Role of PRIP-1, a novel Ins(1,4,5)P₃ binding protein, in Ins(1,4,5)P₃-mediated Ca²⁺ signaling

PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [ins(1,4,5)P₃] binding protein with a domain organization similar to phospholipase C-δ1 (PLC-δ1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P₃. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P₃-mediated Ca²⁺ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1 ^-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca²⁺ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1^-/- mice than in those from wild type mice. The relative amounts of [³H]Ins(1,4,5)P₃ measured in neurons labeled with [³H]inositol was also lower in cells from PRIP-1 ^-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1^-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,S)P₃ is enhanced in cells from PRIP-1^-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1 PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P ₃ to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P₃-mediated Ca²⁺ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P₃.