Sczekan M M, Juliano R L
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599-7365.
J Cell Physiol. 1990 Mar;142(3):574-80. doi: 10.1002/jcp.1041420317.
Using a monoclonal antibody specific for the hamster fibronectin receptor (FnR), we have demonstrated that a portion of the CHO cell FnR population is constitutively endocytosed. Three independent techniques were used to study the internalization: 1) after saturation binding of an anti-FnR antibody (PB1) to cells at 4 degrees C, internalization was initiated by warming to 37 degrees C, and then acid/salt elution of membrane-bound ligand was used to quantitate the internalized 125I-PB1; 2) cell vesicular traffic was pharmacologically disrupted with monensin or chloroquine, and the subsequent reduction of the cell surface pool of FnR was monitored; and 3) selective immunoprecipitation was used to separate surface and internalized 125I-labeled FnR. These experiments indicate that about 30% of the cell surface FnR is endocytosed with a t1/2 of 7 min and that this internalization occurs regardless of the ligation state of the receptor. Other observations indicate that the larger fraction of the cell surface FnR pool (70-75%) is apparently shed from the cell upon ligation with antibody at 37 degrees C. This process occurs much more slowly than receptor internalization and leads to an overall reduction in the amount of cell surface FnR. Our results suggest physically or chemically distinct populations of FnR, one of which is unavailable for internalization and recycling.
利用一种对仓鼠纤连蛋白受体（FnR）具有特异性的单克隆抗体，我们已经证明CHO细胞FnR群体的一部分会持续发生内吞作用。我们使用了三种独立技术来研究内化过程：1）在4℃下抗FnR抗体（PB1）与细胞饱和结合后，通过升温至37℃启动内化，然后用酸/盐洗脱膜结合配体来定量内化的125I-PB1；2）用莫能菌素或氯喹药理学破坏细胞囊泡运输，并监测随后FnR细胞表面池的减少；3）使用选择性免疫沉淀来分离表面和内化的125I标记的FnR。这些实验表明，约30%的细胞表面FnR会发生内吞作用，半衰期为7分钟，并且这种内化作用的发生与受体的连接状态无关。其他观察结果表明，细胞表面FnR池的较大部分（70-75%）在37℃下与抗体连接时显然会从细胞上脱落。这个过程比受体内化慢得多，并导致细胞表面FnR数量的总体减少。我们的结果表明FnR存在物理或化学性质不同的群体，其中之一无法进行内化和再循环。
