Figure 3. PhLP1 augments expression and activity of AC5.
(A) Co-expression of PhLP1 significantly increases AC5 expression in HEK293 cells. **p<0.01 Students’ t-test, n = 3. (B) PhLP1 enhances cAMP levels under basal condition, or in response to stimulation with forskolin (FSK, 1 μM, 5 min) or isoproterenol (ISO, 1 μM, 5 min) in AC5 expressing HEK293 cells. **p<0.01 Student’s t-test, n = 3. (C) PhLP1 has no effect on cAMP generation in HEK293 cells without AC5 overexpression. n = 3 (D) Effects of purified recombinant full length PhLP1 and ΔNT-PhLP1 on adenylyl cyclase activity in striatal membranes. Assays were performed in the absence of GTP. Striatal membranes were pre-incubated with 0.5 μM of purified proteins at 4°C for 20 min and then subjected to adenylyl cyclase activity assay. Data were analyzed by One-Way ANOVA (F[2, 9] = 35.477, p<0.001). **p<0.01, post hoc Tukey’s test. n = 3 (E) Purified recombinant PhLP1 enhances forskolin-stimulated adenylyl cyclase activity in striatal membranes. Membranes were pre-incubated with 0.5 μM of purified proteins at 4°C for 20 min. Membranes were then stimulated with 1 μM forskolin or 80 nM Gαs-GTPγS. Data were analyzed by One-Way ANOVA (F(4, 15) = 52.291, p<0.001).***p<0.001, post hoc Tukey’s test. n = 3 (F) Effect of purified recombinant PhLP1 on the dose response of forskolin-mediated activation of adenylyl cyclase in striatal membranes. The EC50 for forskolin was 32.5 ± 2.7 μM in the control reaction and 8.9 ± 1.6 μM in PhLP1-treated reaction. **p<0.01 Student’s t-test, n = 3. (G) Effect of purified recombinant PhLP1 on the dose response of Gαs-GTPγS-mediated activation of adenylyl cyclase in striatal membranes. The EC50 for Gαs-GTPγS was 16.2 ± 4.7 nM in the control reaction and 22.3 ± 5.7 nM in PhLP1-treated reaction (n = 3 reactions).