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A new cellular model to analyze receptor mediated endocytosis
Biology of the Cell 91 (1999) 535-565
564
HIV-1 LTR ACTlVAMN BY HEAT-SHOCK : A NEW MECHANISM OF ACTNATION OF NF-I& TRANSCRlPTlON FACTOR
ROTUNDRACGAP AND RHO FAMILY SffiNALLING IN DROSOPHILA DEVELOPMENT
KRETZREMY Carole, TANGUAY Robert M. and ARRIGO AndrePatrick
CNRSCEA.
Centre de G&nque Mol&ulaire, Unwerst~ Lyon I, 43 boulevard 1918,69622 Vlleurbanne cede& France
du 1I Novembre
NF-KB is an inducible transcription factor that upregulate the expression of numerous genes such as the human immunodeficiency virus type-l (HIV-l) genes but also cellular genes involved in the immune and inflammatory responses. We report here that heat-shock activates the HIV-l LTR, an activation which is impaired when KB sites present in the LTR were mutated or deleted. Heat-shock also stimulated the transcription of a r&controlled reporter gene. We therefore analyzed the effect of heat-shock on NF-xB transcription factor and observed that the two NF-KB subunits (~65 and ~50) redistributed into the nucleus during heat-shock recovery. However, contrasting with other NF-KB inducers, heat-shock did not trigger NF-KB activation through degradation of its inhibitory subunits (i.e. 1+&3-a, kEf3 and p105), suggesting a new mechanism of activation. SIJMO-1 is a ubiquitin-like protein whose conjugation to its protein substrates can modulate their subcellular local&.&ion. Since NF- KB modification by SUMO-1 has been documented, we tested whether the migration of NF-KB into the nucleus correlated with some modifications of SUMO-1 conjugation to this transcription factor, but the result was negative. At last, since reactive oxygen species seem to be involved in NF-KB activation, we analyzed the involvement of intracellular redox on NF-KB activation by heat-shock. Antioxidant conditions inhibited HIV-1 LTR and &dependent reporter cat construct activation by heat-shock. Of interest, the redistribution of NI-KB into the nucleus after heat-treatment was not inhibited by these conditions. Hence, the transactivation ability or the DNA binding activity of NF-KB appears likely to depend on an intracellular pro-oxidant state induced by heat-shock. Taken together these results suggest that NF-KB activation by heat-shock is under the control of a new mechanism which is dependent on a pro-oxidant state but SIJMO-1 independent.
RAYMOND K. *, GRIFFIN-SHEAR., UMR314.
and FA4NARQUEMARRAS
DBMS, Grenoble,
M.O.
France
Embryogenesis and the development of irnaginal structures in Drosophila are excellent systems to discover and study the function of proteins involved in cellular signalllng. For instance, mutations in genes coding the kinases of the Jun amino-terminal kinases (JNKs) pathway produce embryos with a dorsal hole due to defective dorsal closure, a morphogenetic process which depends on cell deformation and cytoskeletal rearrangements (reviewed in Noselli S. (1998), Trends Genet, 14,33-38). Dominant negative forms of Drac or Dcdc42 reproduce similar defects in dorsal closure when expressed in embryos, supporting the idea that this family of small GTPases regulates the JNKs cascade (Harden, N., Rlcos, M., Ong, Y. M., Chia, W., Lim, L. (1999). J Cell Sci, 112, 273-284). The GTPase Activating Proteins (GAP) catalyze the passage from the active form RX-GTP to the inactive form RX-GDP and therefore negatively regulate RX signalling. The strength and duration of RX signalling is critical for cellular response to extracellular signals and we have examined Rac/Cdc42 regulation in Drosophila by the putative GTPase-activating protein: RotundRacGAP. This protein shows molecular similarities with the RacGAP protein family and is homologous to the human protein MgcRAcGAP expressed in the testes (see abstract, Griffin-Shea et al). We first demonstrated that RotundRacGAP directly interacts with Dcdc42 and, to a lesser extent, with Dracl in yeast. Then, we targeted the expression of RotundRacGAP in Drosophila embryos with or without mutant forms of Dracl or Dcdc42. The defects in dorsal closure resulting from the various genetic combinations tested will be presented and analyzed.
