Purification and characterization of the human type 1 Ins(1,4,5)P3 receptor from platelets and comparison with receptor subtypes in other normal and transformed blood cells.
We report the first purification of a native human form of the Ins(1,4,5)P3 (InsP3) receptor. This receptor, isolated from platelets, has an apparent molecular mass on SDS/PAGE of 252 kDa and is chromatographed by gel filtration as an oligomer of about 1 x 10(6) kDa. [3H]InsP3 bound to a single class of sites on the purified receptor protein with a Kd of 27 nM and a Bmax. of 2.2 nmol/mg of protein. The platelet InsP3 receptor, like the rodent cerebellar receptors, was identified immunochemically as a type 1 receptor, but unlike its brain counterparts bound poorly to concanavalin A and other lectins and was not significantly phosphorylated by protein kinase A. All cultured megakaryocytic leukaemia cell lines (e.g. Dami, CHRF-288 and Meg-01) and HEL cells were also immunopositive for type 1 receptor, which was substantially increased in some cases by DMSO or phorbol 12-myristate 13-acetate (PMA) which induce further megakaryocytic differentiation. Normal mixed lymphocyte and granulocyte fractions and an enriched T-cell fraction from human blood had measurable InsP3-binding activity, but no detectable type 1 protein. In contrast, Jurkat E6-1 (T-cell lymphoma) cells and the transformed B-cell line RPMI 8392 were immunopositive for type 1 receptor. HL-60 (human promyelocytic leukaemia) cells had no detectable type 1 receptor unless they were stimulated to differentiate along monocyte/macrophage lines by PMA. We conclude that: (1) of the major normal blood cells only platelets contain type 1 InsP3 receptors; (2) some neoplastic transformed blood cell lines also express type 1 receptors, in contrast to their normal counterparts; and (3) increased levels of type 1 InsP3 receptor are induced in some transformed cells under conditions that favour their further terminal differentiation.
Department of Pharmacology, University of Connecticut School of Medicine, Farmington 06030, USA.
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O'Rourke
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Matthews
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Feinstein
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D015220:Calcium Channels / Q000302:isolation & purification* / Q000378:metabolism*; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008533:Megakaryocytes / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000302:isolation & purification* / Q000378:metabolism*; D012016:Reference Values; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide