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Vol. 10, Issue 6, 1851-1857, June 1999
12/13 Subunits in Neuronal Cells: Induction of Neurite
Retraction
and
*The Netherlands Cancer Institute, Division of Cellular
Biochemistry, 1066 CX Amsterdam, The Netherlands; and
Neuronal cells undergo rapid growth cone collapse, neurite
retraction, and cell rounding in response to certain G protein-coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been
hampered by the lack of a suitable assay. Furthermore, the nature of
the G protein(s) mediating LPA-induced neurite retraction remains
unknown. We have developed a Rho activation assay that is based on the
specific binding of active RhoA to its downstream effector Rho-kinase
(ROK). A fusion protein of GST and the Rho-binding domain of ROK
pulls down activated but not inactive RhoA from cell lysates. Using
GST-ROK, we show that in N1E-115 neuronal cells LPA activates
endogenous RhoA within 30 s, concomitant with growth cone
collapse. Maximal activation occurs after 3 min when neurite retraction
is complete and the actin cytoskeleton is fully contracted. LPA-induced
RhoA activation is completely inhibited by tyrosine kinase inhibitors
(tyrphostin 47 and genistein). Activated GMedical Research Council Laboratory for Molecular Cell
Biology, University College London, London WC1E 6BT, United Kingdom
12 and
G13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a
G12/13-initiated pathway that involves protein-tyrosine
kinase activity.
Corresponding author. E-mail address:
wmoolen{at}nki.nl.
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L. Zeng, P. Sachdev, L. Yan, J. L. Chan, T. Trenkle, M. McClelland, J. Welsh, and L.-H. Wang Vav3 Mediates Receptor Protein Tyrosine Kinase Signaling, Regulates GTPase Activity, Modulates Cell Morphology, and Induces Cell Transformation Mol. Cell. Biol., December 15, 2000; 20(24): 9212 - 9224. [Abstract] [Full Text] [PDF]
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G. P. v. N. Amerongen, M. A. Vermeer, and V. W. M. van Hinsbergh Role of RhoA and Rho Kinase in Lysophosphatidic Acid-Induced Endothelial Barrier Dysfunction Arterioscler Thromb Vasc Biol, December 1, 2000; 20 (12): e127 - e133. [Abstract] [Full Text] [PDF]
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 A. Gohla, G. Schultz, and S. Offermanns Role for G12/G13 in Agonist-Induced Vascular Smooth Muscle Cell Contraction Circ. Res., August 4, 2000; 87(3): 221 - 227. [Abstract] [Full Text] [PDF]
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R. L. Boshans, S. Szanto, L. van Aelst, and C. D'Souza-Schorey ADP-Ribosylation Factor 6 Regulates Actin Cytoskeleton Remodeling in Coordination with Rac1 and RhoA Mol. Cell. Biol., May 15, 2000; 20(10): 3685 - 3694. [Abstract] [Full Text] [PDF]
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S. Wahl, H. Barth, T. Ciossek, K. Aktories, and B. K. Mueller Ephrin-A5 Induces Collapse of Growth Cones by Activating Rho and Rho Kinase J. Cell Biol., April 17, 2000; 149(2): 263 - 270. [Abstract] [Full Text] [PDF]
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P. Anastasiadis and A. Reynolds The p120 catenin family: complex roles in adhesion, signaling and cancer J. Cell Sci., January 4, 2000; 113(8): 1319 - 1334. [Abstract] [PDF]
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O. Kranenburg, I. Verlaan, and W. H. Moolenaar Dynamin Is Required for the Activation of Mitogen-activated Protein (MAP) Kinase by MAP Kinase Kinase J. Biol. Chem., December 10, 1999; 274(50): 35301 - 35304. [Abstract] [Full Text] [PDF]
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K. Kimura, T. Tsuji, Y. Takada, T. Miki, and S. Narumiya Accumulation of GTP-bound RhoA during Cytokinesis and a Critical Role of ECT2 in This Accumulation J. Biol. Chem., June 2, 2000; 275(23): 17233 - 17236. [Abstract] [Full Text] [PDF]
