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  • Analogous to all GPCRs the

    2022-09-28

    Analogous to all GPCRs, the GLP-1R transduces signals from the outside of the cell to the inside by allosterically converting a ligand-binding event to activation of intracellular effectors. The GLP-1R couples to several intracellular effectors and this pleiotropic coupling is physiologically relevant and can lead to biased agonism, whereby distinct ligands can promote distinct profiles of cellular signaling [23]. Moreover, our previous work reveals that extended GLP-1 peptides are biased agonists at the GLP-1R relative to fully processed peptides, with higher relative efficacy for signaling pathways such as ERK1/2 phosphorylation and limited ability to promote calcium mobilization [24], [25], [26]. GLP-1R activation leading to intracellular signaling relies on a series of polar transmembrane networks within the receptor. Our previous work highlighted that differential engagement of these networks by endogenous ligands, GLP-1(7-36)NH2 and oxyntomodulin and the exogenous ligand exendin-4 may contribute to their observed biased agonism [27], [28], [29]. Here, we extend this work to understand whether GLP-1R engagement by extended GLP-1 or processed GLP-1 peptides results in similar patterns of receptor activation and how this links to differential signaling profiles of these ligands.
    Materials and methods
    Results We have previously shown that networks of conserved polar residues in the transmembrane bundle of the GLP-1R are important for regulating receptor activation and effector coupling. Alanine scanning mutagenesis of 24 conserved polar residues in GLP-1R revealed that these residues are differentially involved in effector coupling and the extent of their involvement differs when GLP-1(7-36)NH2 is compared with oxyntomodulin and the GLP-1 mimetic exendin-4 [27], [28], [29]. In this study, we have performed an extensive comparison of binding and signaling of the 4 forms of GLP-1 (alignment shown in Fig. 1B) at the wildtype GLP-1 receptor and alanine mutants of each of the 24, conserved polar residues in the TM bundle and TM boundaries of the receptor (Fig. 1A and C). These residues have been clustered based on their location and role within the GLP-1R (Fig. 1C, [28]). In our previous study, we used isogenic integration of both wildtype and mutant human GLP-1Rs into flpIN-CHO 3065 using site specific recombination and performed two independent measures of cell surface expression that revealed alanine substitution of many of these key polar residues reduced the cell surface expression; thus residues R2.60190A, H6.52363A, H2.50180A, E3.50247A, T6.42353A, R2.46176 A, R6.37348A, S1.50155A, N2.56182A and N5.40310A all showed statistically significantly lower expression when compared with wild type receptor [27], [28], [29]. These previously characterised cell lines were used to assess affinity and signaling of GLP-1 variants, and the determined values for cell surface expression (re-plotted from original data in Fig. 2) were used to correct for efficacy related effects linked to altered expression. In addition, while all four GLP-1 peptide variants were assessed in parallel, the affinity (pIC50) and efficacy (tauc) data for GLP-1(7-36)NH2 has been published previously in [27], [28], [29], but is discussed here in relation to the other 3 GLP-1 variants.
    Discussion The residues examined in this study are generally conserved in class B GPCRs and are predicted to be involved in regulating conformational transition of the GLP-1R during activation or to be involved in stabilizing receptor structure. Of the residues assessed in this study, R5.40310 is located within the peptide binding pocket and forms a hydrogen bond with the distal N-terminal glutamic acid of exendin-P5 when bound to active GLP-1R, and is important for this peptide to propagate signaling through the receptor to mediate cAMP production [33]. Similarly, an interaction between R5.40310 and the N-terminus of GLP-1(7-36)NH2 is predicted to occur, although there is insufficient density in the active GLP-1:GLP-1R structure to accurately model these side chains [34]. Consistent with these predictions, alanine substitution of R5.40310 heavily reduced the affinity of both processed peptides but, interestingly, this mutation had little effect on the binding affinity of extended peptides, a pattern that was also true for several of the residues of the central polar network such as R2.60190 and H6.52363 (see below and Fig. 9). The positioning of the 3065 peptide backbones in both cryo-EM structures suggests that the extended peptides would probably adopt a slightly different binding pose compared with GLP-1(7-36)NH2 or GLP-1(7-37) and would also need to adopt a turn motif such that the extended N-terminus could exit in the opening between helix 5 and 6 [35]. This likely results in different interactions formed by R5.40310 when these peptides are bound. Nonetheless, this side chain was required for cAMP efficacy of these extended peptides, but not the processed peptides suggesting that while the interactions formed by R5.40310 may differ, this residue is important for receptor function for all four GLP-1 peptides, being required for affinity of the high affinity peptides, but signal propagation for the lower affinity peptides. Given the positioning of the N-terminus of peptides observed in both cryo-EM structures of GLP-1R [32], [33], it is unsurprising that we do not observe any differences in binding affinity attributable to the amide compared with glycine extended carboxy-termini of the peptides at this binding site residue.