Functional selective oxytocin-derived agonists discriminate between individual G protein family subtypes

Abstract

We used a bioluminescence resonance energy transfer biosensor to screen for functional selective ligands of the human oxytocin (OT) receptor. We demonstrated that OT promoted the direct engagement and activation of G(q) and all the G(i/o) subtypes at the OT receptor. Other peptidic analogues, chosen because of specific substitutions in key OT structural/functional residues, all showed biased activation of G protein subtypes. No ligand, except OT, activated G(oA) or G(oB), and, with only one exception, all of the peptides that activated G(q) also activated G(i2) and G(i3) but not G(i1), G(oA), or G(oB), indicating a strong bias toward these subunits. Two peptides (DNalOVT and atosiban) activated only G(i1) or G(i3), failed to recruit β-arrestins, and did not induce receptor internalization, providing the first clear examples of ligands differentiating individual G(i/o) family members. Both analogs inhibited cell proliferation, showing that a single G(i) subtype-mediated pathway is sufficient to prompt this physiological response. These analogs represent unique tools for examining the contribution of G(i/o) members in complex biological responses and open the way to the development of drugs with peculiar selectivity profiles. This is of particular relevance because OT has been shown to improve symptoms in neurodevelopmental and psychiatric disorders characterized by abnormal social behaviors, such as autism. Functional selective ligands, activating a specific G protein signaling pathway, may possess a higher efficacy and specificity on OT-based therapeutics.

FIGURE 1.

BRET measurements of OTR-Gαβ₁γ₂ coupling and activation following OT stimulation. a, BRET² was measured between Rluc (the donor) and GFP¹⁰ (the acceptor), respectively, introduced at the C-terminal tail of OTR (OTR-Rluc) and the N-terminal domain of the Gγ₂ subunit (GFP¹⁰-Gγ₂). OT-induced OTR-Gα coupling places OTR-Rluc and GFP¹⁰-Gγ₂ near each other, which corresponds to an increase in the GFP¹⁰/Rluc BRET ratio. BRET was measured in HEK293 cells co-expressing OTR-RLuc, GFP¹⁰-Gγ₂, and Gβ₁ in the absence (−α, empty bar) or presence of the indicated Gα subunits (n = 4). The results are the differences in the BRET signal with OT (10 μm) or PBS (mean value ± S.D. of three independent experiments). One-way ANOVA followed by Dunnett's test was used to determine the statistical differences between OT-promoted BRET in the presence of the indicated Gα proteins and non-Gα-transfected controls (***, p < 0.001). b, a BRET titration curve was performed in HEK 293 cells transiently transfected to co-express Gα_s-113-Rluc8 and OTR- GFP² or CD4- GFP¹⁰ in combination with β₁γ₂ subunits. The amount of plasmid encoding GFP²-tagged proteins varied (from 0.031 to 8 μg), whereas the amount of Gα_s-113-Rluc8 was kept constant (3 μg). Data are representative of two experiments and were fit using linear regression. c, Gα_s activation was evaluated with BRET in HEK293 cells co-expressing OTR or the positive control V₂R with GFP¹⁰-Gγ₂, Gβ₁, and Gα_s-Rluc8 tagged subunits. Cells were stimulated with OT or AVP at the concentration indicated. The results are the differences in the BRET signal with OT, AVP, or PBS (empty bar) (mean values ± S.D. (error bars) of three independent experiments). One-way ANOVA followed by Dunnett's test was used to determine the statistical differences between ligand-promoted BRET in the presence of the indicated ligand and untreated controls (***, p < 0.001). d, BRET² was measured between Rluc (the donor) and GFP¹⁰ (the acceptor), introduced into the α helical domain of the indicated Gα subunits and the N-terminal domain of Gγ₂ (GFP¹⁰-Gγ₂), respectively. Ligand-induced OTR-Gα activation leads to a conformational rearrangement of the heterotrimeric G protein complex that corresponds to a decrease in the BRET ratio. BRET was measured in HEK293 cells co-expressing OTR, GFP¹⁰-Gγ₂, Gβ₁, and Rluc/Rluc8-tagged Gα subunits: α_q (n = 6), α_i1 (n = 14), α_i2 (n = 5), α_i3 (n = 8), α_oA (n = 3), and α_oB (n = 3). The results are the differences in the BRET signal with OT (10 μm) or PBS and are expressed as mean values ± S.D. One-way ANOVA followed by Dunnett's test was used to determine the statistical differences between OT-promoted BRET in the presence of the indicated Gα proteins and untreated controls (base line) (***, p < 0.001).

FIGURE 2.

BRET measurements of the OT dose-response activation of OTR-Gαβ₁γ₂ complexes in HEK293 cells. BRET was measured in HEK293 cells co-expressing the indicated Gα Rluc/Rluc8 proteins (Gα_q-97-Rluc (a), Gα_i1-91-Rluc (b), Gα_i2-91-Rluc (c), Gα_i3-91-Rluc (d), Gα_oB-91-Rluc8 (e), and Gα_oA-91-Rluc8 (f)), together with OTR, GFP¹⁰-Gγ₂, and Gβ₁. The GFP¹⁰/Rluc ratios were similar for all pairs: 0.66 for Gα_q-97-Rluc, 0.89 for Gα_i1-91-Rluc, 0.65 for Gα_i2-91-Rluc, 0.68 for Gα₃-91-Rluc, 0.59 for Gα_oB-91-Rluc8, and 0.61 for Gα_oA-91-Rluc8. The cells were stimulated with increasing concentrations of OT (from 10⁻¹¹ to 10⁻⁴ m) for 2 min. The results are the differences in the BRET signal in the presence and absence of OT and are expressed as the mean value ± S.D. (error bars) of at least three independent determinations. Calculated EC₅₀ values were 2.16 ± 0. 95, 62.63 ± 39.00, 32.27 ± 11.05, 11.50 ± 7.22, 29.80 ± 19.32, and 91.80 ± 26.00 nm, respectively.

