Activation of RhoA by lysophosphatidic acid and Galpha12/13 subunits in neuronal cells: induction of neurite retraction

Abstract

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.

Figure 1

A new assay to measure Rho activation. (A) Cos7 cells were transfected with expression vectors encoding myc-tagged versions of either wt, activated (L63), or inactive (N19) RhoA. In addition, two activated RhoA mutants with additional point mutations in the effector loop were used: L63A37 and L63G39. Cell lysates were incubated with GST-ROK, and GST-ROK–associating proteins were analyzed by Western blotting using 9E10 monoclonal anti-myc antibody. GST-ROK binds to wt and L63 Rho but not to N19, L63G39, or L63A37. (B) Cos cells were transfected with myc-tagged versions of wt, activated (V12/V14), or inactive (N17/N19) Rho, Rac, or Cdc42. Binding to GST-ROK was then assessed as in A. GST-ROK discriminates between activated and inactive Rho and Cdc42, but Rac is recovered independent of its activation state.

Figure 2

LPA activates endogenous RhoA in N1E-115 cells. (A) N1E-115 cells were cultured overnight in serum-free medium and were subsequently stimulated with LPA (1 μM) or bradykinin (1 μM) for the indicated periods. Cell lysates were prepared and incubated with GST-ROK. The activation state of RhoA was then assessed by anti-RhoA Western blotting. (B) RhoA activation depends on tyrosine kinase activity. Cells were pretreated with either genistein (25 μM, 30 min) or tyrphostin 47 (150 μM, 30 min) before stimulation with LPA (3 min). The activation state of RhoA was then assessed as in A.

Figure 3

Activation of RhoA by Gα₁₂ and Gα₁₃. Cos7 cells were cotransfected with expression vectors encoding activated Gα_i, Gα₁₂, Gα₁₃, or a control vector, together with an expression vector encoding myc-tagged wt RhoA. Twenty-four hours after transfection, the cells were cultured overnight in serum-free medium, and the activation state of RhoA was assessed as in Figure 1.

Figure 4

Gα₁₂ and Gα₁₃, but not Gα_i, inhibit neurite outgrowth and induce cell rounding. (A) N1E-115 cells were transfected with either a control vector or expression vectors encoding activated Gα₁₂, Gα₁₃, or Gα_i, together with an expression vector encoding β-galactosidase. Cells were cultured in serum-free medium overnight, and morphologies were assessed as described. (B) Analysis of the actin cytoskeleton in Gα₁₂- and Gα₁₃-expressing cells. Rhodamine-phalloidin staining reveals that the cortical cytoskeleton in these cells is completely contracted, forcing the cells to round up. (C) Cells were transfected with Gα₁₂ and Gα₁₃ together with either a control vector or a vector encoding (dominant-negative) N19RhoA. Morphologies were then assessed as in A: Gα₁₂- and Gα₁₃-induced inhibition of neurite outgrowth and cytoskeletal contraction are RhoA dependent.