Figure 6.

Direct stimulation by PKC and RhoA are critical for PLD1 activation by G protein-coupled receptors. (A) M3 mAChR-expressing HEK293 cells were transfected with PLD1 alleles as indicated and in vivo assays were performed in the absence (basal) or presence of 1 mM carbachol. The data shown are representative of three similar experiments carried out in duplicate or triplicate. The percentage activation of each mutant was calculated by subtracting its basal activity and the agonist-dependent increase of K898R (which represents the activation of endogenous PLD) from agonist-activated activities of the mutant, and comparing this value with the agonist-activated wild-type PLD1 sample treated similarly. (B) Agonist-stimulated activation of PLD mutants using other G protein-coupled receptors. HEK293 cells were cotransfected with PLD1 mutants and the Edg4 or AT1a receptors. In vivo assays were performed in the absence or presence of 100 nM angiotensin II for the AT1a receptor or 100 μM LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate; 18:1 Lyso PA) for the Edg-4 receptor. The values shown represent the percentage of activation of each mutant compared with that of the wild-type mutant (100%) and were calculated as described above. The experiments were conducted in duplicate or triplicate, and the values shown are representative of two experiments.