PURIFICATION AND CHARACTERIZATION OF THE HUMAN TYPE-1 INS(1,4,5)P-3 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)P-3 (InsP(3)) receptor. This receptor, isolated from platelets, has an apparent molecular mass on SDS/PAGE of 252 kDa and is chromatograp ed by gel filtration as an oligomer of about 1 x 10(6) kDa. [H-3]InsP( 3) bound to a single class of sites on the purified receptor protein w ith a K-d of 27 nM and a B-max. of 2.2 nmol/mg of protein. The platele t InsP, receptor, like the rodent cerebellar receptors, was identity i mmunochemically as a type 1 receptor, but unlike its brain counterpart s bound poorly to concanavaln A and other lectins and was not signific antly phosphorylated by protein kinase A. All cultured megakaryocytic leukaemia cell lines (e.g. Dami, CHRF-288 and Meg-01) and HEL cells we re also immunopositive for type 1 receptor, which was substantially in creased in some cases by DMSO or phorbol 12-myristate 13-acetate (PMA) which induce further megakaryocytic differentiation. Normal mixed lym phocyte and granulocyte fractions and an enriched T-cell fraction from human blood had measurable InsP(3)-binding activity, but no detectabl e 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 detectabl e type 1 receptor unless they were stimulated to differentiate along m onocyte/macrophage lines by PMA. We conclude that: (1) of the major no rmal blood cells only platelets contain type 1 InsP(3) receptors; (2) some neoplastic transformed blood cell lines also express type 1 recep tors, in contrast to their normal counterparts; and (3) increased leve ls of type 1 InsP(3) receptor are induced in some transformed cells un der conditions that favour their further terminal differentiation.