G Alpha(12) Structural Determinants Of Hsp90 Interaction Are Necessary For Serum Response Element-Mediated Transcriptional Activation

The G12/13 class of heterotrimeric G proteins, comprising the alpha-subunits G alpha(12) and G alpha(13), regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets has been reported. To identify G alpha(12) determinants that mediate specific protein interactions, we used a structural and evolutionary comparison between the G12/13, Gs, Gi, and Gq classes to identify "class-distinctive" residues in G alpha(12) and G alpha(13). Mutation of these residues in G alpha(12) to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)-mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of G alpha(12) mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of G alpha(13) exhibited robust SRE activation, suggesting a G alpha(12)-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of G alpha(12) than G alpha(13) signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and G alpha(12):Concertina chimeras revealed G alpha(12)-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify G alpha(12) determinants of SRE activation, implicate G alpha(12):Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of G alpha(12) and G alpha(13) to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.