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Selective determinants of inositol 1,4,5‐trisphosphate and adenophostin A interactions with type 1 inositol 1,4,5‐trisphosphate receptors
Author(s) -
Rossi Ana M,
Sureshan Kana M,
Riley Andrew M,
Potter Barry VL,
Taylor Colin W
Publication year - 2010
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.2010.00947.x
Subject(s) - inositol phosphate , inositol , receptor , agonist , chemistry , inositol trisphosphate receptor , inositol trisphosphate , biochemistry , microbiology and biotechnology , stereochemistry , biology
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.