Characterization of a cytosolic and a luminal Ca2+ binding site in the type I inositol 1,4,5-trisphosphate receptor.
To study the Ca2+ regulation of the inositol 1,4,5-trisphosphate receptor (InsP3R) at the molecular level, we expressed various cytosolic and luminal regions of the mouse type I InsP3R as glutathione S-transferase fusion proteins. 45Ca2+ and ruthenium red overlay studies and Stains-all spectra and staining revealed both a cytosolic and a luminal Ca2+ binding site. The luminal Ca2+ binding site was mapped to the nonconserved acidic subregion of the luminal loop between amino acids 2463 and 2528. A K0.5 of 0.3 microM and a Hill coefficient of 1.1 were determined by 45Ca2+ overlay by quantification of 45Ca2+ binding on blots. The cytosolic Ca2+ binding site was localized in a region just preceding the transmembrane domain M1. The Ca2+ binding was mapped to a 23-amino acid stretch between amino acids 2124 and 2146. This cytosolic region showed a single high affinity site for Ca2+, with a K0.5 of 0. 8 microM and a Hill coefficient of 1.0. Neither of the identified Ca2+ binding regions contained an EF-hand motif. We conclude that the type I InsP3R has at least two quite distinct types of Ca2+ binding sites, which are localized in different structural regions of the protein.
Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium.
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Sienaert
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De Smedt
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JB
Parys
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Missiaen
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Vanlingen
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Sipma
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Casteels
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012430:Ruthenium Red / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D012430:Ruthenium Red; D015544:Inositol 1,4,5-Trisphosphate; D005982:Glutathione Transferase; D002118:Calcium