Selective recognition of inositol phosphates by subtypes of the inositol trisphosphate receptor

• Published in: Biochemical journal 2001-04, Vol.355 (Pt 1), p.59-69
• Main Authors: Nerou, E P, Riley, A M, Potter, B V, Taylor, C W
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Calcium Channels - classification; Calcium Channels - metabolism; Heparin - pharmacology; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate Receptors; Inositol Phosphates - metabolism; Molecular Sequence Data; Receptors, Cytoplasmic and Nuclear - antagonists & inhibitors; Receptors, Cytoplasmic and Nuclear - classification; Receptors, Cytoplasmic and Nuclear - metabolism; Spodoptera; Xenopus
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/0264-6021:3550059

Synthetic analogues of inositol trisphosphate (IP(3)), all of which included structures equivalent to the 4,5-bisphosphate of (1,4,5)IP(3), were used to probe the recognition properties of rat full-length type 1, 2 and 3 IP(3) receptors expressed in insect Spodoptera frugiperda 9 cells. Using equilibrium competition binding with [(3)H](1,4,5)IP(3) in Ca(2+)-free cytosol-like medium, the relative affinities of the receptor subtypes for (1,4,5)IP(3) were type 3 (K(d)=11+/-2 nM)>type 2 (K(d)=17+/-2 nM)>type 1 (K(d)=24+/-4 nM). (1,4,5)IP(3) 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)IP(3) 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)IP(3) (type 1>type 2>type 3), and this probably accounts for the selectivity of (1,4,6)IP(3) 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)IP(4) for type 2 and 3 receptors. Removal of the 3-hydroxy group or its replacement by fluorine or CH(2)OH was best tolerated by type 3 receptors, and accounts for the selectivity of 3-deoxy(1,4,5)IP(3) for type 3 receptors. Our results provide the first systematic analysis of the recognition properties of IP(3) receptor subtypes and have identified the 2- and 3-positions of (1,4,5)IP(3) as key determinants of subtype selectivity.