THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IS LOCALIZED ON SPECIALIZEDSUBREGIONS OF THE ENDOPLASMIC-RETICULUM IN RAT-LIVER

Inositol 1,4,5-trisphosphate (InsP(3)) is involved in the mobilization of Ca2+ from intracellular non-mitochondrial stores. In rat liver, it has been shown that the InsP(3)-binding site copurifies with the plas ma membrane. This suggests that in the liver the InsP(3) receptor (Ins P(3)R) associates with plasma membrane. We studied the subcellular dis tribution of the liver InsP(3)R by measuring the maximal binding capac ity of [H-3]InsP(3) and using antibodies against the 14 C-terminal res idues of the type 1 InsP(3)R. The antibodies recognized a large amount of an InsP(3)R protein of 260 kDa in a membrane fraction which is als o enriched with [H-3]InsP(3)-binding sites and with markers of the bas al, the lateral and the bile-canalicular membrane and the plasma-membr ane Ca2+ pump (PMCA). The fractions enriched in markers of the endopla smic reticulum (ER) and the Ca2+ pump of the ER (SERCA2b) contained lo w levels of InsP(3) receptors. The immunofluorescent labelling of cult ured hepatocytes with anti-InsP(3)R antibodies indicated that the rece ptor is concentrated in the perinuclear area and in some regions near the plasma membrane. The fraction enriched with InsP(3)R is also conta minated with markers of the ER and with SERCA2b. It was exposed to alk aline medium (pH 10.5) to extract endogenous actin and membrane-associ ated proteins before being subfractionated by Percoll-gradient centrif ugation. The alkaline treatment allowed partial separation of the mark ers of the ER from the markers of the plasma membrane. The InsP(3)R wa s recovered in the heavy subfraction, which was also enriched with mar kers for the ER and with the SERCA2b and contained low levels of marke rs of the plasma membrane. These data indicate that the InsP(3)R is ne ither localized on the plasma membrane itself nor homogeneously distri buted on the ER membrane. This supports the view that part of the rece ptor is localized on a specialized sub-region of the ER which interact s with the plasma membrane.