|Abstract||The Lsc RhoGEF is a member of the Dbl-homology family of GTP exchange factors. Lsc has a RhoGEF domain specific for Rho GTPase and an RGS domain specific for G alpha 12/13 subunits. One G protein-coupled receptor activating G alpha 12/13 subunits is the receptor for thromboxane A2 (TXA2) called TP, which is highly expressed in immature thymocytes. To study the role of Lsc downstream TXA2 signaling and TP activation, I analyzed TXA2 dependent activation and development of thymocytes from Lsc knockout mice using the TXA2 analog U46199. We show that U46619 stimulated actin polymerization and cofilin phosphorylation in primary thymocytes, and that these events required Rho, the Rho kinase ROCK and Lsc.
Moreover, in the absence of Lsc, U46619-induced stimulation of phosphorylation of the survival kinase Akt was greatly enhanced and apoptosis of double positive thymocytes in culture was decreased. Additionally, the phosphorylation of several protein kinase C (PKC) isoforms were, like Akt, significantly enhanced in the absence of Lsc. Certain
PKC isoforms are also implicated in the regulation of survival and apoptosis. Supporting these findings, Lsc-/- mice had significantly higher numbers of thymocytes that further increased as mice aged. These results demonstrate that TXA2 induction of thymocyte apoptosis depends on the Rho exchange factor Lsc accompanied by signaling to Rho, ROCK, cofilin and Akt, and that loss of Lsc results in thymic hyperplasia.
The Lsc homolog LARG is more widely expressed than Lsc and, similar to Lsc, is a GEF specific for Rho and is implicated in downstream signaling of G alpha 12/13 subunits via its RGS domain. Many G alpha 12/13-dependent functions are still working in Lsc deficient mice suggesting that other RGS-domain containing GEFs could compensate for Lsc in vivo. Therefore we intended to examine the in vivo functions of LARG by the generation of LARG deficient mice with the future perspective to generate Lsc/LARG double knockout mice. I constructed a targeting vector, which will be used to generate LARG knockout mice, and compensation between LARG and Lsc will be tested.||dc.description.abstract