Jayaraman T, Brillantes A M, Timerman A P, Fleischer S, Erdjument-Bromage H, Tempst P, Marks A R
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029.
J Biol Chem. 1992 May 15;267(14):9474-7.
The calcium release channel (CRC)/ryanodine receptor (RyRec) has been identified as the foot structure of the sarcoplasmic reticulum (SR) and provides the pathway for calcium efflux required for excitation-contraction coupling in skeletal muscle. The CRC has previously been reported to consist of four identical 565-kDa protomers. We now report the identification of a 12-kDa protein which is tightly associated with highly purified RyRec from rabbit skeletal muscle SR. N-terminal amino acid sequencing and cDNA cloning demonstrates that the 12-kDa protein from fast twitch skeletal muscle is the binding protein for the immunosuppressant drug FK506. In humans, FK506 binds to the 12-kDa FK506-binding protein (FKBP12) and blocks calcium-dependent T cell activation. We find that FKBP12 and the RyRec are tightly associated in skeletal muscle SR on the basis of: 1) co-purification through sequential heparin-agarose, hydroxylapatite, and size exclusion chromatography columns; 2) coimmunoprecipitation of the RyRec and FKBP12 with anti-FKBP12 antibodies; and 3) subcellular localization of both proteins to the terminal cisternae of the SR, and not in the longitudinal tubules of SR, in fast twitch skeletal muscle. The molar ratio of FKBP12 to RyRec in highly purified RyRec preparations is approximately 1:4, indicating that one FKBP12 molecule is associated with each calcium release channel/foot structure.
钙释放通道（CRC）/雷诺丁受体（RyRec）已被确定为肌浆网（SR）的足状结构，并为骨骼肌兴奋-收缩偶联所需的钙外流提供途径。此前有报道称CRC由四个相同的565 kDa原聚体组成。我们现在报告鉴定出一种12 kDa的蛋白质，它与从兔骨骼肌SR中高度纯化的RyRec紧密相关。N端氨基酸测序和cDNA克隆表明，快速收缩骨骼肌中的12 kDa蛋白质是免疫抑制药物FK506的结合蛋白。在人类中，FK506与12 kDa的FK506结合蛋白（FKBP12）结合，并阻断钙依赖性T细胞活化。我们发现，基于以下几点，FKBP12和RyRec在骨骼肌SR中紧密相关：1） 通过连续的肝素-琼脂糖、羟基磷灰石和尺寸排阻色谱柱共纯化；2） 用抗FKBP12抗体对RyRec和FKBP12进行共免疫沉淀；3） 在快速收缩骨骼肌中，这两种蛋白质在亚细胞水平上均定位于SR的终池，而非SR的纵管。在高度纯化的RyRec制剂中，FKBP12与RyRec的摩尔比约为1:4，表明每个钙释放通道/足状结构都有一个FKBP12分子与之相关。
