Abstract
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.