Involvement of the C-terminus of the inositol 1,4,5-trisphosphate receptor in Ca2+ release analysed using region-specific monoclonal antibodies.
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.
Nakade
S
S
Institute for Protein Research, Osaka University, Japan.
Maeda
N
N
Mikoshiba
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D002531:Cerebellum / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D051379:Mice; D008817:Mice, Mutant Strains; D008861:Microsomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011956:Receptors, Cell Surface / Q000276:immunology / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D000939:Epitopes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010455:Peptides; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium