RAT BASOPHILIC LEUKEMIA-CELLS AS MODEL SYSTEM FOR INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR-IV, A RECEPTOR OF THE TYPE-II FAMILY - FUNCTIONAL COMPARISON AND IMMUNOLOGICAL DETECTION

This study concerns the detection and analysis of the highly homologous type II-like inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)R-II, -IV and -V). We have particularly investigated RBL-2H3 cells, which at the mRNA level predominantly expressed InsP(3)R-IV [De Smedt H. Missiaen L. Parys JB. et al. (1994) Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. J. Biol. Chem., 269, 21691-21698]. When measured in identical experimental conditions, microsomes from RBL-2H3 cells were characterized by a much higher InsP(3) binding affinity (K-d 3.8 +/- 0.8 nM, B-max 0.40 +/- 0.08 pmol/mg protein) than microsomes from A7r5 cells (K-d 65 +/- 7 nM, B-max 0.65 +/- 0.08 pmol/mg protein) or from cerebellum (K-d 135 +/- 14 nM, B-max 7.35 +/- 1.13 pmol/mg protein). An affinity-purified antibody against the C-terminus of type Ii-like InsP(3)Rs detected, after SDS-PAGE and immunoblotting, a 250 kD protein in RBL-2H3 and C(3)H10T1/2 cells, but not in other cell types, An isoform-specific antibody against the C-terminus of InsP(3)R-I was used to determine the presence of the various InsP(3)R-I splice isoforms at the protein level. The 273 kD (brain), 261 kD (peripheral tissues) and 258 kD (Xenopus oocytes) isoforms were recognized. Expression of InsP(3)R-I in RBL-2H3 cells was very low. Taken together, our results support the hypothesis that InsP(3)R isoforms may differ to a large extent in their affinity for InsP(3) and suggest that RBL-2H3 cells are a useful model for the study of InsP(3)R-IV.