CHARACTERIZATION OF A GTP-INSENSITNE VIP/PHI HIGH AFFlNlTY RECEPTOR
A NEW CELLULAR MODEL TO ANALYZE RECEPTOR MEDIATED ENDOCYTOSIS
PINEAU Hcolas, LELIEVRE Vwent, Thlerry and MULLER Jean-Marc
DE CHASSEY Bemt and LETOURNEUR Francots
GOURSAUD Stephanie,
WASCHEK JamwA,
JANET
Laboratoire de Biologic des lnteracbons Cellolarres CNRS UMR 6558, 40 Av. Recteur Pineau 86 022 Poitiers, France and Department of Psychiatriy, Mental Retardahon Research Center, UCLA, Neuropsychratnc Institute, Los Angeles, CA 90024, USA The 28 amino-acid Vasoactive Intestinal Peptide (VIP) modulates a multitude of biological functions by interacting with receptor sites localized in tissues throughout the body. Besides it biological role as a neuromoduliator, VIP has also been demonstrated to regulate cell growth and differentiation in cell types from tissues as diverse as intestine, lung, lymphocytes, brain or sympathic ganglias (Muller JM et al., (1995) Mol. Neurobiol., 10:115-133). In particular, Hill and coworkers reported that VIP triggers an accelerated cell growth in whole explanted mice embryos at E9.5. During thii period, blockade of VIP effects by an antagonist results in a severe microcephaly of the newborn. Furthermore, based on the ability of guanine nucleotide to inhibit the binding of VIP to its receptor, they demonstrated that 73 % of the VIP binding was GTPinsensitive, suggesting two subtypes of the VIP receptors or different functional states of a single subtype (Gressens I’. et al., (1993) Nature 362:155-158). One main research axis developed in our group concerns the cell signalling pathways and the growth and differentiation events related to VIP and PACAP receptors. We analyzed the effects of VIP, and related peptides PACAP and PHI, on proliferation in a mouse neuroblastoma cell line Neuro2A. We reported that PHI and VIP inhibited proliferation at concentrations as low as 10.‘” M and lo“’ M. In contrast, PACAP action was biphasic (stimulation at subnanomolar doses and inhibition at higher doses). The data obtained indicated that the PHI inhibitory and PACAP stimulatory activities were mediated by a MAP kinase pathway and independent of protein kinase A or C (Lelievre V. et al., (1998) J, Biol. Chem. 273(31):19685-19690). Taken together, the data could suggest the existence of a novel VIP receptor. This led us to characterize this potential receptor using ‘?-VIP and ‘*‘I-PHI binding and cross-linking experiments. Two VIP receptors of 48 and 60 kDa were identified. Both interacted vith VIP with high affinity but only the 48 kDa behave like a high affinity binding site for PHI. Furthermore, the 48 kDa receptor also correspond to a GTP-insensitive species. Considering the property of PHI to discriminate a GTP-insensitive VIP receptor component, we undertook to screen a cDNA library for clones that were able to induce a ‘?PHI binding capacity in COS-7 cells.
Lyon, 27-29 October 1999
IBCP, UPR 412 CNRS 7 passage du Vercors, 69367
Lyon cedex 07 FRANCE
Our group is interested in sorting events taking place within the endocytic compartments. To address these questions, we are making use of the model organism, the amoebae Dictyostelium discoideum While fluid phase endocytosis is well described in these eukaryotic cells, receptor mediated endocytosis has never been reported. Our work demonstrates for the first time that sorting signals based on tyrosine and dileucine residues are recognized by the cellular machinery of Dictyostefium discoideum. Chimeric proteins made of contact site A fused to the tranamembrane domain of P29F8 and different cytoplasmic sorting signals show endocytosis rates comparable to that observed in mammalian cells. In addition, preliminary results suggest, as expected, that clathrin heavy chain is essential in this process. In order to identify new components of the endocytic pathway, we are also currently screening sorting deficient mutants obtained by restriction enzyme mediated plasrnid integration (REMI). The development of this new model for studying receptor mediated endocytosis should give us a unique way to genetically assess this molecular mechanism.
