BACKGROUND AND PURPOSE Adenophostin A (AdA) is a potent agonist of inositol 1,4,5-trisphosphate receptors (IP3R). AdA shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3, 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 IP3.
EXPERIMENTAL APPROACH We examined the structural determinants of AdA binding to type 1 IP3R (IP(3)R1). Chemical synthesis and mutational analysis of IP(3)R1 were combined with 3H-IP3 binding to full-length IP(3)R1 and its N-terminal fragments, and Ca2+ release assays from recombinant IP(3)R1 expressed in DT40 cells.
KEY RESULTS Adenophostin A is at least 12-fold more potent than IP3 in functional assays, and the IP3-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 IP3 (to give inositol 4,5-bisphosphate). Mutation of the only residue (R568) that interacts directly with the 1-phosphate of IP3 decreased similarly (by similar to 30-fold) the affinity for IP3 and AdA, but mutating R504, which has been proposed to form a cation-pi interaction with the adenine of AdA, more profoundly reduced the affinity of IP3R for AdA (353-fold) than for IP3 (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-pi interaction, contributes specifically to AdA binding.
- IP3 receptor
- structure-activity relationship