Activation of RhoA by lysophosphatidic acid and G alpha(12/13) subunits inneuronal 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 su ch as lysophosphatidic acid (LPA). These shape changes are driven by Rho-me diated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable ass ay. Furthermore, the nature of the G protein(s) mediating LPA-induced neuri te retraction remains unknown. We have developed a Rho activation assay tha t is based on the specific binding of active RhoA to its downstream effecto r Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of R OK 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 w ithin 30 s, concomitant with growth cone collapse. Maximal activation occur s after 3 min when neurite retraction is complete and the actin cytoskeleto n is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated G al pha(12) and G alpha(13) subunits mimic LPA both in activating RhoA and in i nducing 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 G(12/13)-initiated p athway that involves protein-tyrosine kinase activity.