Maroteaux INSERM-U 184, CNRS-LGME, 11, rue Humann, 67000 Strasbourg, France The Drosophila melanogaster protein NKD shows 38% homology with the mammalian tachykinin NK-3 receptor within the transmembrane domain region. Stable cell lines expressing this protein are responsive to Locusta migratoria tachykinin but not to other peptides of the tachykinin family. Expression of this gene is detected principally in adult tly heads, but also in the adult body and in embryos. Interestingly, NKD mRNA is detected at very early stages of Drosophila development (6 h). Transgenic flies with 1acZ gene under the NKD promoter show late expression in abdominal ganglia of larval brain where SP or FMRFlike immuno-reactive neurones send projections. Surprisingly, early expression can be detected in embryonic peripheral nervous system precursor cells. Deletions of this promoter, tested in transfections or in transgenic flies, demonstrate the the early pattern of expression is under direct control of one E-box, responding to proneural HLH factors. These results show that neuropeptide receptors are transiently expressed during early embryogenesis. This early expression is under direct regulation by proneural genes in the fly. The role of this expression in the nervous system embryogenesis is investigated using various disruption techniques of our gene. P1/3 Manganese Unmasks Somatostatin’s Stimulation of Striatal Cyclic AMP Accumulation A. Moser Department of Neurology, Medical University of Liibeck, Ratzeburger Allee 160, D-23538 Ltibeck, Germany Since in earlier studies the effect of somatostatin was discussed to be due to an interaction with guanine nucleotide regulated subunits, we examine the interaction between somatostatin and manganese ions in a crude membrane preparation of the rat caudate nucleus. Somatostatin over a broad range of concentrations did not modulate basal adenylate cyclase activity in the presence of different gua-
nine nucleotides. Manganese ions also did not modify basal enzyme activity but inhibited cyclic AMP formation at higher concentrations (0.1 mM). In the presence of MnCl, (1 $A), somatostatin (0.1 pM) significantly stimulated adenylate cyclase activity to 130% when compared to control. Met-encephalin (0.1 pM) was able to antagonize the effect of somatostatin and MnCl,. These effects were, however, only seen in the presence of GTP (10 pM) but not after incubation with guanylylimidodiphosphate (Gppo\lH)p). Since manganese was reported to inhibit the inhibitory guanine nucleotide regulated subunit G-i at low concentrations while met-encephalin activates G-i, these results suggest that manganese unmasks the stimulatory effect of somatostatin acting through G-s and G-i. P1/4 Stimulation of cGMP-dependent Protein Kinase (G-kinase) by PeptideJ. P. Huggins, A. J. Ganzhorn, V. Saudek, J. T. Pelton and R. A. Atkinson MMD, 16 rue d' Ankara, F-67080 Strasbourg Cedex, France G-kinase is present in large quantities in cerebellum and may mediate NO-induced synaptic plasticity changes. The structure of G-kinase Ia-(546576)peptide amide (peptide-546) and its effects on enzyme activity have been studied. The portion of CAMP-dependent protein kinase (A-kinase) analogous to peptideforms part of its peptide substrate binding site. Peptideis a potent Gkinase activator, increasing the turn-over number, stimulating additively with cGMP, and causing G-kinase to enter a superactive state, but not changing the affinity of the enzyme for substrates or cGMP. Helical structure in peptideis observed in 2,2,2&fluoroethanol at concentrations above 25%. The structure is lost only gradually on raising the temperature to 80°C with no clear melting transition. ‘H NMR studies have identified a helical segment from Met’* to Argz6. The N-terminal residues are less structured but show some helical character. Distance geometry and molecular dynamics simulations revealed a helical structure in the C-terminal region preceded by a less well defined proline-rich segment and Nterminal region. Hence, peptidemay adopt a conformation similar to that expected by analogy with Akinase, and we propose that residues 546-576 of G-kinase are involved in enzyme autoinhibition. PlB CCK-8s Induced Increase in Protein Phosphorylation in Rat Glioma C, Cells T. Sch&eberg*, R. Kaulinann*, K. Buchnert and T. Ott* *Institute of Pharmacology and Taxicology, Medical Faculty (Char&), Humboldt University, Berlin and tlnstitute of Biochemistry, Free University, Berlin, Germany
4 While signal transduction mechanisms on CCK, receptor have been comprehensively characterized, little is known about transmembranal signaling associated with CCK, receptor. Recently, we demonstrated high affinity CCK,-type binding sites on rat glioma C, cells whose activation resulted in an increase in [CaZ+li.l Here, we describe CCK-8S induced increase in protein phosphorylation in C, cells. Cells radiolabeled with 32PO:were stimulated with CCK-8s and proteins were separated by SDS-PAGE and visualized by autoradiography. At least a M, = 80000-87000 protein band was enhanced phosphorylated, when cells were treated with CCK-8S (IO-’ M-10-@ M] for 3 min. Further investigations are in progress to clarify the involvement of protein kinases in CCK, receptor activation. 1. Kaufrnann, R., Lindschau, C., Schiineberg, T. et al. Brain Res. 1994 (in press).
