Gα₁₂Structural Determinants of Hsp90 Interaction Are Necessary for Serum Response Element–Mediated Transcriptional Activation

Abstract

The G12/13 class of heterotrimeric G proteins, comprising the a-subunits Ga 12 and Ga 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 Ga 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 Ga 12 and Ga 13 . Mutation of these residues in Ga 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 Ga 12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Ga 13 exhibited robust SRE activation, suggesting a Ga 12 -specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of Ga 12 than Ga 13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Ga 12 :Concertina chimeras revealed Ga 12 -specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Ga 12 determinants of SRE activation, implicate Ga 12 :Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Ga 12 and Ga 13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.

GNA13 expression promotes drug resistance and tumor-initiating phenotypes in squamous cell cancers

“…To elucidate mechanisms that contribute to GNA13-induced TIC-like phenotype, we conducted a series of experiments using promoter-reporter assays downstream of GNA13: NFκB, AP-1, SRE, β-Catenin (TCF) and TEAD (YAP/TAZ) (Supplementary Fig. 6 ) [ 23 , 24 , 36 , 37 ]. Screens in GNA13-knockdown NCC-HN43 cells showed NFκB and AP-1 activity were significantly suppressed (Fig.…”

“…A total of 1 × 10 5 cells were plated in 6-well plates for 24 h. Then, cells were transiently transfected with 1μg of 4X-AP-1-Luciferase reporter or 6X-NF-κB-Luciferase reporter (gifts from Prof. Doris Mayer, Germany) [ 50 ], SRE-Luciferase (gift from Dr. Ted Meigs, North Carolina) [ 24 ], TEAD-Luciferase (gift from Dr. Mathijs Voorhoeve) [ 51 ] or TOP-FLASH-luciferase (gift from Prof. David Virshup) using Lipofectamine (Invitrogen, Germany). A promoter-less pGL3-Basic was used as a negative control.…”

Cellular mechanisms of the 5-HT7receptor-mediated signaling

“…Noteworthy, stimulation of 5-HT 7 receptor led to the dose-dependent increase in SRE-driven gene expression even in the presence of a PKA-inhibitor or pertussis toxin (PTX), suggesting a receptor-mediated SRE activation in a PKA-independent manner (Kvachnina et al, 2005 ). Recent findings also elucidated Rho-independent mechanism of Gα 12 -mediated SRE activation via Hsp90 (Figure 1 ; Montgomery et al, 2014 ). Interaction between Gα 12 and Hsp90 might also be critically involved in a selective transport of the G 12 -protein to the lipid rafts (Waheed and Jones, 2002 ).…”

The Role of Heat Shock Proteins in Regulating Receptor Signal Transduction

“…Regulation of these G proteins by Hsp90 thus has the potential to regulate signal transduction downstream of up to hundreds of GPCRs. Hsp90 was shown to direct mature Ga 12 (but not Ga 13 ) specifically to lipid rafts and the mitochondria, which is required for Ga 12 signaling; this demonstrates an obligate role for Hsp90 in all GPCR signaling evoked by this protein (Vaiskunaite et al, 2001;Waheed and Jones, 2002;Andreeva et al, 2008;Montgomery et al, 2014). Among other potential roles, this Ga 12 targeting was shown to be necessary for tight junction formation in Mardin-Darby canine kidney cells (Sabath et al, 2008).…”