We have studied the effects of monoclonal antibodies that recognize different epitopes of the cerebellar Ins(1,4,5)P3 receptor on Ins(1,4,5)P3-induced Ca(2+)-release activity. Ins(1,4,5)P3 stimulated Ca2+ flux from cerebellar microsomes, and half-maximal Ca2+ release occurred at 112 +/- 8 nM-Ins(1,4,5)P3 [concentration causing half-maximal effect (EC50) = 112.8 nM]. The minimum concentration of Ins(1,4,5)P3 necessary to initiate Ca2+ release (threshold concentration) was 20 +/- 5 nM. A monoclonal antibody (mAb) 18A10 (50 micrograms/ml), which recognizes the C-terminal region of the Ins(1,4,5)P3 receptor, suppressed Ins(1,4,5)P3-induced Ca2+ release: the EC50 and threshold concentration shifted to 460 +/- 56 nM and 61 +/- 6 nM respectively. On the other hand, the antibody at the same concentration raised the affinity of the receptor for binding to Ins(1,4,5)P3, and the Kd value decreased from 43 +/- 12 nM to 25 +/- 4 nM without a change in the number of Ins(1,4,5)P3-binding sites. However, mAbs that recognize the N-terminal domain affected neither Ca2+ release nor Ins(1,4,5)P3 binding. Among the various synthetic peptides, only the 12-residue-long peptide from the most C-terminal portion of the receptor (amino acid residues 2736-2747) reacted strongly with mAb18A10. From these findings, combined with the Immunogold localization of the cerebellar Ins(1,4,5)P3 receptor [Otsu, Yamamoto, Maeda, Mikoshiba & Tashiro (1990) Cell Struct. Funct. 15, 163-173], we concluded that the C-terminus of the Ins(1,4,5)P3 receptor is exposed to the cytoplasmic side of the smooth endoplasmic reticulum and plays an important role in the regulation of both Ins(1,4,5)P3-binding affinity and channel gating.
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July 1991
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Research Article|
July 01 1991
Involvement of the C-terminus of the inositol 1,4,5-trisphosphate receptor in Ca2+ release analysed using region-specific monoclonal antibodies
S Nakade;
S Nakade
*Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita Osaka 565, Japan
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N Maeda;
N Maeda
*Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita Osaka 565, Japan
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K Mikoshiba
K Mikoshiba
*Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita Osaka 565, Japan
†Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1991 The Biochemical Society, London
1991
Biochem J (1991) 277 (1): 125–131.
Citation
S Nakade, N Maeda, K Mikoshiba; Involvement of the C-terminus of the inositol 1,4,5-trisphosphate receptor in Ca2+ release analysed using region-specific monoclonal antibodies. Biochem J 1 July 1991; 277 (1): 125–131. doi: https://doi.org/10.1042/bj2770125
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