Activation of Gαsand Gαq at plasma membrane and early endosomes by V2R monitored by BRET.

(A) Left: Illustration of EbBRET biosensors used to monitor G protein activation at the plasma membrane. Right: Kinetics of the recruitment of mG proteins at the plasma membrane upon stimulation of V2R with 1 μM AVP or vehicle. (B) Left: Illustration of EbBRET biosensors used to monitor G protein activation at the early endosomes. Right: Kinetics of the recruitment of mG proteins to early endosomes upon stimulation of V2R with 1 μM AVP. (C) Left: Illustration of the BRET-based biosensor used to monitor DAG production at the plasma membrane. Right: DAG generated at the plasma membrane upon a 10 minute stimulation of V2R with 1 μM AVP or vehicle in cells pre-treated 30 minutes with 0.1 μM YM254890 or vehicle. (D;) Left: Illustration of the BRET-based biosensor used to monitor DAG production at the early endosomes. Right: DAG generated at early endosomes upon a 10 minute stimulation of V2R with 1 μM AVP in cells pre-treated 30 minutes with 0.1 μM YM254890 or vehicle. (E) Left: Illustration of the BRET-based PKC biosensor used to monitor PKC activation. Right: Activation of PKC upon 10 minutes of stimulation of V2R with 1 μM AVP. For the kinetics, n = 3 (mGs, mGsi, and mGsq) or n = 4 (mG12) independent experiments. For the experiments using the DAG biosensor, n = 7 and n = 5 for measurements at plasma membrane and early endosomes, respectively. For the experiments using the PKC biosensor, n = 5. Asterisks mark statistically significant differences between vehicle and AVP treatments as assessed by two-way ANOVA and Sidak’s post hoc test for multiple comparisons (**P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Activation of Gαs and Gαq at plasma membrane and early endosomes by V2R monitored by confocal microscopy.

(A)Representative confocal microscopy images of cells expressing RFP-Lck, V2R, and Halo-mGs (left panels), Halo-mGsq (middle panels), or Halo-mGsi (right panels) stimulated for 10 minutes with vehicle (upper panels) or 1 μM AVP (bottom panels). (B) Percentages of Lck colocalized with each Halo-mG calculated from 5 representative images. (C) Representative confocal microscopy images of cells expressing RFP-EEA1, V2R, and Halo-mGs (left panels), Halo-mGsq (middle panels), or Halo-mGsi (right panels) stimulated for 45 minutes with vehicle (upper panels) or 1 μM AVP (bottom panels). (D) Percentages of EEA1 colocalized with each Halo-mG calculated from 6 representative images. n = 3 independent experiments for Lck and EEA1. Statistical differences between vehicle and AVP treatments were assessed by two-way ANOVA and Sidak’s post hoc test for multiple comparisons (****P ≤ 0.0001).

Formation of megaplexes with Gαs or Gαq upon stimulation with AVP.

(A)Kinetics of the recruitment of mG proteins at the plasma membrane upon stimulation of V2β2AR with 1 μM AVP. n = 3 for mGs, mGsi, and mGsq, and n = 4 for mG12. (B) Left: Illustration of the EbBRET biosensors used to monitor βarr recruitment to the plasma membrane and early endosomes. Right: Kinetics of the recruitment of βarr1 and βarr2 to the plasma membrane (upper panel) and to early endosomes (bottom panel) upon stimulation of V2R or V2β2AR with 1 μM AVP. n = 3 for all conditions. (C) Left panel: Illustration of the nanoBiT biosensors used to monitor simultaneous coupling of Gα proteins and βarr1 to GPCRs. Right panels: Kinetics of the proximity between SmBiT-βarr1 and LgBiT-mGs (upper panel) or LgBiT-mGsq (bottom panel) upon stimulation of the receptors with 1 μM AVP. n = 3 for all conditions. (D) Representative confocal microscopy images of cells expressing Halo-mGs, strawberry-βarr2, and V2R (left panels), or V2β2AR (right panels) and stimulated for 45 minutes with vehicle (upper panels) or 1 μM AVP (bottom panels). (E) Representative confocal microscopy images of cells expressing Halo-mGsq, strawberry-βarr2, and V2R (left panels), or V2β2AR (right panels) and stimulated for 45 minutes with vehicle (upper panels) or 1 μM AVP (bottom panels). (F) Percentage of βarr2 colocalization with mGs or mGsq upon stimulation with 1μM AVP (6 representative images). (G) Percentage of mGs or mGsq puncta observed that were not colocalized with βarr2 upon stimulation with 1μM AVP (15 representative images). Asterisks mark significant differences between V2R and V2β2AR assessed by two-way ANOVA and Sidak’s post hoc test for multiple comparisons (***P ≤ 0.001, ****P ≤ 0.0001). No statistical difference (ns) was detected between mGs and mGsq.

