Determinants at the N- and C-termini of G?12 required for activation of Rho-mediated signaling.

ABSTRACT: BACKGROUND:Heterotrimeric guanine nucleotide binding proteins of the G12/13 subfamily, which includes the ?-subunits G?12 and G?13, stimulate the monomeric G protein RhoA through interaction with a distinct subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs). The structural features that mediate interaction between G?13 and RhoGEFs have been examined in crystallographic studies of the purified complex, whereas a G?12:RhoGEF complex has not been reported. Several signaling responses and effector interactions appear unique to G?12 or G?13, despite their similarity in amino acid sequence. METHODS:To comprehensively examine G?12 for regions involved in RhoGEF interaction, we screened a panel of G?12 cassette substitution mutants for binding to leukemia-associated RhoGEF (LARG) and for activation of serum response element mediated transcription. RESULTS:We identified several cassette substitutions that disrupt G?12 binding to LARG and the related p115RhoGEF. These G?12 mutants also were impaired in activating serum response element mediated signaling, a Rho-dependent response. Most of these mutants matched corresponding regions of G?13 reported to contact p115RhoGEF, but unexpectedly, several RhoGEF-uncoupling mutations were found within the N- and C-terminal regions of G?12. Trypsin protection assays revealed several mutants in these regions as retaining conformational activation. In addition, charge substitutions near the G?12 N-terminus selectively disrupted binding to LARG but not p115RhoGEF. CONCLUSIONS:Several structural aspects of the G?12:RhoGEF interface differ from the reported G?13:RhoGEF complex, particularly determinants within the C-terminal ?5 helix and structurally uncharacterized N-terminus of G?12. Furthermore, key residues at the G?12 N-terminus may confer selectivity for LARG as a downstream effector.

SUBMITTER: Ritchie BJ 

PROVIDER: S-EPMC3636079 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature