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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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