FIGURE 3.

Inositol phosphate production in OTR-expressing HEK293 cells following OT and OT-derived peptide stimulation. IP1 production was measured using an immunocompetitive HTRF-based assay (HTRF IPOne, Cisbio) in HEK293 cells stably expressing the N-terminally myc-tagged OTR (HEK mycOTR). A total of 100,000 cells were stimulated for 30 min with the OT and OT-derived peptides at a final concentration of 10 μm. The data are expressed as the mean value ± S.D. (error bars). of three independent experiments, each performed in sextuplicate. One-way ANOVA followed by Dunnett's test was used to determine the statistical differences in IP1 production in the presence of the indicated ligand and untreated controls (PBS) (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 4.

BRET measurements of OT and OT-derived ligand activation of OTR-Gαβ₁γ₂ complexes in HEK293 cells. BRET was measured in HEK293 cells co-expressing OTR, GFP¹⁰-Gγ₂, Gβ₁, and the indicated GαRluc constructs: Gα_q-Rluc (a), Gα_i1-Rluc (b), Gα_i2-Rluc (c), Gα_i3-Rluc (d), Gα_oA-Rluc8 (e), and Gα_oB-Rluc8 (f). The cells were stimulated for 2 min with OT and OT-derived peptides at a final concentration of 10 μm; at this dose, OT produced a peak BRET ratio signal in all of the tested Gα proteins. The results are the differences in the BRET signals in the presence and absence of ligands (10 μm) and are expressed as the mean value ± S.D. (error bars) of at least six independent determinations. The statistical significance of the differences between stimulated and unstimulated (PBS) cells was assessed using one-way ANOVA followed by Dunnett's test (*, p < 0.05; ***, p < 0.001).

FIGURE 5.

BRET measurements of DNalOVT and atosiban dose-response activation of OTR-Gαβ₁γ₂ complexes in HEK293 cells. The BRET ratio was recorded in HEK293 cells transfected with cDNAs encoding for OTR, GFP¹⁰-Gγ₂, Gβ₁, and Gα_i1-91-Rluc or Gα_i3-91-Rluc for EC₅₀ determination. The cells were stimulated with increasing concentrations of DNalOVT (a) and atosiban (b) (from 10⁻¹⁰ to 10⁻³ m). The data are expressed as the mean value ± S.D. (error bars) of at least three independent determinations.

FIGURE 6.

BRET measurements of OTR-mediated β-arrestin1 and β-arrestin2 recruitment in HEK293 cells following OT, atosiban, and DNalOVT stimulation. BRET¹ was monitored between Rluc and YFP introduced at the C-terminal of OTR (OTR-RLuc) and the β-arrestins: β-arrestin1-YFP (a) and β-arrestin2-YFP (b). HEK293 cells co-expressing OTR-Rluc and β-arrestins-YFP were stimulated by OT (10 μm), DNalOVT (10 μm), and atosiban (1 mm). Real-time BRET¹ measurements were made every 20 s. The results are the differences in the BRET signals in the presence and absence of agonist and are expressed as the mean value ± S.D. (error bars) of 3–5 independent experiments. c and d, BRET concentration-response curves of OT-induced β-arrestin recruitment in HEK293 cells. HEK293 cells co-expressing OTR-Rluc and β-arrestin1-YFP (c) or β-arrestin2-YFP (d) were treated with OT (10⁻¹⁰ to 10⁻⁴ m). The BRET signal was recorded at maximum plateau level (2 min for β-arrestin2 and 5 min for β-arrestin1). The results are the differences in the BRET signals in the presence and absence of agonist and are expressed as the mean value ± S.D. of three independent experiments. e, imaging of OTR-GFP internalization upon ligand stimulation. The subcellular localization of recombinant OTR C-terminally fused to EGFP (OTR-EGFP) was visualized by means of laser scanning confocal microscopy in stably transfected HEK293 OTR-EGFP cells. The cells were fixed before (CTRL) and after incubation with OT (100 nm), DNalOVT (100 nm), and atosiban (10 μm) for 3 and 30 min at 37 °C. Scale bar, 10 μm.

FIGURE 7.

Atosiban and DNalOVT inhibit cell growth via a PTX-sensitive pathway. Atosiban and DNalOVT effects on cell growth were analyzed in HEK293 cells stably expressing the OTR fused to EGFP (OTR-EGFP) (a) and DU145 human prostate cancer cells endogenously expressing OTR (b). The cells were treated with OT, DNalOVT, and atosiban at a final concentration of 100 nm in the presence or absence of PTX (150 ng/ml). Cell growth was determined by means of an [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS)-based assay after 48 h (HEK293 cells) or 72 h (DU145 cells) of peptide treatment. The results are the percentage variations in the number of cells under treated versus untreated conditions (ctrl); the differences were statistically analyzed using one-way ANOVA followed by Dunnett's test (**, p < 0.01; ***, p < 0.001). Error bars, S.D.