Widmann C, Dolci W, Thorens B
Department of Pharmacology and Toxicology, University of Lausanne, Switzerland.
Biochem J. 1995 Aug 15;310 ( Pt 1)(Pt 1):203-14. doi: 10.1042/bj3100203.
Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.
胰高血糖素样肽-1（GLP-1）是葡萄糖诱导胰岛素分泌的最有效刺激物，其胰腺β细胞受体是G蛋白偶联受体新亚家族的成员，该亚家族包括血管活性肠肽、促胰液素和胰高血糖素的受体。在此，我们使用三种不同方法研究了激动剂诱导的GLP-1受体在受体转染的中国仓鼠肺成纤维细胞中的内化。首先，在4℃下与转染细胞结合的碘化GLP-1在细胞升温至37℃后以2 - 3分钟的半衰期内化。其次，通过使用特异性抗体对受体进行蛋白质印迹检测评估，暴露于GLP-1会导致受体分布从富含质膜的膜部分转移至富含内体的膜部分。第三，将表达GLP-1受体的细胞持续暴露于碘化GLP-1，在20 - 30分钟的延迟时间后，培养基中肽降解产物呈线性积累，表明受体在质膜和内体区室之间持续循环。钾离子耗竭和高渗状态抑制转铁蛋白内吞作用（已知该过程通过有被小窝形成发生）以及GLP-1受体内吞作用。与GLP-1相反，拮抗剂艾塞那肽-（9 - 39）不会导致受体内化。一轮GLP-1受体内化后的表面重新表达发生的半衰期约为15分钟。内化和表面重新表达速率的差异导致在持续暴露于GLP-1时受体在细胞内区室中逐渐重新分布。最后，还发现胰岛素瘤细胞表达的内源性GLP-1受体在激动剂结合后也会内化。我们的数据共同表明，GLP-1受体在激动剂结合后通过类似于单跨膜段受体的途径内化。GLP-1诱导的受体内化途径和动力学特征将有助于理解内化和再循环在该受体信号转导控制中的作用。