P1/6 CC&-type receptors in guinea-pig cortex and continuous cell lines. Comparative [3H]CCK-8S binding studies R. Kaufmann*, T. Schiineberg*, P. Henklein*, M. Boomgaarden *, D. Sohrt -&d T. Ott*
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*Institute of Pharmacology and Toxicology, Medical Faculty (Char&), Humboldt University of Berlin and tInstitute of Biometry and Information Processing, Veterinary Medical Faculty, Free University, Berlin, Germany The question of whether CCK,-type receptors which are localized in different tissues are of the same molecular structure and display the same pharmacological properties seems very interesting but is unresolved so far. We compared CCK, receptor binding of 11 CCK receptor ligands including highly selective CCK,-type and CCK,type agonists (BC 264, C-terminal tetrapeptide Sue-Trp-N(Me)Nle-Asp-Phe-NH,, A 7 1623 and antagonists (L-365,260, PD 135 158, L-364,7 18) on guinea-pig cortex, Jurkat T-cells, rat pituitary GH3 cells and rat glioma C, cells. The rank order of potency of the ligands in inhibiting specific [3H]CCK-8S binding was very similar on CCK,-type receptors in cortex and cell lines. The high correlation of ligand binding profiles of CCK, receptors demonstrated by a computer-assisted statistical analysis suggests that CCK, sites guinea-pig cortex, Jurkat cells, GH3 cells and C, cells share same pharmacological properties. However, a remarkable difference in binding affinity both for CCK-8S and other ligands in Jurkat cells compared with that in the other CCK receptor models was noted. Therefore, differences in the receptor structure may not be excluded. Further investigations should include analyses of receptor signaling andmolecular structure,
APRIL
3
SUPPLEMENT
post-transcriptional level. In the case of the cerebellum, only a completely unspliced mRNA form was detected, in agreement with previous studies in which the presence of CCK, ligand binding sites were not observed. In contrast, a truncated CCK,-R mRNA, lacking 250 bp, was detected in all the studied brain regions except for the cerebellum. This mRNA, for which a cellular function has not been assigned, potentially encodes a protein consisting of 168 amino acids. P1/2 NKD: Neuropeptide Receptor in Neurogenesis P. Rosay, J.-F. Colas and L. Maroteaux INSERM-U 184, CNRS-LGME, 11, rue Humann, 67000 Strasbourg, France The Drosophila melanogaster protein NKD shows 38% homology with the mammalian tachykinin NK-3 receptor within the transmembrane domain region. Stable cell lines expressing this protein are responsive to Locusta migratoria tachykinin but not to other peptides of the tachykinin family. Expression of this gene is detected principally in adult tly heads, but also in the adult body and in embryos. Interestingly, NKD mRNA is detected at very early stages of Drosophila development (6 h). Transgenic flies with 1acZ gene under the NKD promoter show late expression in abdominal ganglia of larval brain where SP or FMRFlike immuno-reactive neurones send projections. Surprisingly, early expression can be detected in embryonic peripheral nervous system precursor cells. Deletions of this promoter, tested in transfections or in transgenic flies, demonstrate the the early pattern of expression is under direct control of one E-box, responding to proneural HLH factors. These results show that neuropeptide receptors are transiently expressed during early embryogenesis. This early expression is under direct regulation by proneural genes in the fly. The role of this expression in the nervous system embryogenesis is investigated using various disruption techniques of our gene. P1/3 Manganese Unmasks Somatostatin’s Stimulation of Striatal Cyclic AMP Accumulation A. Moser Department of Neurology, Medical University of Liibeck, Ratzeburger Allee 160, D-23538 Ltibeck, Germany Since in earlier studies the effect of somatostatin was discussed to be due to an interaction with guanine nucleotide regulated subunits, we examine the interaction between somatostatin and manganese ions in a crude membrane preparation of the rat caudate nucleus. Somatostatin over a broad range of concentrations did not modulate basal adenylate cyclase activity in the presence of different gua-
