Adenophostin A is the most potent known agonist of inositol trisphosphate (IP₃) receptors: it is typically 10-fold more potent than IP₃ in stimulating Ca²⁺ release from intracellular stores, and binds with 10-fold greater affinity in equilibrium competition binding assays (Correa et al. 2001). From analyses of bacterially expressed fragments of the mouse type 1 IP₃ receptor, it is clear that amino acid residues 224-604 are sufficient for high-affinity binding of IP₃ (Yoshikawa et al. 1999). It is unclear whether the same minimal IP₃-binding domain is sufficient to allow high-affinity binding of adenophostin A.

Fragments from the N-terminal region (residues 1-604 and 224-604; R^1-604 and R^224-604) of the rat type 1 IP₃ receptor (lacking the S1 splice site) were expressed as N-terminally tagged His₆ fusion proteins (pTrcHis vector) in E. coli. The expressed proteins were solubilized (Yoshikawa et al. 1999) and expression levels determined using immunoblots with an anti-His₆ antibody (Sigma). For equilibrium competition binding studies, 50 µg of bacterial extract in 200 µl of medium (50 mM Tris, 1 mM EDTA, pH 8.3) containing 1 nM [³H] IP₃ and competing ligand were incubated for 5 min at 2 °C. Reactions were terminated by precipitation with 15 % PEG 8000 followed by centrifugation. Results are means ± S.E.M. of ▓ge│ 3 experiments.

Membranes from rat (humanely killed ) cerebella bound IP₃ with K_d = 6.2 ± 0.1 nM, and after purification with K_d = 2.2 ± 0.48 nM. Adenophostin A bound to both membranes (K_d = 0.6 ± 0.09 nM) and pure type 1 receptors (K_d = 0.3 ± 0.05 nM) with about 10-fold greater affinity than IP₃. The affinity of R^1-604 for IP₃ (K_d = 2.2 ± 0.3 nM) was indistinguishable from that of pure full-length receptors, but its affinity for adenophostin A (K_d = 0.79 ± 0.03 nM) was only 2.8-fold greater than that of IP₃. There was no detectable binding of [³H] IP₃ to R^224-604, although its level of expression was similar to that of R^1-604. After restoration of the S1 splice site (15 residues) to R^224-604, the resulting fusion protein (R (S1⁺)^224-604) bound IP₃ with very high affinity (K_d = 1.1 ± 0.2 nM), and adenophostin A with 3-fold greater affinity (K_d = 0.4 ± 0.05 nM).

We conclude that the S1 splice site of the type 1 IP₃ receptor may influence IP₃ binding and that while the minimal IP₃-binding domain of the receptor (residues 224-604) are sufficient to allow high-affinity binding of adenophostin, additional residues lying beyond the IP₃-binding domain are required for optimal binding of adenophostin A.This work was supported by The Wellcome Trust.

Correa, V., Riley, A.M., Shuto, S., Horne, G., Nerou, E.P., Marwood, R.D., Potter, B.L. & Taylor, C.W. (2001). Mol. Pharmacol. 59, 1206-1215.

Yoshikawa, F., Uchiyama, T., Iwasaki, H., Tomomori-Satoh, C., Tanaka, T., Furuichi, T. & Mikoshiba, K. (1999). Biochem. Biophys. Res. Commun. 257, 792-797.