The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism

Abstract

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Graphical abstract

Figure 1

Agonist Affinity Profiles of GLP-1R ECL Alanine Mutants Reveal the Importance of Individual Residues for Peptide Affinity pKi values for each peptide were derived from radioligand inhibition-binding experiments. Bars represent differences in calculated affinity (pKi) values for each mutant relative to the wild-type receptor for GLP-1 (top), oxyntomodulin (middle), and exendin-4 (bottom). Statistical significance of changes in affinity in comparison with wild-type was determined by one-way analysis of variance and Dunnett’s post-test, and values are indicated with an asterisk (^∗p < 0.05). Data that are statistically significant are colored based on the extent of effect. All values are ± SEM of four to six independent experiments, conducted in duplicate.

Figure 2

Heatmap 3D Representation of the GLP-1R Extracellular Face Based on Affinity-Binding Data Molecular model of the GLP-1R-GLP-1 complex showing the extracellular surface of the TM bundle. Residues that altered affinity of GLP-1 (A), oxyntomodulin (B), and exendin-4 (C) when mutated are highlighted. Teal indicates residues that were assessed and did not alter affinity; yellow (3- to 5-fold), pale orange (5- to 10-fold), orange (10- to 30-fold), and red (>30-fold) are residues that statistically altered affinity.

Figure 3

Peptide-Dependent Effects of ECL Mutations on cAMP Efficacy Differences in the coupling efficiency (logτ_c) for cAMP formation of ECL mutations, compared to the wild-type receptor, by GLP-1 (top), oxyntomodulin (middle), and exendin-4 (bottom). Statistical significance of changes in coupling efficacy was determined by one-way analysis of variance and Dunnett’s post-test, and values are indicated with an asterisk (^∗p < 0.05 compared with wild-type). Data that are statistically significant are colored based on the direction and extent of effect. All values are logτ_c ± SEM of four to six independent experiments, conducted in duplicate.

Figure 4

Peptide-Dependent Effects of ECL Mutations on Efficacy for Ca²⁺ Mobilization Differences in the coupling efficiency (logτ_c) for Ca²⁺ mobilization of ECL mutations, compared to the wild-type receptor, by GLP-1 (top) and exendin-4 (bottom). Statistical significance of changes in coupling efficacy was determined by one-way analysis of variance and Dunnett’s post-test, and values are indicated with an asterisk (^∗p < 0.05 compared with wild-type). Data that are statistically significant are colored based on the extent of effect. All values are logτ_c ± SEM of four to six independent experiments, conducted in duplicate.

Figure 5

Peptide-Dependent Effects of ECL Mutations on pERK1/2 Efficacy Differences in the coupling efficiency (logτ_c) to pERK1/2 of ECL mutations, compared to the wild-type receptor, by GLP-1 (top), oxyntomodulin (middle), and exendin-4 (bottom). Statistical significance of changes in coupling efficacy was determined by one-way analysis of variance and Dunnett’s post-test, and values are indicated with an asterisk (^∗p < 0.05 compared with wild-type). Data that are statistically significant are colored based on the direction and extent of effect. All values are logτ_c ± SEM of four to six independent experiments, conducted in duplicate.

Figure 6

Heatmap 3D Representation of the GLP-1R Extracellular Face Based on Efficacy Data from Three Different Signaling Assays Molecular model of the GLP-1R-GLP-1 complex showing the extracellular surface of the TM bundle. All residues assessed in this study are shown in the center box; the locations of residues in ECL1, ECL2, and ECL3 are highlighted in purple, orange, and blue, respectively. Residues that when mutated altered efficacy are highlighted in (A)–(H). (A–C) cAMP efficacy of GLP-1 (A), oxyntomodulin (B), and exendin-4 (C); (D and E) Ca²⁺ efficacy of GLP-1 (D) and exendin-4 (E); (F–H) pERK1/2 efficacy of GLP-1 (F), oxyntomodulin (G), and pERK1/2 (H). Teal indicates residues that were assessed and did not alter efficacy; yellow (3- to 5-fold), pale orange (5- to 10-fold), orange (10- to 30-fold), and red (>30-fold) are residues that statistically altered efficacy. The 3D heatmaps can be found in Active model S1 (Data S1).

Figure 7

3D Model Illustrating GLP-1R ECL Loop Residues that Are Globally or Selectively Important for GLP-1, Oxyntomodulin, and Exendin-4 Based on statistical significance (p < 0.05) of effect when mutated to alanine, experimentally observed effects on peptide affinity and efficacy can be mapped onto the molecular model to clearly highlight similarities and differences between the three peptide agonists. Residues highlighted in red reduce function (affinity [A] or efficacy [B and C]) of all three peptides, those in pink selectively reduce GLP-1 only, those in yellow selectively reduce exendin-4 only, and those in green selectively reduce oxyntomodulin only. A large number of residues are important for both GLP-1 and exendin but not oxyntomodulin, and these are highlighted in orange. Other colors represent either enhanced function (GLP-1 only/oxyntomoduin only or GLP-1 and exendin) or existence of opposite effects when mutated on oxyntomodulin compared to GLP-1 and exendin-4.

