Selective Uncoupling of Gα12 from Rho-mediated Signaling

Abstract

The heterotrimeric G protein G12 has been implicated in such cellular regulatory processes as cytoskeletal rearrangement, cell-cell adhesion, and oncogenic transformation. Although the activated α-subunit of G12 has been shown to interact directly with a number of protein effectors, the roles of many of these protein-protein interactions in G12-mediated cell physiology are poorly understood. To begin dissecting the specific cellular pathways engaged upon G12 activation, we produced a series of substitution mutants in the regions of Gα12 predicted to play a role in effector binding. Here we report the identification and characterization of an altered form of Gα12 that is functionally uncoupled from signaling through the monomeric G protein Rho, a protein known to propagate several Gα12-mediated signals. This mutant of Gα12 fails to bind the Rho-specific guanine nucleotide exchange factors p115RhoGEF and LARG (leukemia-associated RhoGEF), fails to stimulate Rho-dependent transcriptional activation, and fails to trigger activation of RhoA and the Rho-mediated cellular responses of cell rounding and c-jun N-terminal kinase activation. Importantly, this mutant of Gα12 retains coupling to the effector protein E-cadherin, as evidenced by its ability both to bind E-cadherin in vitro and to disrupt E-cadherin-mediated cell-cell adhesion. Furthermore, this mutant retains the ability to trigger β-catenin release from the cytoplasmic domain of cadherin. This identification of a variant of Gα12 that is selectively uncoupled from one signaling pathway while retaining signaling capacity through a separate pathway will facilitate investigations into the mechanisms through which G12 proteins mediate diverse biological responses.