Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho.
Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.
Jalink
K
K
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.
van Corven
E J
EJ
Hengeveld
T
T
Morii
N
N
Narumiya
S
S
Moolenaar
W H
WH
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D008246:Lysophospholipids / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D016501:Neurites / Q000378:metabolism / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D012266:Ribose / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000502:physiology*; D020742:rhoA GTP-Binding Protein
D000199:Actins; D008246:Lysophospholipids; D000244:Adenosine Diphosphate; D012266:Ribose; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D013917:Thrombin; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D020742:rhoA GTP-Binding Protein