CongressSBCF
564
HIV-1 LTR ACTlVAMN BY HEAT-SHOCK : A NEW MECHANISM OF ACTNATION OF NF-I& TRANSCRlPTlON FACTOR
ROTUNDRACGAP AND RHO FAMILY SffiNALLING IN DROSOPHILA DEVELOPMENT
KRETZREMY Carole, TANGUAY Robert M. and ARRIGO AndrePatrick
CNRSCEA.
Centre de G&nque Mol&ulaire, Unwerst~ Lyon I, 43 boulevard 1918,69622 Vlleurbanne cede& France
du 1I Novembre
NF-KB is an inducible transcription factor that upregulate the expression of numerous genes such as the human immunodeficiency virus type-l (HIV-l) genes but also cellular genes involved in the immune and inflammatory responses. We report here that heat-shock activates the HIV-l LTR, an activation which is impaired when KB sites present in the LTR were mutated or deleted. Heat-shock also stimulated the transcription of a r&controlled reporter gene. We therefore analyzed the effect of heat-shock on NF-xB transcription factor and observed that the two NF-KB subunits (~65 and ~50) redistributed into the nucleus during heat-shock recovery. However, contrasting with other NF-KB inducers, heat-shock did not trigger NF-KB activation through degradation of its inhibitory subunits (i.e. 1+&3-a, kEf3 and p105), suggesting a new mechanism of activation. SIJMO-1 is a ubiquitin-like protein whose conjugation to its protein substrates can modulate their subcellular local&.&ion. Since NF- KB modification by SUMO-1 has been documented, we tested whether the migration of NF-KB into the nucleus correlated with some modifications of SUMO-1 conjugation to this transcription factor, but the result was negative. At last, since reactive oxygen species seem to be involved in NF-KB activation, we analyzed the involvement of intracellular redox on NF-KB activation by heat-shock. Antioxidant conditions inhibited HIV-1 LTR and &dependent reporter cat construct activation by heat-shock. Of interest, the redistribution of NI-KB into the nucleus after heat-treatment was not inhibited by these conditions. Hence, the transactivation ability or the DNA binding activity of NF-KB appears likely to depend on an intracellular pro-oxidant state induced by heat-shock. Taken together these results suggest that NF-KB activation by heat-shock is under the control of a new mechanism which is dependent on a pro-oxidant state but SIJMO-1 independent.
RAYMOND K. *, GRIFFIN-SHEAR., UMR314.
and FA4NARQUEMARRAS
DBMS, Grenoble,
M.O.
France
Embryogenesis and the development of irnaginal structures in Drosophila are excellent systems to discover and study the function of proteins involved in cellular signalllng. For instance, mutations in genes coding the kinases of the Jun amino-terminal kinases (JNKs) pathway produce embryos with a dorsal hole due to defective dorsal closure, a morphogenetic process which depends on cell deformation and cytoskeletal rearrangements (reviewed in Noselli S. (1998), Trends Genet, 14,33-38). Dominant negative forms of Drac or Dcdc42 reproduce similar defects in dorsal closure when expressed in embryos, supporting the idea that this family of small GTPases regulates the JNKs cascade (Harden, N., Rlcos, M., Ong, Y. M., Chia, W., Lim, L. (1999). J Cell Sci, 112, 273-284). The GTPase Activating Proteins (GAP) catalyze the passage from the active form RX-GTP to the inactive form RX-GDP and therefore negatively regulate RX signalling. The strength and duration of RX signalling is critical for cellular response to extracellular signals and we have examined Rac/Cdc42 regulation in Drosophila by the putative GTPase-activating protein: RotundRacGAP. This protein shows molecular similarities with the RacGAP protein family and is homologous to the human protein MgcRAcGAP expressed in the testes (see abstract, Griffin-Shea et al). We first demonstrated that RotundRacGAP directly interacts with Dcdc42 and, to a lesser extent, with Dracl in yeast. Then, we targeted the expression of RotundRacGAP in Drosophila embryos with or without mutant forms of Dracl or Dcdc42. The defects in dorsal closure resulting from the various genetic combinations tested will be presented and analyzed.
