Lièvremont J P, Hill A M, Tran D, Coquil J F, Stelly N, Mauger J P
INSERM U274, Physiologie et Pharmacologie Cellulaire, Université Paris Sud, France.
Biochem J. 1996 Feb 15;314 ( Pt 1)(Pt 1):189-97. doi: 10.1042/bj3140189.
The D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor was localized by immunofluorescence experiments in situ in liver cryosections. Two anti-Ins(1,4,5)P3 receptor antibodies (against the 14 C-terminal residues of the type 1 receptor or against the entire cerebellar receptor) weakly decorated the whole cytoplasm, and a more intense labelling was observed at the periphery of the hepatocytes, particularly beneath the canalicular and the sinusoidal domains of the plasma membrane (PM). Antibodies against calreticulin, the Ca2+ pump (SERCA2b) or endoplasmic reticulum (ER) membranes homogeneously labelled the cytoplasm and the subplasmalemmal area. These data indicate that the ER can be divided into at least two specialized subregions: one is located throughout most of the cytoplasm and contains markers of the rough ER (RER), calreticulin, SERCA2b and a low density of Ins(1,4,5)P3 receptor, and the other is confined to the periphery of the cells and contains calreticulin, Ca2+ pump, RER markers and a high density of Ins(1,4,5)P3 receptor. A membrane fraction enriched in Ins(1,4,5)P3 receptor and in markers of the PM was immuno-adsorbed with the antibody against the C-terminal end of the Ins(1,4,5)P3 receptor and pelleted with Sepharose protein A. The immuno-isolated material was enriched in Ins(1,4,5)P3 receptor, but none of the markers of the ER or of the PM could be detected. This suggests that the Ins(1,4,5)P3 receptor is localized on discrete domains of the ER membrane beneath the canalicular and the sinusoidal membranes, where it was found at higher densities than the other markers.
通过免疫荧光原位实验，在肝脏冰冻切片中定位了D-肌醇1,4,5-三磷酸[Ins(1,4,5)P3]受体。两种抗Ins(1,4,5)P3受体抗体（针对1型受体的14个C末端残基或针对整个小脑受体）对整个细胞质进行了弱染色，并且在肝细胞周边观察到更强的标记，特别是在质膜（PM）的胆小管和窦状区域下方。抗钙网蛋白、Ca2+泵（SERCA2b）或内质网（ER）膜的抗体均匀地标记了细胞质和质膜下区域。这些数据表明，内质网可至少分为两个特化的亚区域：一个位于大部分细胞质中，含有粗面内质网（RER）、钙网蛋白、SERCA2b的标记物以及低密度的Ins(1,4,5)P3受体；另一个局限于细胞周边，含有钙网蛋白、Ca2+泵、RER标记物以及高密度的Ins(1,4,5)P3受体。富含Ins(1,4,5)P3受体和质膜标记物的膜组分用抗Ins(1,4,5)P3受体C末端的抗体进行免疫吸附，并用琼脂糖蛋白A沉淀。免疫分离的物质富含Ins(1,4,5)P3受体，但未检测到内质网或质膜的任何标记物。这表明Ins(1,4,5)P3受体定位于胆小管膜和窦状膜下方内质网膜的离散区域，在这些区域其密度高于其他标记物。
