Locally controlled inhibitory mechanisms are involved in eukaryotic GPCR-mediated chemosensing

Gprotein–coupled receptor (GPCR) signaling mediates a balance of excitatory and inhibitory activities that regulate Dictyostelium chemosensing to cAMP. The molecular nature and kinetics of these inhibitors are unknown. We report that transient cAMP stimulations induce PIP3 responses without a refractory period, suggesting that GPCR-mediated inhibition accumulates and decays slowly. Moreover, exposure to cAMP gradients leads to asymmetric distribution of the inhibitory components. The gradients induce a stable accumulation of the PIP3 reporter PHCrac-GFP in the front of cells near the cAMP source. Rapid withdrawal of the gradient led to the reassociation of G protein subunits, and the return of the PIP3 phosphatase PTEN and PHCrac-GFP to their pre-stimulus distribution. Reapplication of cAMP stimulation produces a clear PHCrac-GFP translocation to the back but not to the front, indicating that a stronger inhibition is maintained in the front of a polarized cell. Our study demonstrates a novel spatiotemporal feature of currently unknown inhibitory mechanisms acting locally on the PI3K activation pathway.

[1]  Pablo A. Iglesias,et al.  Modeling the Cell's Guidance System , 2002, Science's STKE.

[2]  Peter N. Devreotes,et al.  G protein-linked signaling pathways control the developmental program of dictyostelium , 1994, Neuron.

[3]  P. Devreotes,et al.  Eukaryotic Chemotaxis: Distinctions between Directional Sensing and Polarization* , 2003, Journal of Biological Chemistry.

[4]  J. Downward,et al.  Identification of the Ras GTPase-activating protein GAP1m as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo , 1999, Current Biology.

[5]  Hui Ma,et al.  Chemoattractant‐mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium , 1999, The EMBO journal.

[6]  P. Iglesias,et al.  Chemoattractant-induced phosphatidylinositol 3,4,5-trisphosphate accumulation is spatially amplified and adapts, independent of the actin cytoskeleton , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[7]  C. Parent,et al.  The PI3K-Mediated Activation of CRAC Independently Regulates Adenylyl Cyclase Activation and Chemotaxis , 2005, Current Biology.

[8]  J. Segall,et al.  The great escape: when cancer cells hijack the genes for chemotaxis and motility. , 2005, Annual review of cell and developmental biology.

[9]  Joachim Goedhart,et al.  Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches , 2004, Journal of Cell Science.

[10]  Pascale G. Charest,et al.  Feedback signaling controls leading-edge formation during chemotaxis. , 2006, Current opinion in genetics & development.

[11]  Dianqing Wu,et al.  Roles of PLC-β2 and -β3 and PI3Kγ in Chemoattractant-Mediated Signal Transduction , 2000 .

[12]  P. Devreotes,et al.  Cyclic 3',5' AMP relay in Dictyostelium discoideum. II. Requirements for the initiation and termination of the response , 1979, The Journal of cell biology.

[13]  G. Gerisch Chemotaxis in Dictyostelium. , 1982, Annual review of physiology.

[14]  T. Meyer,et al.  A local coupling model and compass parameter for eukaryotic chemotaxis. , 2005, Developmental cell.

[15]  J W Sedat,et al.  Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. , 2000, Science.

[16]  S. Zigmond Chemotaxis by polymorphonuclear leukocytes , 1978, The Journal of cell biology.

[17]  Rong Zeng,et al.  Regulation of PTEN by Rho small GTPases , 2005, Nature Cell Biology.

[18]  P. V. van Haastert,et al.  A diffusion-translocation model for gradient sensing by chemotactic cells. , 2001, Biophysical journal.

[19]  T. Pollard,et al.  Cellular Motility Driven by Assembly and Disassembly of Actin Filaments , 2003, Cell.

[20]  P. Devreotes,et al.  Cyclic 3', 5'-AMP relay in dictyostelium discoideum. IV. Recovery of the cAMP signaling response after adaptation to cAMP , 1980, The Journal of cell biology.

[21]  Richard A. Firtel,et al.  Role of Phosphatidylinositol 3′ Kinase and a Downstream Pleckstrin Homology Domain–Containing Protein in Controlling Chemotaxis inDictyostelium , 2001, The Journal of cell biology.

[22]  R. Firtel,et al.  Signaling pathways controlling cell polarity and chemotaxis. , 2001, Trends in biochemical sciences.

[23]  P. Devreotes,et al.  Receptor-Mediated Activation of Heterotrimeric G-Proteins in Living Cells , 2001, Science.