Figure S1

cAMP and Intracellular Calcium Mobilization Are G Protein-Mediated Signaling Pathways, whereas pERK1/2 Is a Composite of G Protein-Mediated and Non-G Protein-Mediated Events, Related to Figures 3, 4, 5, and 6 pEC₅₀ concentrations of GLP-1 (top), exendin-4 (middle), and oxyntomodulin (bottom) were assessed for cAMP formation (left), calcium mobilization (right), and pERK1/2 (middle) in the absence (control) and presence of selective inhibitors or dominant-negative constructs. This included effectors downstream of Gαs (adenylate cyclase (AC), ddAdo, KH7) and protein kinase A (PKA), H89, KT5720), selective inhibitors of Gβγ, Gαi or Gαq (Gallein, Petussis toxin (PTx) and UBO respectively) and dominant negative versions of β-Arrestins 1 or 2. Data were normalized to % response in the absence of inhibitor/dominant-negative expression of β-arrestin to assess the importance of the various signaling effectors in peptide-mediated cAMP formation, calcium mobilization, and pERK1/2.

Figure S2

Assessment of Biased Agonism Reveals Distinctions in the Pattern of Signaling by GLP-1, Oxyntomodulin, and Exendin-4 at the GLP-1R in Both Recombinant and Natively Expressing Cells, Related to Figures 3, 4, 5, and 6 (A) Alignment showing the degree of sequence conservation between GLP-1, oxyntomodulin, and exendin-4. (B and D) The “web of bias” plots ΔΔτ/K_A values on a logarithmic scale for each ligand, for different signaling pathways in ChoFlpIn recombinant cells and Ins-1/832/3 insulinoma cells. Formation of these values included normalization to the reference ligand GLP-1 and the reference pathway, cAMP accumulation. Note, the plots do not provide information on absolute potency, but on relative efficacy for signaling of individual pathways to that for cAMP. (C) Top panel: Assessment of insulin secretion, apoptosis and proliferation confirms that the Ins-1/832/3 cells are a suitable model for GLP-1R signaling beta islets with GLP-1 promoting glucose-dependent insulin secretion, in addition to promoting proliferation and decreasing apoptosis; these cells were used to reveal bias between GLP-1 and both exendin-4 and oxyntomodulin in insulin secretion, proliferation, and apoptosis (D). Bottom panel: comparison of biased agonism for cAMP promotion and pERK1/2 in ChoFLpIn cells (left), Ins-1/832/3 insulinoma cells in low glucose conditions (2.8 mM) (middle) and Ins-1/832/3 insulinoma cells in high glucose conditions (11 mM) (right). These data revel the biased profile of oxyntomodulin observed in ChoFlpIn cells overexpressing the GLP-1R translates to the natively expressing insulinoma cell line.

Figure S3

Direct Interactions of the GLP-1R ECL Residues and TM Bundle with GLP-1 Predicted from Molecular Modeling, Related to Figures 1 and 2 (A) Top left: Molecular model of GLP-1 (maroon) docked to the GLP-1R with key ECL residues labeled that line the entry to a deep cavity where the N terminus of GLP-1 is predicted to bind. Colors are the heatmaps from binding studies of GLP-1 from Figure 2. Top right: Close up of the peptide and ECL GLP-1R side chains that form direct interactions. Bottom: Sequence alignment of GLP-1, oxyntomodulin (Oxyn) and exendin-4 (Ex) highlighting the absolute sequence conservation within the region of the GLP-1 peptide (blue box) that is predicted to interact with the ECL residues, L201, W297, R299, N300, and R380 in the molecular model. Within the extreme N terminus of the three peptides, only position 3 (position 9 in the unprocessed GLP-1 peptide) differ significantly between GLP-1 and exendin (E) in comparison to oxyntomodulin (Q), as highlight by the red box. (B) E⁹ of GLP-1 (position 3) is predicted to interact with R190^2.60 within the TM bundle cavity. Q³ of oxyntomodulin would not be predicted to form this sat bridge. In support of this, mutation of R190 to A markedly reduced cAMP signaling by GLP-1 (top left graph) but not oxyntomodulin (bottom left graph). To further validate the peptide docking in the GLP-1R model, substitution of E⁹ with Q in the GLP-1 displayed a profile similar to that of oxyntomodulin at the R190A mutant receptor (top right graph). In contrast, substitution of Q³ in oxyntomodulin with E produced a peptide with a similar profile to GLP-1 at R190A, with attenuated cAMP production compared to the wild-type receptor (bottom right graph).

