Cyclic AMP-dependent phosphorylation of an immunoaffinity-purified homotetrameric inositol 1,4,5-trisphosphate receptor (type I) increases Ca2+ flux in reconstituted lipid vesicles.
We established a novel method to isolate a single type of inositol 1,4,5-trisphosphate receptor (IP3R) among the heterogeneous population of receptors to study the regulatory mechanism of Ca2+ release. We raised in the rabbit a polyclonal antibody against synthetic peptide corresponding to amino acids 2736-2747 (pep 6) of type I IP3R (IP3-R-I) that is most abundant in cerebellum. We purified IP3R-I from a 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid solubilized mouse cerebellar microsomal fraction by immunoaffinity chromatography on an anti-pep 6 antibody-Sepharose 4B column with specific elution by the pep 6 peptide (GHPPHMNVNPQQ) of the IP3R-I C terminus. Immunoaffinity-purified IP3R reconstituted into lipid vesicles formed a homotetramer structure. Monoclonal antibody 18A10, which partially blocks the Ca2+ release from cerebellar microsome, almost completely inhibited IP3-induced 45Ca2+ influx into proteoliposomes, whereas monoclonal antibody that recognizes other regions did not inhibit Ca2+ influx. Both the rate and extent of 45Ca2+ influx into proteoliposomes increased 20% after incubation with the catalytic subunit of cyclic AMP-dependent protein kinase, accompanied by stoichiometric phosphorylation of IP3R protein.
Nakade
S
S
Basic Research Laboratory, Minase Research Institute, Ono Pharmaceutical Company, Ltd., Japan.
Rhee
S K
SK
Hamanaka
H
H
Mikoshiba
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000302:isolation & purification* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism*; D002846:Chromatography, Affinity; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010766:Phosphorylation; D011510:Proteolipids / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification* / Q000378:metabolism*; D014461:Ultracentrifugation
D000906:Antibodies; D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008081:Liposomes; D046911:Macromolecular Substances; D010455:Peptides; D011510:Proteolipids; D018160:Receptors, Cytoplasmic and Nuclear; C020485:proteoliposomes; D015544:Inositol 1,4,5-Trisphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium