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PMID: 8002947 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

The Biochemical journal ·Vol. 300 ( Pt 2) ·1994-06-01 ·Pages 419-27

Lièvremont JP, Hill AM, Hilly M, Mauger JP

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.

MeSH Terms
Amino Acid Sequence Animals Binding Sites Calcium Channels/metabolism Cell Membrane Endoplasmic Reticulum/metabolism Female Fluorescent Antibody Technique Hydrogen-Ion Concentration Inositol 1,4,5-Trisphosphate/metabolism Inositol 1,4,5-Trisphosphate Receptors Liver/metabolism Molecular Sequence Data Osmolar Concentration Rats Rats, Wistar Receptors, Cytoplasmic and Nuclear/metabolism Subcellular Fractions/metabolism
Chemicals
Calcium Channels Inositol 1,4,5-Trisphosphate Receptors Receptors, Cytoplasmic and Nuclear Inositol 1,4,5-Trisphosphate
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Lièvremont J P
INSERM U274, Physiologie et Pharmacologie Cellulaire, Université Paris Sud, Orsay, France.
Hill A M
Hilly M
Mauger J P
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj3000419
Published
1994-06-01
Pages
419-27
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1138179
Subset
IM
External Links
PubMed source DOI: 10.1042/bj3000419 PMC Full Text View journal details
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