Figure S4

Modeling of the Predicted Peptide-Binding Cavity in the GLP-1R-GLP-1 Molecular Model, Related to Figures 1 and 3 Molecular model of the GLP-1R-GLP-1 complex reveals the N terminus of the peptide binds in a deep cavity within the TM domain of the GLP-1R. (A) Surface representation of the GLP-1R TM domain viewed from the extracellular face (N terminus removed) highlighting the deep cavity within the TM bundle. Residues lining this cavity are contributed from residues in TM1 (orange), TM2 (yellow), TM3 (green), TM5 (purple), TM6 (red), and TM7 (pale blue). Residues located in the ECLs that line the entry to this cavity are shown in teal. (B), (D), and (F) highlight all the residues provided from these TMs and ECLs that surround the peptide binding cavity shown from three different angles (B from beneath the cavity, D and E from the side of the cavity, 180 degree rotated in E compared to D). Four class B conserved polar residues that reside in a hydrogen bond network in the inactive conformation lining the bottom of this binding cavity are highlighted in dark blue (B–E), where the cavity is shown in gray. The remaining residues are colored according to their location in the TMs using the colors depicted in (A).

Figure S5

MD Simulations of Peptide Interaction with GLP-1 Receptor Models, Related to Figures 1 and 2 In the surface simulation (A–D), the N-terminal GLP-1 peptide fragment was placed such that D¹⁵ of the peptide was within 10 Å of R380 of the receptor, but no specific tethers were used. For this simulation, an open model of the receptor was used based on homology modeling using the CRF1 receptor crystal structure as initial template. The simulation was run for 220 ns. Interaction data for peptide residue E⁹ and receptor residue R190 are shown in the inset panels. The upper inset displays the distance between C_δ (carbon delta) of E⁹ of GLP1 and C_ζ (carbon zeta) of R190 of GLP-1R during the first 110 ns of the MD simulation. The E⁹-R190 hydrogen-bonded salt-bridge engaged at ∼t = 30 ns and then remained stable during the remainder of the 220 ns MD simulation. The lower panel of the inset displays hydrogen bonds between E⁹ of GLP1 and R190 of GLP-1R during the first 110 ns of MD simulation. The donor–acceptor distance cutoff was 3.0 Å, and the angle cutoff was 20°. The E⁹-R190 hydrogen-bonded salt-bridge remained stable during the remainder of the 220 ns MD simulation. Side views from the simulation are illustrated in (A) early time point, and (B) after ∼100 ns where a stable interaction between the ligand and peptide had been formed. Panels (C) and (D) illustrate top views at each of the time points, respectively. In the deep pocket simulation (E–H), MD was performed for a total of 500 ns, commencing with the final model of the full-length peptide bound receptor. The interaction between E⁹ of the peptide and R190 of the receptor remained stable for the duration of the simulation. Inset panels display distances between C_δ (carbon delta) of E⁹ of GLP1 and C_ζ (carbon zeta) of R190 of GLP-1R (upper inset) and hydrogen bonds between the two residues where donor–acceptor distance cutoff was 3.0 Å, and the angle cutoff was 20°. Panels (E) and (F) illustrate side views of early and late time points during the simulation. Panels (G) and (H) illustrate the equivalent top views, respectively. The GLP-1 peptide fragment is displayed in pink (cpk) except for E⁹ and D¹⁵ of the peptide (surface display, ice blue). The receptor is displayed in cartoon form, with the backbone colored by secondary structure. GLP-R residue 190 is displayed as VDW representation (colored by atom). GLP-1R residues that when mutated to alanine decreased GLP-1 peptide affinity are illustrated by quick surface, colored according to the fold-change in affinity (yellow, 3- to 5-fold; light orange, 5- to 10-fold; dark orange, 10- to 30-fold; red, >30-fold). See Movies S1 and S2.

Figure S6

Signaling and Bias Agonism Triggered by Contacts with Peptide Ligands at the Extracellular Surface Is Propagated to the Intracellular Surface through Conformational Changes Involving Key Hydrogen Bond Networks, Related to Figures 2 and 6 Molecular model of full-length GLP-1R showing extended heatmaps highlighting the effects of Ala mutations to ECL loops residues in this study combined with polar residues reported in the literature that alter the efficacy (τ_c) of GLP-1 (left), oxyntomodulin (middle) and exendin-4 (right) for cAMP (A), iCa²⁺ (B), and pERK1/2 (C). Residues that statistically altered efficacy 3- to 5-fold are in yellow, 5- to 10-fold are pale orange, 10- to 30-fold are in dark orange, with those in red having greater than 30-fold loss in efficacy.

Figure S7

Residues within ECL3 Are Important for pERK1/2, Related to Figure 6 (A) Mapping of effects on pERK1/2 efficacy by GLP-1, oxyntomodulin, and exendin-4 when individual residues are mutated to alanine reveals a commonality in the principal site for signaling to this pathway that is localized to ECL3, although there are only a few residues within this site that, when mutated, have the same effect for all three peptides. (B) Concentration response curves for cAMP accumulation (left) and pERK1/ (right) for GLP-1 (GLP-1(7-36)NH₂) and its truncated metabolite (GLP-1(9-36)NH₂). Data are normalized to the response elicited forskolin (cAMP) or FBS (pERK1/2) and fitted to a three parameter logistic equation (Equation 1 in SI). All values are means ± SEM of three independent experiments, conducted in duplicate. These data reveal that removal of the first two residues of GLP-1 results in a peptide that is unable to generate a cAMP response but maintains the ability to promote pERK1/2.