Proteolysis of type I inositol 1,4,5-trisphosphate receptor in WB rat liver cells.
Proteolysis of type I inositol 1,4,5-trisphosphate receptor in WB rat liver cells.
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WB 大鼠肝细胞中 I 型肌醇 1,4,5-三磷酸受体的蛋白水解。
DOI：
10.1042/bj20030828
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Joseph,SureshK
中科院分区：
文献类型：
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作者：
Khan,MTariq;Joseph,SureshK
通过脉冲追踪分析或通过分析放线菌酮添加后的免疫反应性InsP 3Rs，比较I型InsP 3Rs [d-肌肌醇1，4，5-三磷酸受体]的基础降解，表明与成熟InsP 3Rs的大池相比，新合成的放射性InsP 3Rs的小池降解相对较快。针对受体IL-3（第三管腔内环）内肽序列的抗体（Ab）（IL-3 Ab）用于鉴定可能在细胞中积累的受保护的蛋白水解片段。IL-3抗体识别WB大鼠肝细胞和A7 R5平滑肌细胞中的56 kDa片段。凝胶过滤实验表明，56 kDa的片段是单体的，并基于对其他抗体的反应性，缺失了受体的胞质溶胶暴露的N-和C-末端片段。加入溶酶体蛋白酶抑制剂氯喹导致56 kDa条带迅速消失。半胱氨酸蛋白酶抑制剂亮抑酶肽、N-乙酰基-L-亮氨酰-L-亮氨酰-L-蛋氨酸和N-乙酰基-亮氨酰-亮氨酰-正亮氨酸模拟了这种作用。Lactacystin和NH 4Cl的效果较差。只有当细胞用NH 4Cl处理时，含有C-末端的16 kDa的第二片段才积累，而不是用任何其他测试的抑制剂。未观察到N-末端反应性片段。我们建议，成熟的InsP 3R四聚体解离成单体和56 kDa的片段是一个裂解中间体的单体代表的6个跨膜结构域。血管紧张素II刺激的WB细胞中InsP 3R的下调已被证明涉及泛素/蛋白酶体途径。血管紧张素-II处理WB细胞既不导致任何新片段的积累，也不增加56或16 kDa片段的水平。我们的结论是基础和激动剂刺激的降解InsP 3Rs发生不同的途径。激动剂介导的途径涉及整个受体分子从内质网膜的协同去除和蛋白水解，而不会出现中间的管腔内片段。
A comparison of the basal degradation of type I InsP3Rs [d-myo-inositol 1,4,5-trisphosphate receptor], measured by pulse–chase analysis or by analysis of immunoreactive InsP3Rs after cycloheximide addition, indicated that the small pool of newly synthesized radioactive InsP3Rs degraded relatively rapidly compared with the large pool of mature InsP3Rs. An antibody (Ab) against a peptide sequence within the IL-3 (third intraluminal loop) of the receptor (IL-3 Ab) was used to identify protected proteolytic fragments that may accumulate in cells. The IL-3 Ab recognized a 56 kDa fragment in both WB rat liver cells and A7R5 smooth-muscle cells. Gel filtration experiments indicated that the 56 kDa fragment was monomeric and, based on reactivity to other Abs, was missing the cytosol-exposed N- and C-terminal segments of the receptor. The addition of the lysosomal protease inhibitor chloroquine resulted in the rapid disappearance of the 56 kDa band. This effect was mimicked by the cysteine protease inhibitors leupeptin,N-acetyl-l-leucyl-l-leucyl-l-methioninal andN-acetyl-leucyl-leucyl-norleucinal. Lactacystin and NH4Cl were less effective. A second fragment of 16 kDa containing the C-terminus accumulated only when the cells were treated with NH4Cl, and not with any of the other inhibitors tested. No N-terminal-reactive fragments were observed. We propose that mature InsP3R tetramers dissociate into monomers and that the 56 kDa fragment is a cleavage intermediate of the monomer representing the six transmembrane domains. Angiotensin-II-stimulated down-regulation of InsP3Rs in WB cells has been shown to involve the ubiquitin/proteasome pathway. Angiotensin-II treatment of WB cells neither resulted in the accumulation of any new fragments nor increased the levels of the 56 or 16 kDa fragments. We conclude that basal and agonist-stimulated degradations of InsP3Rs occur by different pathways. The agonist-mediated pathway involves the concerted removal and proteolysis of the entire receptor molecule from the endoplasmic reticulum membrane without the appearance of intermediate intraluminal fragments.