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

Parys, J. B.; de Smedt, H.; Missiaen, L.; Bootman, M. D.; Sienaert, I. and Casteels, R. (1995). 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. Cell Calcium, 17(4) pp. 239–249.

URL: http://www.sciencedirect.com/science/article/pii/0...

Abstract

This study concerns the detection and analysis of the highly homologous type II-like inositol 1,4,5-trisphosphate (InsP₃) receptors (InsP₃R-II, -IV and -V). We have particularly investigated RBL-2H3 cells, which at the mRNA level predominantly expressed InsP₃R-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 InsP₃ binding affinity (K_d 3.8 ± 0.8 nM, B_max 0.40 ± 0.08 pmol/mg protein) than microsomes from A7r5 cells (Kd 65 ± 7 nM, B_max 0.65 ± 0.08 pmol/mg protein) or from cerebellum (Kd 135 ± 14 nM, B_max 7.35 ± 1.13 pmol/mg protein). An affinity-purified antibody against the C-terminus of type II-like InsP₃Rs detected, after SDS-PAGE and immunoblotting, a 250 kD protein in RBL-2H3 and C₃H10T1/2 cells, but not in other cell types. An isoform-specific antibody against the C-terminus of InsP₃R-I was used to determine the presence of the various InsP₃R-I splice isoforms at the protein level. The 273 kD (brain), 261 kD (peripheral tissues) and 256 kD (Xenopus oocytes) isoforms were recognized. Expression of InsP₃R-I in RBL-2H3 cells was very low. Taken together, our results support the hypothesis that InsP₃R isoforms may differ to a large extent in their affinity for InsP₃ and suggest that RBL-2H3 cells are a useful model for the study of InsP₃R-IV.

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