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R. Bhattacharyya and P. B. Wedegaertner Galpha 13 Requires Palmitoylation for Plasma Membrane Localization, Rho-dependent Signaling, and Promotion of p115-RhoGEF Membrane Binding J. Biol. Chem., May 12, 2000; 275(20): 14992 - 14999. [Abstract] [Full Text] [PDF]
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A. Hahn, J. Heusinger-Ribeiro, T. Lanz, S. Zenkel, and M. Goppelt-Struebe Induction of Connective Tissue Growth Factor by Activation of Heptahelical Receptors. MODULATION BY Rho PROTEINS AND THE ACTIN CYTOSKELETON J. Biol. Chem., November 22, 2000; 275(48): 37429 - 37435. [Abstract] [Full Text] [PDF]
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H. Togashi, K.-i. Nagata, M. Takagishi, N. Saitoh, and M. Inagaki Functions of a Rho-specific Guanine Nucleotide Exchange Factor in Neurite Retraction. POSSIBLE ROLE OF A PROLINE-RICH MOTIF OF KIAA0380 IN LOCALIZATION J. Biol. Chem., September 15, 2000; 275(38): 29570 - 29578. [Abstract] [Full Text] [PDF]
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F. P. G. van Horck, M. R. Ahmadian, L. C. Haeusler, W. H. Moolenaar, and O. Kranenburg Characterization of p190RhoGEF, A RhoA-specific Guanine Nucleotide Exchange Factor That Interacts with Microtubules J. Biol. Chem., February 9, 2001; 276(7): 4948 - 4956. [Abstract] [Full Text] [PDF]
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D. A. Linseman, K. A. Heidenreich, and S. K. Fisher Stimulation of M3 Muscarinic Receptors Induces Phosphorylation of the Cdc42 Effector Activated Cdc42Hs-associated Kinase-1 via a Fyn Tyrosine Kinase Signaling Pathway J. Biol. Chem., February 16, 2001; 276(8): 5622 - 5628. [Abstract] [Full Text] [PDF]
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J. van der Wal, R. Habets, P. Varnai, T. Balla, and K. Jalink Monitoring Agonist-induced Phospholipase C Activation in Live Cells by Fluorescence Resonance Energy Transfer J. Biol. Chem., April 27, 2001; 276(18): 15337 - 15344. [Abstract] [Full Text] [PDF]
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S. A. Sagi, T. M. Seasholtz, M. Kobiashvili, B. A. Wilson, D. Toksoz, and J. H. Brown Physical and Functional Interactions of Galpha q with Rho and Its Exchange Factors J. Biol. Chem., April 27, 2001; 276(18): 15445 - 15452. [Abstract] [Full Text] [PDF]
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S. Siehler, Y. Wang, X. Fan, R. T. Windh, and D. R. Manning Sphingosine 1-Phosphate Activates Nuclear Factor-kappa B through Edg Receptors. ACTIVATION THROUGH Edg-3 AND Edg-5, BUT NOT Edg-1, IN HUMAN EMBRYONIC KIDNEY 293 CELLS J. Biol. Chem., December 21, 2001; 276(52): 48733 - 48739. [Abstract] [Full Text] [PDF]
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A. Gilchrist, J. F. Vanhauwe, A. Li, T. O. Thomas, T. Voyno-Yasenetskaya, and H. E. Hamm Galpha Minigenes Expressing C-terminal Peptides Serve as Specific Inhibitors of Thrombin-mediated Endothelial Activation J. Biol. Chem., July 6, 2001; 276(28): 25672 - 25679. [Abstract] [Full Text] [PDF]
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C. D. Wells, S. Gutowski, G. Bollag, and P. C. Sternweis Identification of Potential Mechanisms for Regulation of p115 RhoGEF through Analysis of Endogenous and Mutant Forms of the Exchange Factor J. Biol. Chem., July 27, 2001; 276(31): 28897 - 28905. [Abstract] [Full Text] [PDF]
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C.-S. Shi, S. Sinnarajah, H. Cho, T. Kozasa, and J. H. Kehrl G13alpha -mediated PYK2 Activation. PYK2 IS A MEDIATOR OF G13alpha -INDUCED SERUM RESPONSE ELEMENT-DEPENDENT TRANSCRIPTION J. Biol. Chem., August 4, 2000; 275(32): 24470 - 24476. [Abstract] [Full Text] [PDF]
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T. M. Seasholtz, T. Zhang, M. R. Morissette, A. L. Howes, A. H. Yang, and J. H. Brown Increased Expression and Activity of RhoA Are Associated With Increased DNA Synthesis and Reduced p27Kip1 Expression in the Vasculature of Hypertensive Rats Circ. Res., September 14, 2001; 89(6): 488 - 495. [Abstract] [Full Text] [PDF]
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