Spatiotemporal GLP-1 and GIP receptor signaling and trafficking/recycling dynamics induced by selected receptor mono- and dual-agonists

Abstract

Objective: We assessed the spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics of GIPR mono-agonists, GLP-1R mono-agonists including semaglutide, and GLP-1/GIP dual-agonists MAR709 and tirzepatide.

Methods: Receptor G protein recruitment and internalization/trafficking dynamics were assessed using bioluminescence resonance energy transfer (BRET)-based technology and live-cell HILO microscopy.

Results: Relative to native and acylated GLP-1 agonists, MAR709 and tirzepatide showed preserved maximal cAMP production despite partial Gα_s recruitment paralleled by diminished ligand-induced receptor internalization at both target receptors. Despite MAR709's lower internalization rate, GLP-1R co-localization with Rab11-associated recycling endosomes was not different between MAR709 and GLP-1R specific mono-agonists.

Conclusions: Our data indicated that MAR709 and tirzepatide induce unique spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics relative to native peptides, semaglutide, and matched mono-agonist controls. These findings support the hypothesis that the structure of GLP-1/GIP dual-agonists confer a biased agonism that, in addition to its influence on intracellular signaling, uniquely modulates receptor trafficking.

Keywords: Biased agonism; Dual-agonists; GIPR; GLP-1R; Receptor Internalization; Receptor Trafficking.

Figure 1

Schematic and structure of the tested GLP-1R and GIPR ligands. GLP-1R mono-agonists comprised of human GLP-1 (7–36 amide), semaglutide, and fatty acyl-GLP-1 (a pharmacokinetically-matched His1 and Val10 mutant of MAR709) (left panel). GIPR mono-agonists include human GIP (1–42) and fatty acyl-GIP (a pharmacokinetically matched Ile7 mutant of MAR709) (middle panel). GLP-1/GIP dual-agonist MAR709 and tirzepatide (right panel).

Figure 2

Ligand-induced G protein recruitment at GLP-1R and GIPR. Ligand-induced (1 μM) recruitment of Nluc-tagged Gα_s(A), Gα_q(B), Gα_i(C), and Gα_12/13(D) to GFP-tagged GLP-1R in HEK293T cells. Ligand-induced (1 μM) recruitment of Nluc-tagged Gα_s(E), Gα_q(F), Gα_i(G), and Gα_12/13(H) with GFP-tagged GIPR⁺ HEK293T cells. The positive iAUC (+iAUC) representation of vehicle and baseline-corrected 30 min response to each agonist is expressed as mean ± SEM. Bonferroni's test, ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.0005 using one-way ANOVA vs GLP-1 (7–36 amide) or GIP (1–42). Three independent experiments were performed with at least two technical replicates per group.

Figure 3

Dose-dependent effects of ligands on Gα_srecruitment and cAMP production. Dose–response curves (A) and temporal resolution (1 μM stimulation) (B) of ligand-induced BRET changes between Nluc-tagged Gα_s recruitment to GFP-tagged GLP-1R. Dose–response curves of ligand-induced cAMP production GLP-1R⁺ HEK293T cells (C). Dose–response curves (D) and temporal resolution (1 μM stimulation) (E) of ligand-induced Gα_s recruitment to the GIPR. Dose–response curves of ligand-induced cAMP production GIPR⁺ HEK293T cells (F). +iAUC representation of vehicle and baseline-corrected 60 min (Gα_s recruitment) or 25 min (cAMP generation) temporal responses to each agonist is expressed as mean ± SEM. Three independent experiments were performed with at least two technical replicates per group.

Figure 4

Ligand-induced GLP-1R internalization. Dose–response curves (A) and temporal resolution (1 μM stimulation) (B) of ligand-induced hGLP-1-Rluc8 internalization as measured by loss of BRET with plasma membrane marker Venus-KRAS. Live HILO imaging of GLP-1R-GFP internalization in HEK293T cells at baseline and approximately 15 min after drug (1 μM) treatment (representative image from n = 4 experiments) (C). Dose–response curves for β-arrestin 1-Rluc8 (D), β-arrestin 2-Rluc8 (E), and Gα_q-Nluc recruitment (F) to GLP-1R-GFP. The + iAUC representation of vehicle and baseline-corrected 60 min (GLP-1R internalization and Gα_q recruitment) or 30 min (β-arrestin1/2 recruitment) temporal response to each agonist is expressed as mean ± SEM. Three independent experiments were performed with at least two technical replicates per group.

