Inositol 1,4,5-trisphophate receptor : localization and regulation

1.用抗小鼠小脑P400蛋白的单克隆抗体检测了1，4，5-三磷酸肌醇受体（IP 3R）的定位。其中一种抗体4C 11与小鼠内皮细胞、平滑肌细胞和表皮角质形成细胞中的240 kD蛋白质反应。免疫金标记的蛋白质观察几乎只在小窝，小未涂层inpocketings的质膜。培养的牛主动脉内皮细胞用膜不可渗透的生物素化试剂处理，用非离子去污剂提取的膜蛋白与链霉亲和素-琼脂糖混合。通过链霉亲和素树脂回收240-kD蛋白，证明其暴露于细胞表面.在小鼠转化角质形成细胞（PAM 212）和牛主动脉内皮细胞中，发现了沿着肌动蛋白丝的240-kD蛋白，用兔抗质膜Ca^<2+>-泵ATP酶的多克隆抗体进行免疫金电镜观察，发现该蛋白定位于多种小鼠细胞包括内皮细胞和平滑肌细胞的细胞膜小窝. 240-kD蛋白的功能目前尚不清楚，但考虑到其与IP 3 R的相似性，该蛋白可能与Ca^2+通过质膜的内流有关。本实验结果表明，小窝参与了Ca^2+通过质膜的进出。

1. Localization of inositol 1,4,5-trisphosphate receptor (IP3R) was examined by using monoclonal antibodies raised to mouse cerebellar P400 protein. One of the antibodies, 4C11, reacted with a 240-kD protein in endothelial cells, smooth muscle cells, and epidermal keratinocytes of the mouse. Immunogold labeling for the protein was observed almost exclusively in caveolae, small uncoated inpocketings of the plasma membrane.2. Cultured bovine aortic endothelial cells were treated with a membrane-impermeable biotinylating reagent, and membrane proteins extracted with non-ionic detergent were mixed with streptavidin-agarose. The 240-kD protein was recovered by the streptavidin resin, thus proved to be exposed to the cell surface.3. The 240-kD protein was observed along actin filaments in mouse transformed keratinocytes (PAM212) and bovine aortic endothelial cells.By immunogold electron microscopy using rabbit polyclonal antibody to plasmalemmal Ca^<2+>-pump ATPase, the protein was localized to caveolae of various mouse cells including endothelial cells and smooth muscle cells.5. Functions of the 240-kD protein are not known at present, but considering its similarity to IP3R, the protein may be related to Ca^<2+> influx through the plasma membrane. The results obtained in the present experiments implicate that caveolae are engaged in in and out of Ca^<2+> through the plasma membrane.