Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 13400 East Shea Blvd., Scottsdale, AZ 85259, USA.
Am J Physiol Endocrinol Metab. 2010 Jul;299(1):E62-8. doi: 10.1152/ajpendo.00067.2010. Epub 2010 Apr 20.
The family B G protein-coupled glucagon-like peptide 1 (GLP-1) receptor is an important drug target for treatment of type 2 diabetes. Like other family members, the GLP-1 receptor is a glycosylated membrane protein that contains three potential sites for N-linked glycosylation within the functionally important extracellular amino-terminal domain. However, the roles for each potential site of glycosylation in receptor biosynthesis, trafficking, and function are not known. In this work, we demonstrated that tunicamycin inhibition of glycosylation of the GLP-1 receptor expressed in CHO cells interfered with biosynthesis and intracellular trafficking, thereby eliminating natural ligand binding. To further investigate the roles of each of the glycosylation sites, site-directed mutagenesis was performed to eliminate these sites individually and in aggregate. Our results showed that mutation of each of the glycosylation sites individually did not interfere with receptor expression on the cell surface, ligand binding, and biological activity. However, simultaneous mutation of two or three glycosylation sites resulted in almost complete loss of GLP-1 binding and severely impaired biological activity. Immunostaining studies demonstrated receptor biosynthesis but aberrant trafficking, with most of the receptor trapped in the endoplasmic reticulum and golgi compartments and little of the receptor expressed on the cell surface. Interestingly, surface expression, ligand binding, and biological activity of these mutants improved significantly when biosynthesis was slowed using low temperature (30 degrees C). These data suggest that N-linked glycosylation of the GLP-1 receptor is important for its normal folding and trafficking to the cell surface.
家族 B G 蛋白偶联胰高血糖素样肽 1（GLP-1）受体是治疗 2 型糖尿病的重要药物靶点。像其他家族成员一样，GLP-1 受体是一种糖基化的膜蛋白，在功能重要的细胞外氨基末端结构域内含有三个潜在的 N-连接糖基化位点。然而，每个潜在糖基化位点在受体生物合成、运输和功能中的作用尚不清楚。在这项工作中，我们证明了在 CHO 细胞中表达的 GLP-1 受体的衣霉素抑制糖基化会干扰生物合成和细胞内运输，从而消除天然配体结合。为了进一步研究每个糖基化位点的作用，我们进行了定点突变，单独和集体消除这些位点。我们的结果表明，单独突变每个糖基化位点不会干扰细胞表面受体的表达、配体结合和生物学活性。然而，同时突变两个或三个糖基化位点几乎完全消除了 GLP-1 的结合，并严重损害了生物学活性。免疫染色研究表明受体的生物合成，但出现异常的运输，大多数受体被困在内质网和高尔基体区室中，很少有受体表达在细胞表面。有趣的是，当使用低温（30°C）减缓生物合成时，这些突变体的表面表达、配体结合和生物学活性显著改善。这些数据表明，GLP-1 受体的 N-连接糖基化对于其正常折叠和运输到细胞表面是重要的。
