Llewelyn Roderick H, Llewellyn D H, Campbell A K, Kendall J M
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK.
Cell Calcium. 1998 Oct;24(4):253-62. doi: 10.1016/s0143-4160(98)90049-5.
Calreticulin is a Ca2+ binding protein located primarily in the endoplasmic reticulum (ER) lumen of non-excitable cells, where it is considered to be involved mainly in Ca2+ storage and buffering. However, there is increasing evidence to implicate the protein in other facets of Ca2+ signalling. In this study, we sought to establish more clearly the role of the protein in the regulation of intracellular Ca2+ signalling. Generating HeLa cells stably transfected with GFP-tagged calreticulin (GFPCRT) allowed to us to select cells by FACS in which calreticulin was expressed at ten times its endogenous levels. Using transiently expressed aequorin as a Ca2+ indicator in these cells, we investigated the role of calreticulin in intracellular Ca2+ storage, IP3-mediated Ca2+ release, and capacitative Ca2+ entry. The data showed that the capacity of the ionomycin-sensitive Ca2+ store was doubled in over-expressing cells, indicating that although calreticulin has a role in Ca2+ storage within the lumen, other lumenal proteins are also likely to be involved. No difference was observed in the release of Ca2+ from the IP3-sensitive store in response to prolonged single stimulation with histamine in the absence of extracellular Ca2+, but use of short, sequential pulses of histamine and ATP revealed that calreticulin may exert an effect upon IP3-mediated Ca2+ release. Two different experimental approaches indicated that calreticulin participates in the regulation of capacitative Ca2+ entry. In the presence of extracellular Ca2+, the histamine-generated cytosolic Ca2+ signal was significantly lower in GFPCRT cells than those in control cells. Induction of capacitative Ca2+ entry by complete emptying of the store using the SERCA pump inhibitor, cyclopiazonic acid also showed that the influx component was significantly reduced in the GFPCRT cells. Use of ER-targeted apoaequorin acting as a luciferase demonstrated that the resting ER free [Ca2+] in the GFPCRT cells was lower than that in control cells. These data implicate calreticulin in the control of IP3-mediated Ca2+ release and capacitative Ca2+ entry, which may involve direct interaction with Ca2+ signalling components or control of ER free [Ca2+].
钙网蛋白是一种主要位于非兴奋性细胞内质网（ER）腔中的Ca2+结合蛋白，在那里它主要被认为参与Ca2+的储存和缓冲。然而，越来越多的证据表明该蛋白还参与Ca2+信号传导的其他方面。在本研究中，我们试图更清楚地确定该蛋白在调节细胞内Ca2+信号传导中的作用。通过生成稳定转染了绿色荧光蛋白标记的钙网蛋白（GFPCRT）的HeLa细胞，使我们能够通过荧光激活细胞分选术（FACS）选择钙网蛋白表达水平是其内源水平十倍的细胞。在这些细胞中使用瞬时表达的水母发光蛋白作为Ca2+指示剂，我们研究了钙网蛋白在细胞内Ca2+储存、IP3介导的Ca2+释放和钙池调控性Ca2+内流中的作用。数据表明，在过表达细胞中，离子霉素敏感的Ca2+储存能力增加了一倍，这表明尽管钙网蛋白在ER腔内的Ca2+储存中起作用，但其他腔内蛋白可能也参与其中。在无细胞外Ca2+的情况下，用组胺进行长时间单次刺激时，未观察到IP3敏感储存中Ca2+释放的差异，但使用短时间、连续的组胺和ATP脉冲显示钙网蛋白可能对IP3介导的Ca2+释放产生影响。两种不同的实验方法表明钙网蛋白参与钙池调控性Ca2+内流的调节。在存在细胞外Ca2+的情况下，GFPCRT细胞中组胺产生的胞质Ca2+信号明显低于对照细胞。使用SERCA泵抑制剂环匹阿尼酸完全排空储存来诱导钙池调控性Ca2+内流也表明，GFPCRT细胞中的内流成分明显减少。使用内质网靶向的脱辅基水母发光蛋白作为荧光素酶表明，GFPCRT细胞中内质网静息游离[Ca2+]低于对照细胞。这些数据表明钙网蛋白参与IP3介导的Ca2+释放和钙池调控性Ca2+内流的控制，这可能涉及与Ca2+信号传导成分的直接相互作用或内质网游离[Ca2+]的控制。
