Figure 6.

Impact of LARG expression on Gα₁₃ activity, as indicated by FRET between Gα₁₃-YFP and Gβ-Cer. A) Alteration of FRET between Gα₁₃-YFP and Gβ-Cer induced by 25 nM U-46619 was compared in cells in absence (black) or presence of mRFP-LARG (red, both n = 16). The bar graphs depict the average decrease in F₅₃₅/F₄₈₀ of the last 5 time points before withdrawal of U-46619 (means ± sem). P = 0.0358, unpaired Student’s t test. B) In similar experiments as described in A, relaxation kinetics of FRET between Gα₁₃-YFP and Gβ-Cer after withdrawal of agonist (either 10 nM or 1 µM, as indicated) were compared in the absence (black trace, n = 22) or presence of mRFP-LARG (red, n = 5). To reduce the impact of endogenous RH-RhoGEFs under control conditions (black data), endogenous LARG expression was attenuated by siRNA treatment. C) Analysis of relaxation halftimes of the experiments shown in B revealed much slower kinetics in the presence of mRFP LARG (means ± sem). P < 0.0001, unpaired Student’s t test. D) The agonist concentration-dependent alteration in FRET between Gα₁₃-YFP and Gβ-Cer was compared in cells overexpressing mRFP-LARG (red trace), transfected with mock-siRNA (blue trace), or in cells with less endogenous LARG because of siRNA treatment (black trace). Traces were corrected for bleaching and blotted as means ± sem of 10 (blue trace) and 11 cells (black and red traces). E) The siRNA pool, consisting of siRNA against LARG, PDZ-RhoGEF, and p115-RhoGEF, reduced endogenous LARG expression significantly compared to mock-siRNA pool. P < 0.05 (paired Student’s t test), as shown for 7 individual Western blots (means ± sem) and a representative blot. Transfection of mRFP-LARG resulted in an ∼5-fold increase in LARG expression. Protein samples originate from the transfections measured in D and Fig. 7D. Traces are means ± sem (A, B, D).