The inositol 1,4,5-trisphosphate receptor is localized on specialized sub-regions of the endoplasmic reticulum in rat liver.
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.
Lièvremont
J P
JP
INSERM U274, Physiologie et Pharmacologie Cellulaire, Université Paris Sud, Orsay, France.
Hill
A M
AM
Hilly
M
M
Mauger
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000378:metabolism*; D002462:Cell Membrane; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate