The role of an n-terminal aspartic acid residue (D67), highly conserved in family B GPCRs, in the synthesis and trafficking of the GLP-1 receptor

Yan Huang1, Graeme Robb2, Naichang Li3,1, Gary Willars1. 1Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, LE1 9HN, United Kingdom, 2Discovery R&D, CVGI, Mereside, Alderley Park, Cheshire SK10 4TG, United Kingdom, 3Department of Biochemistry, Tianjin Medical University, Tianjin, 300070, China.

The incretin hormone, glucagon like peptide-1 (GLP-1), mediates a range of antidiabetogenic effects through the GLP-1 receptor (GLP-1R), a Family B GPCR, which is a target for the treatment of type 2 diabetes. The extended GLP-1R N-terminus contains the sequence NRTFD (N63-D67). Synthetic NRTFD is a GLP-1R agonist with the aspartic acid residue (equivalent to D67) being critical (Dong et al., 2008). These data suggested that orthosteric agonists may cause conformational changes in the receptor resulting in the interaction of the endogenous sequence, NRTFD, with the body of the receptor to provide agonism. Interestingly, the residue equivalent to D67 is conserved in all liganded Family B GPCRs. Further, the crystal structure of the GLP-1R N-terminus shows D67 to form a network of interactions with nearby residues of the N-terminal domain (Underwood et al., 2010). These may be essential for the structure of this region and here we have, therefore, examined the role of D67 in the synthesis, expression and function of the GLP-1R.

Based on C-terminal HA- or EGFP-tagged wild-type (WT) GLP-1Rs (WTGLP-1R-HA or WTGLP-1R-EGFP), D67A, D67E and D67R mutants were generated and transiently expressed in HEK293 cells. Previously we demonstrated that epitope tags of WTGLP-1R-HA and WTGLP-1R-EGFP immunoblot as two bands (47kDa & 64kDa/66kDa & 93kDa respectively) and only the heavier bands, representing mature fully glycosylated receptors, are at the plasma membrane (Huang et al., 2010). Here, only the lower band of each D67 mutant was apparent, reflecting an immature but full-length receptor. Confocal imaging of WTGLP-1R-EGFP showed strong plasma membrane fluorescence but the D67A and D67E counterparts had only cytosolic fluorescence. Consistent with this, GLP-1 7-36 amide increased cAMP by WT (basal 26±5, stimulated 750±75 pmol/mg protein, mean±sem, n = 3) but not mutant receptors (<45 pmol/mg protein). Treatment of cells with a proteasome inhibitor, MG132 (10μM, 10h), partially restored plasma membrane expression assessed by immunoblotting, confocal imaging and cAMP responses to the small-molecule allosteric agonist, compound 2 (Knudsen et al., 2007) (62-85% of WT). MG132 partially restored GLP-1 7-36 amide-mediated cAMP elevation in D67E (47±5% WT, mean±sem, n = 3) but not D67A or D67R mutants.

The crystal structure of the N-terminal domain of the GLP-1R with either orthosteric agonist or antagonist bound indicates a hydrogen bonding network of D67 with Y69, A70, W72, R102 and R121. Using a molecular dynamics minimisation protocol we showed these interactions to be maintained following removal of ligand and solvent molecules. However, mutations of D67 removed functionality or meaningful interactions, suggesting loss of secondary structure. This may prevent appropriate post-translational processing and receptor trafficking to the plasma membrane and account for the lack of expression of the mutated receptors. Mutation is also predicted to allow the first helix (residues 30-60) to approach a loop containing D67. This helix forms a major binding interaction with GLP-1 and any conformational change may, therefore, influence orthosteric ligand binding accounting for reduced receptor function.

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