The calmodulin-binding domain in the mouse type 1 inositol 1,4,5-trisphosphate receptor.
The calmodulin-binding domain in the mouse type 1 inositol 1,4,5-trisphosphate receptor.
复制标题
小鼠 1 型肌醇 1,4,5-三磷酸受体中的钙调蛋白结合域。
作者：
Maki K. Yamada;Atsushi;Miyawaki;Kazuki Saito;Terumi Nakajima;M. Yamamoto;Y. Ryo;T. Furuichi;K. Mikoshiba
我们测定了与小鼠1型INS(1，4，5)P3受体(IP3R1)钙调素(CaM)结合能力有关的氨基酸序列。我们从缺失的cDNA中表达了IP3R1的不同部分，并检测了它们与CaM的结合能力。结果表明，从Lys-1564到Arg-1585的序列是结合所必需的。Ala取代Trp-1576后，全长IP3R1失去了与CaM结合的能力。抗IP3R1 1564-1585残基的抗体抑制小脑IP3R1与CaM的结合。当加入Ca(2+)-CaM时，对应于1564-1585残基的多肽的荧光光谱发生了移动。根据荧光光谱的变化，我们估算了多肽与CaM之间的解离常数(Kd)为0.7微米。Kd的亚微摩尔值表明CaM和IP3R1在细胞内确实存在相互作用。还考察了其他类型的IP3R与CaM的结合能力。2IP3R的一部分，包括与IP3R1的CaM结合域序列相同的区域，也与CaM结合，而表达的全长3型IP3R不结合CaM。
We determined the amino acid sequence responsible for the calmodulin (CaM)-binding ability of mouse type 1 Ins(1,4,5)P3 receptor (IP3R1). We expressed various parts of IP3R1 from deleted cDNA and examined their CaM-binding ability. It was shown that the sequence stretching from Lys-1564 to Arg-1585 is necessary for the binding. The full-length IP3R1 with replacement of Trp-1576 by Ala lost its CaM-binding ability. Antibody against residues 1564-1585 of IP3R1 inhibited cerebellar IP3R1 from binding CaM. The fluorescence spectrum of the peptide that corresponds to residues 1564-1585 shifted when Ca(2+)-CaM was added. From the change in the fluorescence spectrum, we estimated the dissociation constant (KD) between the peptide and CaM to be 0.7 microM. The submicromolar value of KD suggests an actual interaction between CaM and IP3R1 within cells. The CaM-binding ability of other types of IP3Rs was also examined. A part of the type 2IP3R, including the region showing sequence identity with the CaM-binding domain of IP3R1, also bound CaM, while the expressed full-length type 3 IP3R did not.