Selective determinants of inositol 1,4,5-trisphosphate and adenophostin A interactions with type 1 inositol 1,4,5-trisphosphate receptors

BACKGROUND AND PURPOSE: Adenophostin A (AdA) is a potent agonist of inositol 1,4,5-trisphosphate receptors (IP(3)R). AdA shares with IP(3) the essential features of all IP(3)R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP(3), but the basis of its increased affinity is unclear. Hitherto, the 2′-phosphate of AdA has been thought to provide a supra-optimal mimic of the 1-phosphate of IP(3). EXPERIMENTAL APPROACH: We examined the structural determinants of AdA binding to type 1 IP(3)R (IP(3)R1). Chemical synthesis and mutational analysis of IP(3)R1 were combined with (3)H-IP(3) binding to full-length IP(3)R1 and its N-terminal fragments, and Ca(2+) release assays from recombinant IP(3)R1 expressed in DT40 cells. KEY RESULTS: Adenophostin A is at least 12-fold more potent than IP(3) in functional assays, and the IP(3)-binding core (IBC, residues 224–604 of IP(3)R1) is sufficient for this high-affinity binding of AdA. Removal of the 2′-phosphate from AdA (to give 2′-dephospho-AdA) had significantly lesser effects on its affinity for the IBC than did removal of the 1-phosphate from IP(3) (to give inositol 4,5-bisphosphate). Mutation of the only residue (R568) that interacts directly with the 1-phosphate of IP(3) decreased similarly (by ∼30-fold) the affinity for IP(3) and AdA, but mutating R504, which has been proposed to form a cation-π interaction with the adenine of AdA, more profoundly reduced the affinity of IP(3)R for AdA (353-fold) than for IP(3) (13-fold). CONCLUSIONS AND IMPLICATIONS: The 2′-phosphate of AdA is not a major determinant of its high affinity. R504 in the receptor, most likely via a cation-π interaction, contributes specifically to AdA binding.