PDZ-RhoGEF and LARG Are Essential for Embryonic Development and Provide a Link between Thrombin and LPA Receptors and Rho Activation

Wenling Li,Daniel Martin,Hiroki Chikumi,J.Silvio Gutkind,Constantinos M Mikelis,Todd R Palmby,Hiroshi Yagi,Roman Szabo,Shigetomo Fukuhara,May Simaan,Thomas H Bugge,Rebeca Galisteo,Ruth Lyons,Yoh-Suke Mukouyama

doi:10.1074/jbc.m112.428599

Abstract

G protein-coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), lymphoid blast crisis (Lbc), and Dbl. PRG, LARG, and p115 share the presence of a regulator of G protein signaling homology (RGS) domain. There is limited information on the biological roles of this RGS-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension. Here, we generated knock-out mice for Prg and observed that these mice do not display any overt phenotype. However, deficiency in Prg and Larg leads to complex developmental defects and early embryonic lethality. Signaling from Gα11/q-linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all three RGS-containing RhoGEFs. However, a combined lack of Prg, Larg, and p115 expression abolished signaling through Gα12/13 to Rho and thrombin-induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by Gα12/13-coupled GPCRs, which may likely play a critical role during embryonic development.

Highlights

Many Rho GEFs have been proposed to link G protein-coupled receptors (GPCRs) to Rho activation
There is limited information on the biological roles of this regulator of G protein signaling homology (RGS)-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension
No compensatory changes were observed on Prg expression in mouse embryonic fibroblasts (MEFs) isolated from LargϪ/Ϫ mice or on Larg expression levels in fibroblasts derived from PrgϪ/Ϫ mice (Fig. 1C and see below)

Summary

Background

Many Rho GEFs have been proposed to link GPCRs to Rho activation. Results: PDZ-RhoGEF and LARG double deficiency leads to embryonic lethality and, along with p115 RhoGEF, are required for thrombin-induced cell migration, proliferation, and RhoA activation. Members of the G␣q and G␣12 subfamilies have been shown to regulate the activation of several small GTPases of the Ras and Rho families (reviewed in Ref. 7) In this regard, multiple Rho GEFs that have been proposed to link G12/13-coupled GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), and lymphoid blast crisis (Lbc), its longer transcript fused to an AKAP (A kinase anchor protein), AKAP-Lbc [8, 9], proto-Dbl [10, 11], and Lfc [12]. Thrombin was not able to induce cell proliferation and directional migration and failed to initiate nuclear signaling through JNK and p38 These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by G12/13-coupled GPCRs, which may likely play a critical role during embryonic development

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION