Synthetic analogues of inositol trisphosphate (IP3), all of which included structures equivalent to the 4,5-bisphosphate of (1,4,5)IP3, were used to probe the recognition properties of rat full-length type 1, 2 and 3 IP3 receptors expressed in insect Spodoptera frugiperda 9 cells. Using equilibrium competition binding with [3H](1,4,5)IP3 in Ca2+-free cytosol-like medium, the relative affinities of the receptor subtypes for (1,4,5)IP3 were type 3 (Kd = 11±2nM)>type 2 (Kd = 17±2nM) > type 1 (Kd = 24±4nM). (1,4,5)IP3 binding was reversibly stimulated by increased pH, but the subtypes differed in their sensitivity to pH (type 1 > type 2>type 3). For all three subtypes, the equatorial 6-hydroxy group of (1,4,5)IP3 was essential for high-affinity binding, the equatorial 3-hydroxy group significantly improved affinity, and the axial 2-hydroxy group was insignificant; a 1-phosphate (or in its absence, a 2-phosphate) improved binding affinity. The subtypes differed in the extents to which they tolerated inversion of the 3-hydroxy group of (1,4,5)IP3 (type 1>type 2>type 3), and this probably accounts for the selectivity of (1,4,6)IP3 for type 1 receptors. They also differed in their tolerance of inversion, removal or substitution (by phosphate) of the 2-hydroxy group (types 2 and 3>type 1), hence the selectivity of (1,2,4,5)IP4 for type 2 and 3 receptors. Removal of the 3-hydroxy group or its replacement by fluorine or CH2OH was best tolerated by type 3 receptors, and accounts for the selectivity of 3-deoxy(1,4,5)IP3 for type 3 receptors. Our results provide the first systematic analysis of the recognition properties of IP3 receptor subtypes and have identified the 2- and 3-positions of (1,4,5)IP3 as key determinants of subtype selectivity.

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