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

Abstract

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.

Fig. 1.

Effects of tunicamycin on glucagon-like peptide-1 (GLP-1) binding to human GLP-1 receptor-bearing Chinese hamster ovary (GLP-1R-CHO) cells. The GLP-1R-CHO cells were incubated for 40 h at 37°C with a range of concentrations of tunicamycin before being incubated for 1 h with the radioligand ¹²⁵I-GLP-1 in the presence of increasing concentrations of unlabeled GLP-1. A representative competition-binding curve is shown for each condition.

Fig. 2.

Effects of single mutations of the GLP-1 receptor glycosylation sites on ligand binding and biological activity. Left: binding curves for increasing concentrations of the natural GLP-1 ligand to displace binding of the radioligand ¹²⁵I-GLP-1 to COS-1 cells transfected with the wild-type (WT) and single Asn-to-Leu mutant receptors (N63L, N82L, and N115L). Cells were grown at 37°C after transfection. Values are calculated as percentages of maximal saturable binding observed in the absence of the competitor. They are expressed as means ± SE of duplicate data from 3 independent experiments. Right: intracellular cAMP responses in these cells (grown at 37°C) to stimulation by increasing concentrations of GLP-1. Values are expressed as the means ± SE of at least 3 independent experiments, with data normalized relative to maximal responses. Basal and maximal cAMP levels were similar for each of the GLP-1 receptor constructs tested (4.5 ± 0.9 and 191 ± 49 pmol/1,000,000 cells, respectively).

Fig. 3.

Effects of multiple mutations of the GLP-1 receptor glycosylation sites on ligand binding and biological activity. Left: binding curves for increasing concentrations of the natural GLP-1 ligand to displace binding of the radioligand ¹²⁵I-GLP-1 to COS-1 cells transfected with the WT and multiple Asn-to-Leu mutant receptors (N63L/N82L, N61L/N115L, N82L/N115L, and N63L/N82L/N115L). Cells were grown at 37°C after transfection. Values are calculated as percentages of maximal saturable binding observed in the absence of the competitor. They are expressed as means ± SE of duplicate data from 3 independent experiments. Right: intracellular cAMP responses in these cells (grown at 37°C) to stimulation by increasing concentrations of GLP-1. Values are expressed as means ± SE of at least 3 independent experiments, with data normalized relative to maximal responses.

Fig. 4.

Morphological localization of GLP-1 receptor constructs. Shown are representative examples of anti-GLP-1 receptor antibody immunostaining of COS-1 cells transfected with the WT and Asn-to-Leu mutant GLP-1 receptors with (top and upper middle rows) and without (lower middle and bottom rows) saponin treatment and cells transfected with the empty pcDNA3 eukaryotic expression vector. Also shown are COS-1 cells transfected with the WT GLP-1 receptor but immunostained only with Alexa 488-conjugated goat anti-mouse IgG. Cells used in this figure were grown at 37°C after transfection. Images are representative of 3 repetitions.

Fig. 5.

Functional characterization of multiple Asn-to-Leu mutant receptors in COS-1 cells grown at low temperature. Left: binding curves for increasing concentrations of the natural GLP-1 ligand to displace binding of the radioligand ¹²⁵I-GLP-1 to COS-1 cells transfected with the WT and the indicated multiple Asn-to-Leu mutant receptors. Cells were grown at 30°C after transfection. Values are calculated as percentages of maximal saturable binding for each of the receptor constructs observed in the absence of the competitor. They are expressed as means ± SE of duplicate data from 3 independent experiments. Right: intracellular cAMP responses in these cells (grown at 30°C) to stimulation by increasing concentrations of GLP-1. Values are expressed as means ± SE of at least 3 independent experiments, with data normalized relative to maximal responses observed for the WT GLP-1 receptor. Basal and maximal cAMP levels for the WT GLP-1 receptor were 5.1 ± 1.1 and 204 ± 53 pmol/1,000,000 cells, respectively.

Fig. 6.

Morphological localization of multiple Asn-to-Leu mutant receptors in COS-1 cells grown at low temperature. Shown are representative examples of anti-GLP-1 receptor antibody immunostaining of COS-1 cells transfected with the WT and the indicated multiple Asn-to-Leu mutant GLP-1 receptors. Shown also are COS-1 cells transfected with the WT GLP-1 receptor but immunostained only with Alexa 488-conjugated goat anti-mouse IgG. Cells used in this experiment were grown at 30°C after transfection.