The inositol 1,4,5-trisphosphate receptor is localized on specialized sub-regions of the endoplasmic reticulum in rat liver

Abstract

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.

High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

“…Distinction among individual ER cisternae had therefore not been possible. Immunocytochemistry and subcellular fractionation studies, carried out in many nonmuscle cell types Volpe et al, 1991;Sharp et al, 1992;Villa et al, 1993;Lievremont et al, 1994;Caspersen and Treiman, 1995) including PC12 (Rooney and Meldolesi, 1996), documented heterogeneities in the distribution of various ER proteins participating in Ca2+ homeostasis, including pumps and channels (see Pozzan et al, 1994). Whether such molecular heterogeneities give rise to heterogeneous distributions of calcium within the ER had however not been established.…”

Capacitative calcium entry

“…Evidence for such an association has come from cell fractionation studies which revealed that InsP3 receptors often appear in the plasma membrane fraction [78][79][80][81]. Further fractionation revealed that these InsP3 receptors were located in an ER membrane component that was tightly bound to the plasma membrane but could be dissociated by treatments which disrupt the cytoskeleton [79,81].…”

RhoA Interaction with Inositol 1,4,5-Trisphosphate Receptor and Transient Receptor Potential Channel-1 Regulates Ca2+ Entry

“…We also determined whether actin polymerization (another Rhodependent event) participates in the Ca 2ϩ entry response. We treated HPAEC with C3 transferase and latrunculin-A (which inhibits actin polymerization by binding with actin monomers (54)) to address the contribution of Rho and actin polymerization, respectively, in mediating association of TRPC1 and IP 3 R. Lysates of control cells and cells treated with C3 transferase or latrunculin were immunoprecipitated using anti-IP 3 R antibody, and Western blotted using anti-TRPC1 or IP 3 R Abs. In addition, GFP-Rho-transfected, or cells pretreated without or with C3 transferase or latrunculin were fixed to assess actin filament polymerization.…”