Association of the inositol (1,4,5)-trisphosphate receptor ligand binding site with phosphatidylinositol (4,5)-bisphosphate and Adenophostin A

The inositol 1,4,5-trisphosphate receptor (InsP₃R) is activated by InsP₃ binding to amino-terminal ligand binding domain (InsP₃R-N). Recently we reported functional coupling of phosphatidylinositol (4,5)-bisphosphate (PIP₂) to the InsP₃R. Specific binding of PIP₂ to InsP₃R-N domain was postulated as a part of the InsP₃R-PIP₂ functional coupling model. Here we utilized bacterially expressed and purified InsP₃R-N domain to characterize its binding specificity for InsP₃, Adenophostin A (AdA) and the water, soluble PIP₂ analog dioctanoyl-(4,5)PIP₂ (ShPIP₂). Obtained data led us to conclude that specific InsP₃, AdA, and ShPIP₂ binding sites are located within the InsP₃R-N domain, that the extra receptor binding element responsible for enhanced binding of AdA is an integral part of the InsP₃R-N domain, that ShPIP₂ is able to displace InsP₃ from the InsP₃R-N, but InsP₃ or AdA is unable to completely displace ShPIP₂. These results support the InsP₃R-PIP₂ functional coupling model and provide novel insights into InsP₃R ligand specificity. (C) 2000 Academic Press.