CHARACTERIZATION OF A GTP-INSENSITNE VIP/PHI HIGH AFFlNlTY RECEPTOR
A NEW CELLULAR MODEL TO ANALYZE RECEPTOR MEDIATED ENDOCYTOSIS
PINEAU Hcolas, LELIEVRE Vwent, Thlerry and MULLER Jean-Marc
DE CHASSEY Bemt and LETOURNEUR Francots
GOURSAUD Stephanie,
WASCHEK JamwA,
JANET
Laboratoire de Biologic des lnteracbons Cellolarres CNRS UMR 6558, 40 Av. Recteur Pineau 86 022 Poitiers, France and Department of Psychiatriy, Mental Retardahon Research Center, UCLA, Neuropsychratnc Institute, Los Angeles, CA 90024, USA The 28 amino-acid Vasoactive Intestinal Peptide (VIP) modulates a multitude of biological functions by interacting with receptor sites localized in tissues throughout the body. Besides it biological role as a neuromoduliator, VIP has also been demonstrated to regulate cell growth and differentiation in cell types from tissues as diverse as intestine, lung, lymphocytes, brain or sympathic ganglias (Muller JM et al., (1995) Mol. Neurobiol., 10:115-133). In particular, Hill and coworkers reported that VIP triggers an accelerated cell growth in whole explanted mice embryos at E9.5. During thii period, blockade of VIP effects by an antagonist results in a severe microcephaly of the newborn. Furthermore, based on the ability of guanine nucleotide to inhibit the binding of VIP to its receptor, they demonstrated that 73 % of the VIP binding was GTPinsensitive, suggesting two subtypes of the VIP receptors or different functional states of a single subtype (Gressens I’. et al., (1993) Nature 362:155-158). One main research axis developed in our group concerns the cell signalling pathways and the growth and differentiation events related to VIP and PACAP receptors. We analyzed the effects of VIP, and related peptides PACAP and PHI, on proliferation in a mouse neuroblastoma cell line Neuro2A. We reported that PHI and VIP inhibited proliferation at concentrations as low as 10.‘” M and lo“’ M. In contrast, PACAP action was biphasic (stimulation at subnanomolar doses and inhibition at higher doses). The data obtained indicated that the PHI inhibitory and PACAP stimulatory activities were mediated by a MAP kinase pathway and independent of protein kinase A or C (Lelievre V. et al., (1998) J, Biol. Chem. 273(31):19685-19690). Taken together, the data could suggest the existence of a novel VIP receptor. This led us to characterize this potential receptor using ‘?-VIP and ‘*‘I-PHI binding and cross-linking experiments. Two VIP receptors of 48 and 60 kDa were identified. Both interacted vith VIP with high affinity but only the 48 kDa behave like a high affinity binding site for PHI. Furthermore, the 48 kDa receptor also correspond to a GTP-insensitive species. Considering the property of PHI to discriminate a GTP-insensitive VIP receptor component, we undertook to screen a cDNA library for clones that were able to induce a ‘?PHI binding capacity in COS-7 cells.
Lyon, 27-29 October 1999
IBCP, UPR 412 CNRS 7 passage du Vercors, 69367
Lyon cedex 07 FRANCE
Our group is interested in sorting events taking place within the endocytic compartments. To address these questions, we are making use of the model organism, the amoebae Dictyostelium discoideum While fluid phase endocytosis is well described in these eukaryotic cells, receptor mediated endocytosis has never been reported. Our work demonstrates for the first time that sorting signals based on tyrosine and dileucine residues are recognized by the cellular machinery of Dictyostefium discoideum. Chimeric proteins made of contact site A fused to the tranamembrane domain of P29F8 and different cytoplasmic sorting signals show endocytosis rates comparable to that observed in mammalian cells. In addition, preliminary results suggest, as expected, that clathrin heavy chain is essential in this process. In order to identify new components of the endocytic pathway, we are also currently screening sorting deficient mutants obtained by restriction enzyme mediated plasrnid integration (REMI). The development of this new model for studying receptor mediated endocytosis should give us a unique way to genetically assess this molecular mechanism.
CongressSBCF