Figure 5

Ligand-induced GIPR internalization. Dose–response curves (A) and temporal resolution (1 μM stimulation) (B) of ligand-induced hGIPR-Rluc8 internalization. Live HILO imaging of GIPR-GFP internalization in HEK293T cells at baseline and approximately 15 min after drug (1 μM) treatment (C). Dose–response curves for β-arrestin 2-Rluc8 (D) and Gα_q-Nluc recruitment (E). The + iAUC representation of vehicle and baseline-corrected 20 min (GIPR internalization), 30 min (β-arrestin1/2 recruitment), or 60 min (Gα_q recruitment) temporal response to each agonist is expressed as mean ± SEM. Three independent experiments were performed with at least two technical replicates per group.

Figure 6

Ligand-induced GLP-1R endosomal trafficking. Ligand-induced co-localization of GLP-1R-Rluc8 with Venus-Rab5 early endosomes (A–C), Venus-Rab7 late endosomes (D–F), and Venus-Rab11 recycling endosomes (G–I). The + iAUC representation of vehicle and baseline-corrected temporal response to each agonist is expressed as mean ± SEM. Bonferroni's test, ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.0005 using one-way ANOVA vs GLP-1 (7–36 amide). Six independent experiments were performed with at least two technical replicates per group.

Figure 7

Ligand-induced GIPR endosomal trafficking. Ligand-induced co-localization of GIPR with Venus-Rab5⁺ early endosomes (A and B), Venus-Rab7⁺ late endosomes (C and D), and Venus-Rab11⁺ recycling endosomes (E and F). The + iAUC representation of vehicle and baseline-corrected temporal response to each agonist is expressed as mean ± SEM. Bonferroni's test, ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.0005 using one-way ANOVA vs GIP (1–42). Six independent experiments were performed with at least two technical replicates per group.

Supplementary Table 1

Structure and identifiers of GLP-1 and GIP mono- and dual-agonists. Amino acid sequence, in-paper abbreviations, and external identifiers of the GLP-1 and GIP-derived compounds used.

Supplementary Figure 1

Schematic of the endosomal marker associated with GLP-1R/GIPR internalization and trafficking. Markers indicative of endosomal trafficking include Rab5 (early endosome), Rab7 (late endosome), and Rab11 (recycling endosome).

Supplementary Figure 2

Co-localization of GLP-1R with Gα_sand Rab5, Rab7, or Rab11 in GLP-1R⁺HEK293T cells. Ligand-induced HEK293T GLP-1R-mediated proximal co-localization of Nluc-tagged Gα_s with Venus-tagged Rab5 (A-C), Gα_s and Rab7 (D-F), or Gα_s and Rab11 (G-I) over 60 min. The +iAUC representation of vehicle and baseline-corrected temporal response to each agonist expressed as mean ± SEM. Bonferroni’s test, ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.0005 using one-way ANOVA vs GLP-1 (7-36 amide). Three independent experiments were performed with at least two technical replicates per group.

Supplementary Figure 3

Co-localization of GIPR with Gα_sand Rab5, Rab7, or Rab11 in GIPR⁺HEK293T cells. Ligand-induced HEK293T GIPR-mediated proximal co-localization of Nluc-tagged Gα_s and Venus-Rab5 (A-C), Gα_s and Rab7 (D and E), or Gα_s and Rab11 (F-H) over 60 min. The +iAUC representation of vehicle and baseline-corrected temporal response to each agonist is expressed as mean ± SEM. Bonferroni’s test, ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.0005 using one-way ANOVA vs GIP (1-42). Three independent experiments were performed with at least two technical replicates per group.

Supplementary Figure 4

CAMYEL cAMP sensor evidenced lack of saturation at 10 μM of GLP-1R agonist. Time course (A) and fitted dose-response curve (B) of cAMP-positive control forskolin as measured by the intracellular sensor CAMYEL in HEK293T cells. Stimulation with 10 μM of agonist or forskolin in GLP-1R+ HEK293T cells (C). The +iAUC representation of vehicle and baseline-corrected temporal response to each agonist is expressed as mean ± SE. Three independent experiments were performed with at least two technical replicates per group.

Supplementary Figure 5

GLP-1R internalization and Rab5 co-localization was differentially effected by the dual-agonists (1μM) in Min6 cells. Agonist-induced internalization of hGLP-1R-Rluc8 in Min6 cells for 60 min (A). Agonist-induced hGLP-1R-Rluc8 co-localization with Venus-Rab5⁺ (B), Venus-Rab7⁺ (C), and Venus-Rab11⁺ (D) endosomes in Min6 cells for 60 min. Temporal response for each agonist is expressed as mean ± SE. Tukey's test, ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < 0.0005 using one-way ANOVA vs GLP-1 (7-36 amide). Six independent experiments were performed with at least two technical replicates per group.

Supplementary Figure 6

Degree of Rab-specific GLP-1R co-localization was positively associated with the degree of GLP-1R-mediated endosomal Gαs recruitment in HEK293T cells. Linear regression between Gα_s recruitment and GLP-1R co-localization at the Rab5- (A), Rab7- (B), and Rab11-positive (C) endosomal compartments.