Figure 8.

The increase in Ca²⁺ release in IP₃R1 by PKA phosphorylation is regulated by receptor fragmentation in a cleavage region-specific manner. A, cells were loaded with 1 μm Fluo-2/AM for 1 h, followed by a FlexStation assay to monitor the change in [Ca²⁺]_i. Preincubation of cells stably expressing IP₃R1 WT with forskolin significantly increased the amplitude of Ca²⁺ release in response to PAR2 activation. B and D, this increase was observed at all agonist concentrations tested for IP₃R1 WT (B) and at high agonist concentrations tested for IP₃R1 I–III+IV–V (tryp) (D). C and E, forskolin had no effect on the amplitude of Ca²⁺ release in cells expressing IP₃R1 (S1589A, S1755A) (C) or IP₃R1 I-IV+V (calp) (E). F and G, pretreatment of forskolin results in IP₃R1 phosphorylation at residue Ser-1755 in both full-length and fragmented receptors. Statistics were performed using one-way ANOVA followed by Dunnett post-test. Experiments were repeated three times for each set of IP₃R1 or complementary receptor fragments. WB, Western blot.