nine nucleotides. Manganese ions also did not modify basal enzyme activity but inhibited cyclic AMP formation at higher concentrations (0.1 mM). In the presence of MnCl, (1 $A), somatostatin (0.1 pM) significantly stimulated adenylate cyclase activity to 130% when compared to control. Met-encephalin (0.1 pM) was able to antagonize the effect of somatostatin and MnCl,. These effects were, however, only seen in the presence of GTP (10 pM) but not after incubation with guanylylimidodiphosphate (Gppo\lH)p). Since manganese was reported to inhibit the inhibitory guanine nucleotide regulated subunit G-i at low concentrations while met-encephalin activates G-i, these results suggest that manganese unmasks the stimulatory effect of somatostatin acting through G-s and G-i. P1/4 Stimulation of cGMP-dependent Protein Kinase (G-kinase) by PeptideJ. P. Huggins, A. J. Ganzhorn, V. Saudek, J. T. Pelton and R. A. Atkinson MMD, 16 rue d' Ankara, F-67080 Strasbourg Cedex, France G-kinase is present in large quantities in cerebellum and may mediate NO-induced synaptic plasticity changes. The structure of G-kinase Ia-(546576)peptide amide (peptide-546) and its effects on enzyme activity have been studied. The portion of CAMP-dependent protein kinase (A-kinase) analogous to peptideforms part of its peptide substrate binding site. Peptideis a potent Gkinase activator, increasing the turn-over number, stimulating additively with cGMP, and causing G-kinase to enter a superactive state, but not changing the affinity of the enzyme for substrates or cGMP. Helical structure in peptideis observed in 2,2,2&fluoroethanol at concentrations above 25%. The structure is lost only gradually on raising the temperature to 80°C with no clear melting transition. ‘H NMR studies have identified a helical segment from Met’* to Argz6. The N-terminal residues are less structured but show some helical character. Distance geometry and molecular dynamics simulations revealed a helical structure in the C-terminal region preceded by a less well defined proline-rich segment and Nterminal region. Hence, peptidemay adopt a conformation similar to that expected by analogy with Akinase, and we propose that residues 546-576 of G-kinase are involved in enzyme autoinhibition. PlB CCK-8s Induced Increase in Protein Phosphorylation in Rat Glioma C, Cells T. Sch&eberg*, R. Kaulinann*, K. Buchnert and T. Ott* *Institute of Pharmacology and Taxicology, Medical Faculty (Char&), Humboldt University, Berlin and tlnstitute of Biochemistry, Free University, Berlin, Germany
4 While signal transduction mechanisms on CCK, receptor have been comprehensively characterized, little is known about transmembranal signaling associated with CCK, receptor. Recently, we demonstrated high affinity CCK,-type binding sites on rat glioma C, cells whose activation resulted in an increase in [CaZ+li.l Here, we describe CCK-8S induced increase in protein phosphorylation in C, cells. Cells radiolabeled with 32PO:were stimulated with CCK-8s and proteins were separated by SDS-PAGE and visualized by autoradiography. At least a M, = 80000-87000 protein band was enhanced phosphorylated, when cells were treated with CCK-8S (IO-’ M-10-@ M] for 3 min. Further investigations are in progress to clarify the involvement of protein kinases in CCK, receptor activation. 1. Kaufrnann, R., Lindschau, C., Schiineberg, T. et al. Brain Res. 1994 (in press).
P1/6 CC&-type receptors in guinea-pig cortex and continuous cell lines. Comparative [3H]CCK-8S binding studies R. Kaufmann*, T. Schiineberg*, P. Henklein*, M. Boomgaarden *, D. Sohrt -&d T. Ott*
NEUROPEPTIDES
*Institute of Pharmacology and Toxicology, Medical Faculty (Char&), Humboldt University of Berlin and tInstitute of Biometry and Information Processing, Veterinary Medical Faculty, Free University, Berlin, Germany The question of whether CCK,-type receptors which are localized in different tissues are of the same molecular structure and display the same pharmacological properties seems very interesting but is unresolved so far. We compared CCK, receptor binding of 11 CCK receptor ligands including highly selective CCK,-type and CCK,type agonists (BC 264, C-terminal tetrapeptide Sue-Trp-N(Me)Nle-Asp-Phe-NH,, A 7 1623 and antagonists (L-365,260, PD 135 158, L-364,7 18) on guinea-pig cortex, Jurkat T-cells, rat pituitary GH3 cells and rat glioma C, cells. The rank order of potency of the ligands in inhibiting specific [3H]CCK-8S binding was very similar on CCK,-type receptors in cortex and cell lines. The high correlation of ligand binding profiles of CCK, receptors demonstrated by a computer-assisted statistical analysis suggests that CCK, sites guinea-pig cortex, Jurkat cells, GH3 cells and C, cells share same pharmacological properties. However, a remarkable difference in binding affinity both for CCK-8S and other ligands in Jurkat cells compared with that in the other CCK receptor models was noted. Therefore, differences in the receptor structure may not be excluded. Further investigations should include analyses of receptor signaling andmolecular structure,