Parameters related to AVP dose-response curves of the mGs and mGsq recruitment to the plasma membrane or early endosomes in parental and Δbarr1/2 cells.

ΔEbBRET values were fitted using four parameters equation with the bottom fixed at zero. n = 4 biological replicates for each condition. Statistical differences between parental and Δβarr1/2 cells for AVP-induced maximal efficacy and potency were assessed by comparing independent fits with a global fit that shares the selected parameter using extra sum-of-squares F test (**P ≤ 0.01, ****P ≤ 0.0001).

Contribution of megaplex to endosomal Gαs and Gαq signaling.

(A) AVP dose-response curves of the recruitment of mGs (left panel) and mGsq (right panel) to the plasma membrane in parental and Δβarr1/2 cells expressing V2R upon 10 minutes of stimulation. (B) AVP dose-response curves of the recruitment of mGs (left panel) and mGsq (right panel) to early endosomes in parental and Δβarr1/2 cells expressing V2R upon 45 minutes of stimulation. (C) Left: Illustration of EbBRET biosensors used to monitor AVP-mediated internalization of the V2R. Right: Kinetics of V2R internalization upon stimulation with AVP 0.1 μM. Asterisks mark significant differences from zero as assessed by one sample t test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). (D) Transduction coefficients of mGs (left panel) or mGsq (right panel) recruitment to the plasma membrane and early endosomes in V2R-or V2β2AR-expressing cells. Asterisks mark significant differences between the V2R and V2β2AR as assessed by two-way ANOVA and Sidak’s post hoc test for multiple comparisons (*P ≤ 0.05). No statistical difference (ns) was detected between the V2R and V2β2AR at the plasma membrane. n = 4 biological replicates for all experiments.

Parameters related to AVP dose-response curves of the recruitment of mGs and mGsq to the plasma membrane and early endosomes by the V2R or V2β2AR.

ΔEbBRET values from the dose-response curves were fitted using four parameters equation with the bottom fixed at zero. n = 4 biological replicates for each condition. Statistical differences between V2R and V2β2AR for AVP-mediated maximal efficacy and potency were assessed by comparing independent fits with a global fit that shares the selected parameter using extra sum-of-squares F test (%P ≤ 0.05, %%P ≤ 0.01, %%%P ≤ 0.001). Statistical differences between V2R and V2β2AR for Log(t/Ka) values were assessed by two-way ANOVA and Sidak’s post hoc test for multiple comparisons (%P ≤ 0.05). PM, plasma membrane; EE, early endosomes.

Updated model of V2R signaling.

At the plasma membrane, AVP binding to V2R results in receptor-mediated Gαs or Gαqactivation. This initial G protein activation at the plasma membrane is followed by V2R internalization into early endosomes. This internalization occurs primarily in a βarr-dependent manner, leading to the formation of a megaplex with Gαsor Gαq/11 and robust activation of these G proteins from endosomes. Additionally, a minor population of V2R internalize in a βarr-independent fashion, which also leads to minor but significant Gαs or Gαq/11 activation from endosomes.