title: 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.
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 13400 East Shea Blvd., Scottsdale, AZ 85259, USA.
authors.forename: Quan
authors.initials: Q
authors.lastname: Chen
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Miller
authors.forename: Maoqing
authors.initials: M
authors.lastname: Dong
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D052216:Glucagon-Like Peptide 1 / Q000096:biosynthesis / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000096:biosynthesis / Q000378:metabolism*; D017510:Protein Folding; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology*
chemical_list: D010446:Peptide Fragments; D014415:Tunicamycin; C055453:glucagon-like peptide 1 (7-36)amide; D052216:Glucagon-Like Peptide 1
references.citation: Biochemistry. 2000 May 30;39(21):6514-20
references.pmid: 10828967
references.citation: Annu Rev Biochem. 1988;57:785-838
references.pmid: 3052290
references.citation: J Biol Chem. 1984 Aug 25;259(16):10414-21
references.pmid: 6088499
references.citation: J Biol Chem. 1994 Aug 5;269(31):19738-44
references.pmid: 8051054
references.citation: Hum Reprod Update. 1999 Jul-Aug;5(4):356-64
references.pmid: 10465525
references.citation: Gastroenterology. 1993 Nov;105(5):1534-43
references.pmid: 8224659
references.citation: Biochim Biophys Acta. 2001 May 28;1539(1-2):131-9
references.pmid: 11389975
references.citation: J Mol Neurosci. 2008 Nov;36(1-3):279-85
references.pmid: 18401761
references.citation: Biochemistry. 1996 Feb 13;35(6):1745-52
references.pmid: 8639654
references.citation: Am J Physiol Endocrinol Metab. 2001 Nov;281(5):E1015-21
references.pmid: 11595658
references.citation: Mol Endocrinol. 2008 Jun;22(6):1489-99
references.pmid: 18372345
references.citation: J Biol Chem. 2009 Dec 4;284(49):34135-44
references.pmid: 19815559
references.citation: Pharmacol Ther. 2007 Mar;113(3):546-93
references.pmid: 17306374
references.citation: Biochemistry. 2009 Dec 15;48(49):11773-85
references.pmid: 19886671
references.citation: Endocrinology. 1999 Nov;140(11):5102-11
references.pmid: 10537138
references.citation: Anal Biochem. 1980 Sep 1;107(1):220-39
references.pmid: 6254391
references.citation: Peptides. 1994;15(4):675-81
references.pmid: 7937345
references.citation: Br J Pharmacol. 2010 Jan;159(1):237-51
references.pmid: 20002095
references.citation: J Biol Chem. 1998 Dec 18;273(51):34558-67
references.pmid: 9852126
references.citation: Biol Chem. 2004 Jan;385(1):49-57
references.pmid: 14977046
references.citation: J Biol Chem. 1996 Jun 21;271(25):14944-9
references.pmid: 8663161
references.citation: Pancreas. 1996 Aug;13(2):130-9
references.pmid: 8829180
references.citation: FEBS Lett. 2000 Dec 15;486(3):320-4
references.pmid: 11119727
references.citation: J Biol Chem. 2010 Jan 1;285(1):723-30
references.pmid: 19861722
references.citation: Biochemistry. 1999 Feb 9;38(6):1866-72
references.pmid: 10026267
references.citation: Biochemistry. 1999 Jul 6;38(27):8621-7
references.pmid: 10393537
references.citation: Biochem Biophys Res Commun. 2006 Feb 10;340(2):395-402
references.pmid: 16364240
references.citation: Biochemistry. 1996 Dec 10;35(49):15890-5
references.pmid: 8961954
references.citation: Mol Endocrinol. 2004 Jul;18(7):1768-77
references.pmid: 15105436
references.citation: J Biol Chem. 2008 Apr 25;283(17):11340-7
references.pmid: 18287102