Molecular basis of the isoform-specific ligand-binding affinity of inositol 1,4,5-trisphosphate receptors
Molecular basis of the isoform-specific ligand-binding affinity of inositol 1,4,5-trisphosphate receptors
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DOI：
10.1074/jbc.m609833200
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发表时间：
2007-04-27
影响因子：
4.8
通讯作者：
Mikoshiba, Katsuhiko
中科院分区：
文献类型：
--
作者：
Iwai, Miwako;Michikawa, Takayuki;Mikoshiba, Katsuhiko
1，4，5-三磷酸肌醇（IP 3）受体（IP 3R）的三种亚型IP（3）R1、IP（3）R2和IP（3）R3具有不同的IP 3结合亲和力和协同性。在这里，我们报告了三种小鼠IP 3R类型的氨基末端604个残基显示出49.5 +/- 10.5、14.0 +/- 3.5和163.0 +/- 44.4 nM的Kd值，这接近于先前从全长IP（3）Rs分析中估计的固有IP 3结合亲和力。相比之下，IP（3）R1和IP（3）R2的残基224-604以及IP（3）R3的残基225-604（其含有IP 3结合核心结构域但不含抑制子结构域）显示出几乎相同的IP 3结合亲和力，K-d值类似于2nM。添加100倍过量的抑制结构域没有改变IP 3结合核心结构域的IP 3结合亲和力。人工嵌合蛋白，其中的抑制结构域融合到IP 3结合的核心结构域从不同的亚型表现出IP 3结合的亲和力显着不同的抑制结构域和IP 3结合的核心结构域的天然组合组成的蛋白质。系统诱变分析表明，对3型受体特异性IP 3结合亲和力至关重要的氨基酸残基涉及Glu-39、Ala-41、Asp-46、Met-127、Ala-154、Thr-155、Leu-162、Trp-168、Asn-173、Asn-176和瓦尔-179。这些结果表明IP（3）Rs的IP 3结合亲和力通过氨基末端抑制结构域对IP 3结合核心结构域的IP 3结合亲和力的分子内衰减而特异性调节。此外，IP 3R亚型之间配体敏感性的功能多样性至少源于抑制结构域上鉴定的结构差异。
Three isoforms of the inositol 1,4,5-trisphosphate ( IP3) receptor ( IP3R), IP(3)R1, IP(3)R2, and IP(3)R3, have different IP3-binding affinities and cooperativities. Here we report that the amino-terminal 604 residues of three mouse IP3R types exhibited K-d values of 49.5 +/- 10.5, 14.0 +/- 3.5, and 163.0 +/- 44.4 nM, which are close to the intrinsic IP3-binding affinity previously estimated from the analysis of full-length IP(3)Rs. In contrast, residues 224-604 of IP(3)R1 and IP(3)R2 and residues 225-604 of IP(3)R3, which contain the IP3-binding core domain but not the suppressor domain, displayed an almost identical IP3-binding affinity with a K-d value of similar to 2 nM. Addition of 100-fold excess of the suppressor domain did not alter the IP3-binding affinity of the IP3-binding core domain. Artificial chimeric proteins in which the suppressor domain was fused to the IP3-binding core domain from different isoforms exhibited IP3-binding affinity significantly different from those of the proteins composed of the native combination of the suppressor domain and the IP3-binding core domain. Systematic mutagenesis analyses showed that amino acid residues critical for type-3 receptor-specific IP3-binding affinity are involved in Glu-39, Ala-41, Asp-46, Met-127, Ala-154, Thr-155, Leu-162, Trp-168, Asn-173, Asn-176, and Val-179. These results indicate that the IP3-binding affinity of IP(3)Rs is specifically tuned through the intramolecular attenuation of IP3-binding affinity of the IP3-binding core domain by the aminoterminal suppressor domain. Moreover, the functional diversity in ligand sensitivity among IP3R isoforms originates from at least the structural difference identified on the suppressor domain.