Activation of RhoA by lysophosphatidic acid and Galpha12/13 subunits in neuronal cells: induction of neurite retraction.
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 Galpha12 and Galpha13 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.
Kranenburg
O
O
The Netherlands Cancer Institute, Division of Cellular Biochemistry, 1066 CX Amsterdam, The Netherlands.
Poland
M
M
van Horck
F P
FP
Drechsel
D
D
Hall
A
A
Moolenaar
W H
WH
D000818:Animals; D001681:Biological Assay; D002455:Cell Division / Q000502:physiology; D003599:Cytoskeleton / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D016501:Neurites / Q000378:metabolism*; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein
D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein