Adenophostin A (AdA) is a potent agonist of inositol 1,4,5-trisphosphate receptors (IP 3R). AdA shares with IP 3 the essential features of all IP 3R 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.
We examined the structural determinants of AdA binding to type 1 IP 3R (IP 3R1). Chemical synthesis and mutational analysis of IP 3R1 were combined with 3H-IP 3 binding to full-length IP 3R1 and its N-terminal fragments, and Ca 2+ release assays from recombinant IP 3R1 expressed in DT40 cells.
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 3R1) 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 3R for AdA (353-fold) than for IP 3 (13-fold).