Alternative GTPase‐deficient mutants of Gα12/13 show differences in signaling potency and effector binding

Abstract

Heterotrimeric G proteins are implicated in tumorigenic signaling in a variety of cancer types. Although G protein α subunits of the Gs, Gi, and Gq subfamilies have been found in tumors as mutationally activated, GTPase‐deficient variants, the G12/13 subfamily has the unusual property of participating in cancer progression as an overexpressed, wildtype form. Recent work by Maziarz et al. (2020) identified two “hotspot” mutations at the Arg200 position of Gα13 in bladder tumor samples, demonstrating that mutational activation of G12/13 subfamily proteins may also play a role in cancer progression. Although corresponding mutations at this Arg residue in Gα12 have not been located in tumors, the recent findings for Gα13 suggest that such Gα12 mutants may be identified in future work. Most in vitro signaling studies employing constitutively active Gα12 and/or Gα13 have utilized a Gln‐to‐Leu (QL) substitution in the switch II region to eliminate GTPase activity. Therefore, we sought to compare these QL variants of Gα12 and Gα13 to the recently reported Arg mutants in several G12/13‐specific signaling readouts. We engineered the previously identified hotspot Gα13 mutants (R200K and R200G) along with corresponding mutants of Gα12 (R203K and R203G). Our preliminary findings from reporter gene assays in HEK293 cells suggest that Gα12.R203K and Gα12.R203G are more potent than the Gα12.QL variant in driving two pathways implicated in Gα12‐mediated tumor progression: serum response factor (SRF) and YAP/TAZ. Gα13.R200K and Gα13.R200G appear to stimulate SRF and TEAD reporters at a similar strength to Gα13.QL, consistent with the findings of Maziarz et al. In addition, we investigated binding of these mutants to the RGS‐homology RhoGEFs LARG, p115RhoGEF, and PDZ‐RhoGEF. Unexpectedly, Gα13.R200G showed a RhoGEF binding profile that was different from Gα13.QL, suggesting these GTPase‐deficient mutants differ in their affinity for specific effector proteins. We currently are examining the comparative binding of these mutants to other targets such as epithelial cadherin (E‐cadherin) and radixin. Further, we found these activated Gα12/13 mutants to cause a decrease in cellular levels of E‐cadherin. Interestingly, these mutants appear to have differential effects on E‐cadherin levels compared to QL variants of Gα12/13. We have found this difference to be most pronounced between Gα12 Arg and QL mutants, in comparison to the Gα13 mutants. Due to our recent finding that Gα12 and Gα13 cause an increase in levels of calreticulin, a protein reported to down‐regulate E‐cadherin gene expression, we are using RNA interference to test the hypothesis that Gα12 and/or Gα13 modulate E‐cadherin levels in a calreticulin‐dependent manner.