Role of N-linked glycosylation in biosynthesis, trafficking, and function of the human glucagon-like peptide 1 receptor.

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.