Figure 3.

Gβγ signaling and Ca²⁺ transient may serve as two major inputs from the G_i and EGFR, respectively, to trigger the synergistic JNK activation. (a) Cos-7 cells expressing JNK-HA with or without Gβ₁ and Gγ₂ subunits were stimulated in the absence or presence of EGF (100 ng ml⁻¹) for 30 min. (b) ORL₁R and JNK-HA were co-transfected into Cos-7 cells, followed by individual or simultaneous stimulation with OFQ (100 nM) and thapsigargin (Thap, 5 μM) for 30 min before determining the JNK activity. Data shown represent the mean±s.e. from three separate experiments, and dotted lines indicate the corresponding basal activities. (a, b) ^*Transient expression of Gβ₁γ₂ subunit and the treatment with EGF, OFQ or thapsigargin significantly increased the JNK activity as compared to the basal (Bonferroni's corrected t-test, one-way ANOVA, P<0.05). ^#Co-administration of Gβ₁γ₂ with EGF, or OFQ with thapsigargin induced JNK activations synergistically as compared to their individual responses (Bonferroni's corrected t-test, two-way ANOVA, P<0.05). (c) Cos-7 cells expressing JNK-HA with either G_i-coupled SSTR₁ or D₂R were also capable of inducing a synergistic JNK activation upon co-treatment with their agonists (100 nM somatostatin for SSTR₁ and 10 μM dopamine for D₂R) and thapsigargin. (d) Cos-7 cells co-transfected with the cDNAs of JNK-HA with or without Gβ₁ and Gγ₂ subunits were stimulated in the absence or presence of thapsigargin (Thap, 5 μM, 30 min) before determining the JNK activity. (c, d) Both expression of JNK-HA and the phosphorylation of GST-c-Jun are illustrated. Data of JNK activity (fold-induction) represent the averaged values of two separate experiments.