Lièvremont J P, Hill A M, Hilly M, Mauger J P
INSERM U274, Physiologie et Pharmacologie Cellulaire, Université Paris Sud, Orsay, France.
Biochem J. 1994 Jun 1;300 ( Pt 2)(Pt 2):419-27. doi: 10.1042/bj3000419.
Inositol 1,4,5-trisphosphate (InsP3) is involved in the mobilization of Ca2+ from intracellular non-mitochondrial stores. In rat liver, it has been shown that the InsP3-binding site co-purifies with the plasma membrane. This suggests that in the liver the InsP3 receptor (InsP3R) associates with plasma membrane. We studied the subcellular distribution of the liver InsP3R by measuring the maximal binding capacity of [3H]InsP3 and using antibodies against the 14 C-terminal residues of the type 1 InsP3R. The antibodies recognized a large amount of an InsP3R protein of 260 kDa in a membrane fraction which is also enriched with [3H]InsP3-binding sites and with markers of the basal, the lateral and the bile-canalicular membrane and the plasma-membrane Ca2+ pump (PMCA). The fractions enriched in markers of the endoplasmic reticulum (ER) and the Ca2+ pump of the ER (SERCA2b) contained low levels of InsP3 receptors. The immunofluorescent labelling of cultured hepatocytes with anti-InsP3R antibodies indicated that the receptor is concentrated in the perinuclear area and in some regions near the plasma membrane. The fraction enriched with InsP3R is also contaminated with markers of the ER and with SERCA2b. It was exposed to alkaline medium (pH 10.5) to extract endogenous actin and membrane-associated proteins before being subfractionated by Percoll-gradient centrifugation. The alkaline treatment allowed partial separation of the markers of the ER from the markers of the plasma membrane. The InsP3R was recovered in the heavy subfraction, which was also enriched with markers for the ER and with the SERCA2b and contained low levels of markers of the plasma membrane. These data indicate that the InsP3R is neither localized on the plasma membrane itself nor homogeneously distributed on the ER membrane. This supports the view that part of the receptor is localized on a specialized sub-region of the ER which interacts with the plasma membrane.
肌醇1,4,5 -三磷酸（InsP3）参与从细胞内非线粒体储存库中动员Ca2+。在大鼠肝脏中，已表明InsP3结合位点与质膜共同纯化。这表明在肝脏中InsP3受体（InsP3R）与质膜相关联。我们通过测量[3H]InsP3的最大结合能力并使用针对1型InsP3R的14个C末端残基的抗体，研究了肝脏InsP3R的亚细胞分布。这些抗体在一个膜组分中识别出大量260 kDa的InsP3R蛋白，该膜组分还富含[3H]InsP3结合位点以及基底膜、侧膜、胆小管膜和质膜Ca2+泵（PMCA）的标志物。富含内质网（ER）标志物和ER的Ca2+泵（SERCA2b）的组分中InsP3受体水平较低。用抗InsP3R抗体对培养的肝细胞进行免疫荧光标记表明，该受体集中在核周区域和质膜附近的一些区域。富含InsP3R的组分也被ER标志物和SERCA2b污染。在通过Percoll梯度离心进行亚分级分离之前，将其暴露于碱性介质（pH 10.5）中以提取内源性肌动蛋白和膜相关蛋白。碱性处理使ER标志物与质膜标志物部分分离。InsP3R在重亚组分中回收，该重亚组分也富含ER标志物和SERCA2b，并且质膜标志物水平较低。这些数据表明InsP3R既不定位在质膜本身上，也不均匀地分布在内质网膜上。这支持了这样一种观点，即部分受体定位在内质网的一个与质膜相互作用的特殊亚区域上。