[24]  H. Meinhardt Orientation of chemotactic cells and growth cones: models and mechanisms. , 1999, Journal of cell science.

[25]  P. Devreotes,et al.  Temporal and spatial regulation of chemotaxis. , 2002, Developmental cell.

[26]  C. Parent,et al.  A cell's sense of direction. , 1999, Science.

[27]  R. Lefkowitz,et al.  Keeping G Proteins at Bay: A Complex Between G Protein-Coupled Receptor Kinase 2 and Gßγ , 2003, Science.

[28]  P. Hawkins,et al.  Roles of PI3Ks in leukocyte chemotaxis and phagocytosis. , 2002, Current opinion in cell biology.

[29]  P. V. van Haastert,et al.  Chemotactic antagonists of cAMP inhibit Dictyostelium phospholipase C. , 1993, Journal of cell science.

[30]  C. Parent,et al.  A G alpha-dependent pathway that antagonizes multiple chemoattractant responses that regulate directional cell movement. , 2004, Genes & development.

[31]  Jingsong Xu,et al.  Divergent Signals and Cytoskeletal Assemblies Regulate Self-Organizing Polarity in Neutrophils , 2003, Cell.

[32]  P. Murphy The molecular biology of leukocyte chemoattractant receptors. , 1994, Annual review of immunology.

[33]  Thomas D Pollard,et al.  Cellular Motility Driven by Assembly and Disassembly of Actin Filaments , 2003, Cell.

[34]  Carole A. Parent,et al.  PI 3-Kinases and PTEN How Opposites Chemoattract , 2002, Cell.

[35]  T. Yanagida,et al.  Single-Molecule Analysis of Chemotactic Signaling in Dictyostelium Cells , 2001, Science.

[36]  Z. Li,et al.  Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction. , 2000, Science.

[37]  C. Parent,et al.  Localization of the G Protein βγ Complex in Living Cells During Chemotaxis , 2000 .

[38]  P. Devreotes,et al.  Chemotaxis in eukaryotic cells: a focus on leukocytes and Dictyostelium. , 1988, Annual review of cell biology.

[39]  Zigmond Sh Chemotaxis by polymorphonuclear leukocytes. , 1978 .

[40]  M. Meier-Schellersheim,et al.  Quantitative imaging of single live cells reveals spatiotemporal dynamics of multistep signaling events of chemoattractant gradient sensing in Dictyostelium. , 2004, Molecular biology of the cell.

[41]  P. Devreotes,et al.  Tumor Suppressor PTEN Mediates Sensing of Chemoattractant Gradients , 2002, Cell.

[42]  P. Devreotes,et al.  Chemotaxis: signalling the way forward , 2004, Nature Reviews Molecular Cell Biology.

[43]  Tian Jin,et al.  Key Role of Local Regulation in Chemosensing Revealed by a New Molecular Interaction-Based Modeling Method , 2006, PLoS Comput. Biol..

[44]  Toshio Yanagida,et al.  Single Molecule Analysis of Chemotactic Signaling in Dictyostelium Cells , 2001 .

[45]  Richard A. Firtel,et al.  Spatial and Temporal Regulation of 3-Phosphoinositides by PI 3-Kinase and PTEN Mediates Chemotaxis , 2002, Cell.

[46]  William F. Loomis,et al.  RasC Plays a Role in Transduction of Temporal Gradient Information in the Cyclic-AMP Wave of Dictyostelium discoideum , 2004, Eukaryotic Cell.

[47]  W. Rappel,et al.  Directional sensing in eukaryotic chemotaxis: a balanced inactivation model. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[48]  C. Parent,et al.  Localization of the G protein betagamma complex in living cells during chemotaxis. , 2000, Science.

[49]  Richard A. Firtel,et al.  Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement , 2004, The Journal of cell biology.

[50]  R. Firtel,et al.  Control of cell polarity and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa. , 2001, Molecular cell.

[51]  A. Levchenko,et al.  Models of eukaryotic gradient sensing: application to chemotaxis of amoebae and neutrophils. , 2001, Biophysical journal.

[52]  D. Murphy,et al.  G Protein Signaling Events Are Activated at the Leading Edge of Chemotactic Cells , 1998, Cell.

[53]  P. Devreotes,et al.  Cyclic 3', 5'-AMP relay dictyostelium discoideum. V. Adaptation of the cAMP signaling response during cAMP stimulation , 1980, The Journal of cell biology.