title: Differential modulation of inositol 1,4,5-trisphosphate receptor type 1 and type 3 by ATP.
abstract: Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP(3)R) results in a more pronounced Ca(2+)release in the presence of inositol 1,4,5-trisphosphate (IP(3)). Two recently published studies demonstrated a different ATP sensitivity of IP(3)-induced Ca(2+)release in cell types expressing different IP(3)R isoforms. Cell types expressing mainly IP(3)R3 were less sensitive to ATP than cell types expressing mainly IP(3)R1 (Missiaen L, Parys JB, Sienaert I et al. Functional properties of the type 3 InsP(3)receptor in 16HBE14o- bronchial mucosal cells. J Biol Chem 1998;273: 8983-8986; Miyakawa T, Maeda A, Yamazawa T et al. Encoding of Ca(2+)signals by differential expression of IP(3)receptor subtypes. EMBO J 1999;18: 1303-1308). In order to investigate the difference in ATP sensitivity between IP(3)R isoforms at the molecular level, microsomes of Sf9 insect cells expressing full-size IP(3)R1 or IP(3)R3 were covalently labeled with ATP by using the photoaffinity label 8-azido[alpha-(32)P]ATP. ATP labeling of the IP(3)R was measured after immunoprecipitation of IP(3)Rs with isoform-specific antibodies, SDS-PAGE and Phosphorimaging. Unlabeled ATP inhibited covalent linking of 8-azido[alpha-(32)P]ATP to the recombinant IP(3)R1 and IP(3)R3 with an IC(50)of 1.6 microM and 177 microM, respectively. MgATP was as effective as ATP in displacing 8-azido[alpha-(32)P]ATP from the ATP-binding sites on IP(3)R1 and IP(3)R3, and in stimulating IP(3)-induced Ca(2+)release from permeabilized A7r5 and 16HBE14o- cells. The interaction of ATP with the ATP-binding sites on IP(3)R1 and IP(3)R3 was different from its interaction with the IP(3)-binding domains, since ATP inhibited IP(3)binding to the N-terminal 581 amino acids of IP(3)R1 and IP(3)R3 with an IC(50)of 353 microM and 4.0 mM, respectively. The ATP-binding sites of IP(3)R1 bound much better ATP than ADP, AMP and particularly GTP, while IP(3)R3 displayed a much broader nucleotide specificity. These results therefore provide molecular evidence for a differential regulation of IP(3)R1 and IP(3)R3 by ATP.
authors.affiliation: Laboratorium voor Fysiologie, K U Leuven Campus Gasthuisberg, Herestraat 49, Leuven, B-3000, Belgium.Karlien.Maes@med.kuleuven.ac.be
authors.forename: K
authors.initials: K
authors.lastname: Maes
authors.forename: L
authors.initials: L
authors.lastname: Missiaen
authors.forename: P
authors.initials: P
authors.lastname: De Smet
authors.forename: S
authors.initials: S
authors.lastname: Vanlingen
authors.forename: G
authors.initials: G
authors.lastname: Callewaert
authors.forename: J B
authors.initials: JB
authors.lastname: Parys
authors.forename: H
authors.initials: H
authors.lastname: De Smedt
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D004926:Escherichia coli; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051379:Mice; D020033:Protein Isoforms; D011817:Rabbits; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D000697:Central Nervous System Stimulants; D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D002110:Caffeine; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002118:Calcium