The interaction of calmodulin with alternatively spliced isoforms of the type-I inositol trisphosphate receptor.
The interaction of calmodulin with alternatively spliced isoforms of the type-I inositol trisphosphate receptor.
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钙调蛋白与 I 型肌醇三磷酸受体的选择性剪接异构体的相互作用。
DOI：
10.1074/jbc.275.4.2305
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Joseph,SK
中科院分区：
文献类型：
--
作者：
Lin,C;Widjaja,J;Joseph,SK
将神经元I型肌醇1，4，5-三磷酸受体（IP 3R）同种型（I型长，氨基酸1314 -1905）调节结构域的592个氨基酸片段和外周组织中存在的相应552个氨基酸可变剪接形式（I型短，氨基酸1693-1733缺失）表达为谷胱甘肽S-转移酶融合蛋白。这些结构域包括一个假定的钙调蛋白（CaM）结合域和两个蛋白激酶A磷酸化位点。长和短的融合蛋白都保留了以Ca 2+依赖的方式结合CaM的能力，如通过CaM-Sepharose色谱法或丹磺酰-CaM荧光测定所测量的。两种测定均表明，在饱和浓度的CaM下，短融合蛋白结合的CaM量是长形式的两倍。此外，钙调素-琼脂糖凝胶的短形式的结合被抑制磷酸化与蛋白激酶A，而长形式的结合不受影响。在COS细胞中表达编码I型长型、I型短型和III型IP 3R亚型的全长cDNA，并测量[3 H] IP 3结合透化细胞的Ca 2+敏感性。I型长亚型对Ca 2+抑制的敏感性（IC 50 = 0.55 μm）高于I型短亚型（IC 50 = 5.7 μm）或III型亚型（IC 50 = 3 μm）。与融合蛋白的研究一致，全长I型短结合更多的CaM-Sepharose，并且这种结合比I型长IP 3R在更大程度上被蛋白激酶A磷酸化抑制。虽然III型IP 3R不直接结合CaM-Sepharose，但I/III型IP 3R的异源寡聚体保留了与CaM相互作用的能力。我们的结论是，I型IP 3 R中SII剪接位点的缺失导致Ca 2+、CaM和蛋白激酶A对选择性剪接亚型的差异调节。
A 592-amino acid segment of the regulatory domain of the neuronal type-I inositol 1,4,5-trisphosphate receptor (IP3R) isoform (type-I long, amino acids1314–1905) and the corresponding 552-amino acid alternatively spliced form present in peripheral tissues (type-I short, amino acids 1693–1733 deleted) were expressed as glutathioneS-transferase fusion proteins. These domains encompass a putative calmodulin (CaM) binding domain and two protein kinase A phosphorylation sites. Both long and short fusion proteins retained the ability to bind CaM in a Ca2+-dependent manner as measured by CaM-Sepharose chromatography or a dansyl-CaM fluorescence assay. Both assays indicated that the short fusion protein bound twice the amount of CaM than the long form at saturating concentrations of CaM. In addition, the binding of the short form to CaM-Sepharose was inhibited by phosphorylation with protein kinase A, whereas the binding of the long form was unaffected. Full-length cDNAs encoding type-I long, type-I short, and type-III IP3R isoforms were expressed in COS cells, and the Ca2+sensitivity of [3H]IP3binding to permeabilized cells was measured. The type-I long isoform was more sensitive to Ca2+inhibition (IC50= 0.55 μm) than the type-I short (IC50= 5.7 μm) or the type-III isoform (IC50= 3 μm). In agreement with studies on the fusion proteins, the full-length type-I short bound more CaM-Sepharose, and this binding was inhibited to a greater extent by protein kinase A phosphorylation than the type-I long IP3R. Although type-III IP3Rs did not bind directly to CaM-Sepharose, hetero-oligomers of type-I/III IP3Rs retained the ability to interact with CaM. We conclude that the deletion of the SII splice site in the type-I IP3R results in the differential regulation of the alternatively spliced isoforms by Ca2+, CaM, and protein kinase A.
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