Selective recognition of inositol phosphates by subtypes of the inositol trisphosphate receptor
Selective recognition of inositol phosphates by subtypes of the inositol trisphosphate receptor
复制标题
DOI：
10.1042/bj3550059
复制
发表时间：
2001-04-01
影响因子：
4.1
通讯作者：
Taylor, CW
中科院分区：
文献类型：
--
作者：
Nerou, EP;Riley, AM;Taylor, CW
利用合成的三磷酸肌醇(IP3)类似物(与(1，4，5)IP3的4，5-二磷酸等同)，研究了在昆虫细胞中表达的大鼠1型、2型和3型IP3受体的识别特性。通过与[H-3](1，4，5)IP3在无钙条件下的平衡竞争结合，(1，4，5)IP3受体亚型的相对亲和力为3型(K-d=11+/-2 nM)和gt；2型(K-d=17+/-2 nM)和gt；1型(K-d=24+/-4 nM)。(1，4，5)IP3结合被pH升高可逆地刺激，但不同亚型对pH的敏感性不同(类型1和类型2和类型3)。对于所有三种亚型，(1，4，5)IP3的赤道6-羟基是高亲和力所必需的，赤道3-羟基显著提高了亲和力，轴向2-羟基不显著：1-磷酸(或在不存在时，2-磷酸)提高了结合亲和力。不同亚型对(1，4，5)IP3的3-羟基倒置的容忍程度不同，这可能解释了(1，4，6)IP3对类型1受体的选择性。它们对2-羟基(2型和3型)的翻转、去除或取代(通过磷酸取代)的耐受性也不同，因此对2型和3型受体的(1，2，3，5)IP4的选择性也不同。3型受体对3-羟基的去除或用氟或CH2OH取代3-羟基的耐受性最好，这解释了3-脱氧(1，4，5)IP3对3型受体的选择性。我们的结果首次系统地分析了IP3受体亚型的识别特性，并确定(1，4，5)IP3的2-和3-位是亚型选择性的关键决定因素。
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 [H-3](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 (K-d = 11+/-2 nM) > type 2 (K-d = 17+/-2 nM) > type 1 (K-d = 24+/-4 nM). (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 (1,2,3,5)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.