Detergent solubility of the inositol trisphosphate receptor in rat brain membranes. Evidence for association of the receptor with ankyrin.
Rat cerebellar membranes sedimenting at 10,000 x g contain significantly greater amounts of [3H]D-myo inositol 1,4,5-trisphosphate- (IP3) binding sites that are resistant to extraction by Triton X-100 than membranes sedimenting at 100,000 x g. Scatchard analysis of the Triton X-100-resistant binding site revealed the presence of a single binding site with an 8-fold higher affinity for IP3 than present in 10,000 x g membranes. The Triton X-100-resistant binding site displayed high specificity for Ins(1,4,5)P3 and was sensitive to inhibition by both heparin and calcium. A polyclonal antibody to the C terminus of the IP3 receptor (IP3R) and biotinylated concanavalin A recognized the same 235 kDa band in Western blots of membrane and Triton X-100-insoluble fractions. The ligand binding activity of the IP3R could be measured in immunoprecipitates obtained from detergent-soluble extracts treated with IP3R antibody. An antibody to chick erythrocyte ankyrin also immunoprecipitated [3H]IP3-binding sites and immunoreactive IP3R protein. These effects of ankyrin antibody were prevented by preadsorption to erythrocyte ghosts and were not reproduced by spectrin antibodies. Cross-reactivity of the ankyrin antibody with the IP3R was excluded by the demonstration of selective loss of immunoreactive ankyrin protein when IP3R antibody immunoprecipitates were boiled in SDS buffers or when membranes were alkali-treated. These results suggest that a population of IP3R in brain may be attached to an isoform of ankyrin. This may mediate interactions of the IP3R with the cytoskeleton and account for observations regarding the detergent insolubility of the protein.
Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107.
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D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D015220:Calcium Channels; D002531:Cerebellum / Q000378:metabolism*; D003208:Concanavalin A / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D003902:Detergents; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D051379:Mice; D017830:Octoxynol; D011092:Polyethylene Glycols; D011233:Precipitin Tests; D051381:Rats; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D012995:Solubility
D017487:Ankyrins; D015220:Calcium Channels; D003902:Detergents; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D003208:Concanavalin A; D011092:Polyethylene Glycols; D017830:Octoxynol