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Ca2+ and calmodulin differentially modulate myo-inositol 1,4, 5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms.

ABSTRACT: We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP(3)R1), IP(3)R2 and IP(3)R3 as recombinant proteins [ligand-binding site 1 (lbs-1), lbs-2, lbs-3] in the soluble fraction of Escherichia coli. These recombinant proteins contain the complete IP(3)-binding domain and bound IP(3) and adenophostin A with high affinity. Ca(2+) and calmodulin were previously found to maximally inhibit IP(3) binding to lbs-1 by 42+/-6 and 43+/-6% respectively, and with an IC(50) of approx. 200 nM and 3 microM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca(2+) inhibited IP(3) binding to lbs-3 with an IC(50) of approx. 700 nM (37+/-4% inhibition at 5 microM Ca(2+)), while IP(3) binding to lbs-2 was not affected by increasing [Ca(2+)] from 100 nM to 25 microM. Calmodulin (10 microM) inhibited IP(3) binding to lbs-3 by 37+/-4%, while IP(3) binding to lbs-2 was inhibited by only 11+/-2%. The inhibition of IP(3) binding to lbs-3 by calmodulin was dose-dependent (IC(50) approximately 2 microM). We conclude that the IP(3)-binding domains of the various IP(3)R isoforms differ in binding characteristics for IP(3) and adenophostin A, and are differentially modulated by Ca(2+) and calmodulin, suggesting that the various IP(3)R isoforms can have different intracellular functions.

SUBMITTER: Vanlingen S

PROVIDER: S-EPMC1220851 | biostudies-other | 2000 Mar

REPOSITORIES: biostudies-other

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Project description:The Ins(1,4,5)P3 regioisomers, Ins(1,4,6)P3 and Ins(1,3,6)P3, which can mimic the 1,4,5-arrangement on the inositol ring of Ins(1,4,5)P3, were examined for Ca2+ release by using four types of saponin-permeabilized cell possessing various abundances of receptor subtypes, with special reference to the relation of potency to receptor subtype. Ins(1,4,6)P3 and Ins(1,3,6)P3 were weak agonists in rat basophilic leukaemic cells (RBL cells), which possess predominantly subtype II receptors, with respective potencies of 1/200 and less than 1/500 that of Ins(1,4,5)P3 [the EC50 values were 0.2, 45 and more than 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively]. Similar rank order potencies were also evaluated for the displacement of [3H]Ins(1,4,5)P3 bound to RBL cell membranes by these regioisomers. However, they caused Ca2+ release from GH3 rat pituitary cells possessing predominantly subtype I receptors more potently; Ins(1,4,6)P3 and Ins(1,3,6)P3 evoked release at respective concentrations of only one-third and one-twentieth that of Ins(1,4,5)P3 (the EC50 values were 0.4, 1.2 and 8 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In COS-1 African green-monkey kidney cells, with the relative abundances of 37% of the subtype II and of 62% of the subtype III receptor, potencies of 1/40 and approx. 1/200 for Ins(1, 4,6)P3 and Ins(1,3,6)P3 respectively were exhibited relative to Ins(1,4,5)P3 (the EC50 values were 0.4, 15 and approx. 80 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In HL-60 human leukaemic cells, in spite of the dominant presence of subtype I receptors (71%), similar respective potencies to those seen with COS-1 cells were exhibited (the EC50 values were 0.3, 15 and approx. 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). These results indicate that these regioisomers are the first ligands that distinguish between receptor subtypes; the present observations are of significance for the future design of molecules with enhanced selectivity.

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Ca2+ and calmodulin differentially modulate myo-inositol 1,4, 5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms.

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