Endoplasmic reticulum Ca2+ is important for the proteolytic processing and intracellular transport of proinsulin in the pancreatic beta-cell
Endoplasmic reticulum Ca2+ is important for the proteolytic processing and intracellular transport of proinsulin in the pancreatic beta-cell
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DOI：
10.1042/bj3230445
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发表时间：
1997-04-15
影响因子：
4.1
通讯作者：
Hutton, JC
中科院分区：
文献类型：
--
作者：
Guest, PC;Bailyes, EM;Hutton, JC
通过对分离的大鼠朗格汉斯胰岛进行脉冲追踪放射性标记，研究了细胞内 Ca2+ 在分泌颗粒前蛋白的蛋白水解加工和细胞内运输中的作用。通过用 EGTA 消耗培养基中的 Ca2+ 以及用 Ca2+ 通道拮抗剂维拉帕米、硝苯地平和 NiCl2 阻断 Ca2+ 转运至细胞，可以抑制胰岛素原的转化。内质网 Ca2+-ATP 酶抑制剂毒胡萝卜素也会降低胰岛素原转化，表明该过程需要将 Ca2+ 转运至内质网。这一发现得到了支持，即当脉冲标记之前或期间 Ca2+ 耗尽时，胰岛素原加工会受到抑制，但在蛋白质转运到内质网后区室之后不会受到抑制。类似地，在放射性标记时重新引入培养基 Ca2+ 可逆转胰岛素原加工的抑制，但在追踪孵育 15 分钟后不会逆转。 Ca2+ 耗竭还降低了激素原转化酶 PC1、PC2 和羧肽酶 H 的蛋白水解成熟度。分泌实验表明，胰岛素原转运到分泌颗粒的速率和程度受到 Ca2+ 耗竭的轻微抑制，而转化酶的速率和程度则受到明显阻碍。因此，Ca2+耗竭对胰岛素原转化的抑制不仅与成熟PC1和PC2的Ca2+依赖性有关，而且还与转化酶的蛋白水解成熟及其转移至分泌颗粒过程中对内质网Ca2+的需求有关。结果还表明，颗粒中激素原加工所需的 Ca2+ 通过内质网进入分泌途径。
The role of intracellular Ca2+ in the proteolytic processing and intracellular transport of secretory granule proproteins was investigated by pulse-chase radiolabelling of isolated rat islets of Langerhans. The conversion of proinsulin was inhibited by depletion of medium Ca2+ with EGTA and by blocking the transport of Ca2+ into cells with the Ca2+-channel antagonists verapamil, nifedipine and NiCl2. Proinsulin conversion was also reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, indicating that the process requires transport of Ca2+ into the endoplasmic reticulum. This was supported by the finding that proinsulin processing was inhibited when Ca2+ was depleted before or during pulse-labelling, but not after transport of the protein to post-endoplasmic-reticulum compartments. Similarly, the inhibition of proinsulin processing was reversed by re-introduction of medium Ca2+ around the time of radiolabelling, but not after 15 min of chase incubation. Ca2+ depletion also decreased proteolytic maturation of the prohormone convertases PC1, PC2 and carboxypeptidase H. Secretion experiments suggested that the rate and extent of proinsulin transport into secretory granules were inhibited marginally by Ca2+ depletion, whereas those of the convertases were markedly impeded. Inhibition of proinsulin conversion by Ca2+ depletion was thus not simply related to the Ca2+-dependencies of mature PC1 and PC2, but also to a requirement for endoplasmic reticulum Ca2+ in proteolytic maturation of the convertases and in their transfer to secretory granules. The results also suggest that the Ca2+ required for prohormone processing in the granules enters the secretory pathway via the endoplasmic reticulum.