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

ABSTRACT: 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.

SUBMITTER: Nerou EP

PROVIDER: S-EPMC1221712 | biostudies-other | 2001 Apr

REPOSITORIES: biostudies-other

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Project description:The Ins(1,4,5)P3 regioisomers, Ins(1,4,6)P3 and Ins(1,3,6)P3, which can mimic the 1,4,5-arrangement on the inositol ring of Ins(1,4,5)P3, were examined for Ca2+ release by using four types of saponin-permeabilized cell possessing various abundances of receptor subtypes, with special reference to the relation of potency to receptor subtype. Ins(1,4,6)P3 and Ins(1,3,6)P3 were weak agonists in rat basophilic leukaemic cells (RBL cells), which possess predominantly subtype II receptors, with respective potencies of 1/200 and less than 1/500 that of Ins(1,4,5)P3 [the EC50 values were 0.2, 45 and more than 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively]. Similar rank order potencies were also evaluated for the displacement of [3H]Ins(1,4,5)P3 bound to RBL cell membranes by these regioisomers. However, they caused Ca2+ release from GH3 rat pituitary cells possessing predominantly subtype I receptors more potently; Ins(1,4,6)P3 and Ins(1,3,6)P3 evoked release at respective concentrations of only one-third and one-twentieth that of Ins(1,4,5)P3 (the EC50 values were 0.4, 1.2 and 8 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In COS-1 African green-monkey kidney cells, with the relative abundances of 37% of the subtype II and of 62% of the subtype III receptor, potencies of 1/40 and approx. 1/200 for Ins(1, 4,6)P3 and Ins(1,3,6)P3 respectively were exhibited relative to Ins(1,4,5)P3 (the EC50 values were 0.4, 15 and approx. 80 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In HL-60 human leukaemic cells, in spite of the dominant presence of subtype I receptors (71%), similar respective potencies to those seen with COS-1 cells were exhibited (the EC50 values were 0.3, 15 and approx. 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). These results indicate that these regioisomers are the first ligands that distinguish between receptor subtypes; the present observations are of significance for the future design of molecules with enhanced selectivity.

Project description:We have compared the properties of the [3H]Ins(1,4,5)P3-binding sites from a number of tissues in an attempt to determine if heterogeneity exists within the Ins(1,4,5)P3-receptor family. The binding of Ins(1,4,5)P3 was characterized in detail by using membranes prepared from human uterine smooth muscle and bovine adrenal cortex. Ins(1,4,5)P3 exhibited an approx. 5 times greater affinity for the binding site in adrenal cortex (KD = 9.81 +/- 1.92 nM) compared with uterine smooth muscle (KD = 37.1 +/- 1.8 nM). The binding was dependent on pH in both tissues, with a maximum at pH 8.3; at this pH various inositol phosphates and nucleotides competed for the binding sites with similar potencies on both tissues. However, the binding of Ins(1,4,5)P3 to the uterine smooth-muscle membranes was Ca2(+)-sensitive, whereas that to the bovine adrenal cortex was not; furthermore, heparin displaced the binding of Ins(1,4,5)P3 in the uterus with an IC50 value (concn. of displacer giving 50% inhibition of specific binding) of 3.9 micrograms/ml (2.5, 6.4; lower, upper range), compared with a value of 22 (13, 30) micrograms/ml in adrenal cortex. In view of the ability of Ins(1,4,5)P3 and heparin to distinguish between these binding sites, their effect on other tissues was examined. Ins(1,4,5)P3 showed a similar affinity for receptors located in the bovine cerebellum to those in the bovine adrenal cortex, but heparin displaced Ins(1,4,5)P3 binding with a 5-fold greater affinity from the cerebellum. Ins(1,4,5)P3 had a 2-fold greater affinity for its receptor with human platelets, as compared with human uterus, but heparin was unable to distinguish between these sites. In guinea-pig ileum, Ins(1,4,5)P3 displayed a similar affinity for the receptors in the longitudinal muscle compared with the circular muscle, but heparin could distinguish between these sites. These data show that small differences exist between tissues, but no clear picture is apparent. It is possible that these results reflect tissue-dependent factors such as phosphorylation, the presence of calmedin etc., rather than the presence of receptor subtypes or species difference.

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

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