Vanlingen S, Sipma H, De Smet P, Callewaert G, Missiaen L, De Smedt H, Parys J B
Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium.
Biochem J. 2000 Mar 1;346 Pt 2(Pt 2):275-80.
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.
我们已将1型肌醇1,4,5 -三磷酸受体（IP(3)R1）、IP(3)R2和IP(3)R3的N端581个氨基酸作为重组蛋白[配体结合位点1（lbs - 1）、lbs - 2、lbs - 3]在大肠杆菌的可溶部分中表达。这些重组蛋白包含完整的IP(3)结合结构域，并能以高亲和力结合IP(3)和腺嘌呤核苷三磷酸（adenophostin A）。先前发现Ca(2+)和钙调蛋白分别能最大程度地抑制IP(3)与lbs - 1的结合，抑制率分别为42±6%和43±6%，IC(50)分别约为200 nM和3 μM [西普马、德·斯梅特、西纳尔特、范林根、米西亚恩、帕里斯和德·斯梅德特（1999年）《生物化学杂志》274, 12157 - 12562]。我们现在报告，Ca(2+)抑制IP(3)与lbs - 3的结合，IC(50)约为700 nM（在5 μM Ca(2+)时抑制率为37±4%），而IP(3)与lbs - 2的结合不受[Ca(2+)]从100 nM增加到25 μM的影响。钙调蛋白（10 μM）抑制IP(3)与lbs - 3的结合，抑制率为37±4%，而IP(3)与lbs - 2的结合仅被抑制11±2%。钙调蛋白对IP(3)与lbs - 3结合的抑制呈剂量依赖性（IC(50)约为2 μM）。我们得出结论，各种IP(3)R亚型的IP(3)结合结构域在与IP(3)和腺嘌呤核苷三磷酸（adenophostin A）的结合特性上存在差异，并受到Ca(2+)和钙调蛋白的不同调节，这表明各种IP(3)R亚型可能具有不同的细胞内功能。
