G-protein-coupled receptor signaling to phospholipase D1 mediated by G 12-type G proteins, LIM-kinase and cofilin

The M3 muscarinic acetylcholine receptor (mAChR), a typical G protein-coupled receptor, expressed in HEK-293 cells stimulates phospholipase D in a pertussis toxin (PTX)-insensitive manner and the PLD response to the M3 mAChR requires ARF and Rho GTPases and the Rho-activated Rho-kinase. However, Rho-kinase did not phosphorylate PLD enzymes directly, suggesting additional components mediate the PLD regulation by Rho/Rho-kinase. In this thesis, first, by transient expression of a-subunits of the PTX-resistant G proteins, Gq, G12 and G13 (wild-type, constitutively active and dominant-negative mutants), evidence is provided that the M3 mAChR specifically couples to PLC via Gaq and to PLD via the G12-type G proteins, Ga12 and Ga13, which are apparently both required for full PLD stimulation. These data were confirmed by expression (transient or by infection with recombinant adenoviruses) of RGS4 or Lsc-RGS, which act as specific GTPase-activating proteins for Gaq- and Ga12-type G proteins, respectively. Second, the expression of catalytically inactive PLD1 reduced specifically PLD stimulation by M3 mAChR, whereas its counterpart of PLD2 inhibited only PLD stimulation by PKC and EGF, suggesting that the G protein-coupled receptors signals primarily to PLD1, whereas the tyrosine kinase receptors signal via PKC to PLD2. Third, expression of wild-type and constitutively active LIM-kinase, a Rho-kinase effector, potentiated PLD stimulation by the M3 mAChR, whereas kinase-deficient LIM-kinase had the opposite effect. Purified recombinant LIM-kinase stimulated PLD activity in cell membranes, similar as but not additive with activated RhoA or Rho-kinase. In addition, PLD stimulation by constitutively active LIM-kinase, but not by wild-type LIM-kinase, was resistant to inactivation of Rho and Rho-kinase, whereas PLD stimulation by constitutively active Rho-kinase was fully abolished by kinase-deficient LIM-kinase. Furthermore, LIM-kinase neither directly interacts with nor phosphorylates PLD enzymes, suggesting that some undefined component is involved in PLD regulation by Rho-kinase/LIM-kinase. We found that expression of wild-type cofilin, an actin depolymerization factor (LIM-kinase substrate) potentiated PLD stimulation by the M3 mAChR, whereas the nonphosphorylatable cofilin mutant, S3A cofilin, reduced the receptor response. And this PLD stimulation by cofilin was suppressed by inactivation of Rho or Rho-kinase. In vitro, cofilin protein, but not its S3A mutant, specifically interacts with PLD1 and strongly increases the activity of PLD1 upon phosphorylation by LIM-kinase. In addition, expression of wild-type cofilin, but not S3A cofilin, specifically redistributed PLD1 to the plasma membrane. Taken together, we demonstrated that stimulation of PLD by G protein-coupled receptors, known to involve ARF and Rho GTPases and the Rho-activated Rho-kinase, is mediated specifically by heterotrimeric G proteins of the G12-subtype (Ga12 and Ga13), the Rho-kinase effector, LIM-kinase, and the LIM-kinase substrate, cofilin, which apparently in its phosphorylated form interacts with and stimulates PLD1 activity.



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