Prolonged Interactions Between Gα13 And Leukemia-Associated RhoGEF Are Associated With High Sensitivity Of Receptor-Induced LARG Activation

E-L Bodmann, A-L Müller, M Bünemann. Institut fuer Pharmakologie und Klinische Pharmazie, Philipps-Universtität Marburg, Germany

RhoA-GTPases control diverse cellular functions and are activated by trimeric G-proteins via RhoGEFs (1), for example Leukemia associated RhoGEF (LARG). In this study we focus on the activation of LARG by Gα13 after stimulation of thromboxane A2 receptors (Txa2-R). Currently not much is known about the kinetics of this pathway due to the lack of specific inhibitors and exclusively Gα13-coupled receptors (2). We used Förster resonance energy transfer (FRET) to measure Gα13-activation and interaction of Gα13 with LARG in single living cells. To this end we labeled the respective proteins with cyan and yellow fluorophores and proved their functional integrity in a Serum response factor (SRF)-dependent luciferase assay.

The activation of Gα13 occurred rapidly upon stimulation with U46619, a Txa2-R agonist, whereas the inactivation of the complex took about 41.22±5.42 s (t1/2±S.E.M., n=15), as measured by FRET between Gα13 and Gβ1γ2. Thus the speed of inactivation of Gα13 was in the same range as previously measured for other G proteins (3, 4). Surprisingly, for LARG dissociation from Gα13 after withdrawal of U46619 was much slower, since we could not detect a major decline of FRET in the time frame of these live-cell experiments. Furthermore we measured the concentration-response-relationship for the Gα13 activation as well as for the Gα13-LARG interaction after stimulation of TXA2-R with U46619. The dose-response-relationship of the LARG-Gα13-interaction was left-shifted compared to the Gα13 activation by about two orders of magnitude (EC50= 0.3 nM and 32.5 nM, respectively; F-test p<0.0001). As the dissociation of LARG from Gα13 after withdrawal of agonist was much slower than the Gα13-inactivation, we propose that prolonged interaction of G protein and effector underlies the hypersensitivity observed for LARG.

(1) Bustelo X R et al, Bioessays 29:356, 2007

(2) Latek D et al, Acta Biochim. Pol. 59: 515, 2012

(3) Bodmann E-L et al, Biochem J 458:131, 2014

(4) Frank M et al, J. Biol. Chem. 280:24584, 2005