Determinants at the N- and C-termini of Gα(12) required for activation of Rho-mediated signaling

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