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

Abstract

BACKGROUND AND PURPOSE

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.

EXPERIMENTAL APPROACH

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.

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 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).

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.