Biased signaling favoring gi over β-arrestin promoted by an apelin fragment lacking the C-terminal phenylalanine

Abstract

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or Gi protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in BP decrease may be as a Gi-independent but β-arrestin-dependent signaling pathway that could involve MAPKs. For this purpose, we have used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates Gi and promotes β-arrestin recruitment to the receptor, K16P had a much reduced ability to promote β-arrestin recruitment while maintaining its Gi activating property, revealing the biased agonist character of K16P. We further show that both β-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity, whereas the K16P-promoted ERK1/2 activity is entirely Gi-dependent. In addition to providing new insights on the structural basis underlying the functional selectivity of apelin peptides, our study indicates that the β-arrestin-dependent ERK1/2 activation and not the Gi-dependent signaling may participate in K17F-induced BP decrease.

Keywords: Apelin; Apelin Receptor; Arrestin; Cell Signaling; Extracellular Signal-regulated Kinase (ERK); G Protein; G Protein-coupled Receptor (GPCR); Mitogen-activated Protein Kinase (MAPK); Pertussis Toxin.

FIGURE 1.

Gα_i activation and adenylyl cyclase inhibition by ApelinR upon K17F and K16P stimulation. A, effects of increasing concentrations of K17F or K16P on Gα_i activation monitored by BRET2 measurements of the dissociation between Gα_i-RlucII and Gγ₅-GFP10 upon a 5-min stimulation of HEK293T cells expressing the wild-type ApelinR. B, ligand-promoted changes in cAMP production in HEK293T cells expressing the wild-type ApelinR monitored by BRET2 using the EPAC biosensor (46) after forskolin (Forsk) treatment (10⁻⁵ m) alone or in combination with 10⁻⁶ m of either K17F or K16P. Forskolin and ligands (where applicable) were added 15 min prior to BRET2 measurements. The BRET2 values were subtracted from the BRET2 obtained with vehicle-treated cells to yield ligand-induced Δ BRET2 ratios. The data represent the means ± S.E. from three independent experiments. **, p < 0.01; ***, p < 0.001; n.s., nonsignificant.

FIGURE 2.

β-Arrestin recruitment to the wild-type ApelinR after stimulation with K17F or K16P. The effects of increasing concentrations of K17F or K16P on the recruitment of β-arrestin 1-Rluc (A, upper panel) or β-arrestin 2-Rluc (B, upper panel) to wild-type ApelinR-YFP were measured by BRET1. The effects of time on the recruitment of β-arrestin 1-Rluc (A, lower panel) or β-arrestin 2-Rluc (B, lower panel) to wild-type ApelinR-YFP were measured by BRET1 following stimulation with vehicle or 10⁻⁶ m of either K17F or K16P. The data in the upper panels represent the means ± S.E. of three independent experiments, whereas the lower panels are a representative illustration of three independent experiments.

FIGURE 3.

Apelin receptor-mediated ERK1/2 activation induced by K17F and K16P. A and B, time course of apelin receptor-mediated ERK1/2 phosphorylation induced by 10⁻⁷ m K17F or K16P. A, CHO cells stably expressing wild-type apelin receptor were treated with 10⁻⁷ m of K17F or K16P for 120 min. ERK1/2 phosphorylation was detected by Western blotting analysis using anti-phospho-ERK1/2 antibody (p-ERK), and total ERK1/2 was detected by total anti-ERK1/2 antibody (total ERK) in the same immunoblot for loading control. B, quantification of the bands corresponding to 44 and 42 kDa was performed by densitometry using ImageJ software. The data are expressed as percentages of pERK/total ERK. C and D, dose response of apelin receptor-mediated ERK1/2 phosphorylation induced by K17F and K16P stimulation. C, cells were treated with the indicated concentrations of K17F and K16P for 10 min, and ERK1/2 phosphorylation was measured. D, quantification of Western blotting bands was performed by densitometry analysis. The data correspond to the means ± S.E. from at least three independent experiments.

FIGURE 4.

Apelin receptor-mediated ERK1/2 activation induced by K17F or K16P in the presence or absence of pertussis toxin. A and C, time course of apelin receptor-mediated ERK1/2 phosphorylation induced by K17F (A) or K16P (C) without or with PTX. CHO cells stably expressing the rat apelin receptor were pretreated with PTX (25 ng/ml) for 16 h and were treated with 10⁻⁷ m of K17F (A) or K16P (C) for 120 min. ERK1/2 phosphorylation was detected by Western blotting analysis using anti-phospho-ERK1/2 antibody (p-ERK) and anti-ERK1/2 antibody (total ERK) for loading control. B and D, quantification of Western blotting bands for K17F (B) or K16P (D) corresponding to 44 and 42 kDa was obtained by densitometry analysis. The data are expressed as percentages of pERK/total ERK and correspond to the means ± S.E. from at least three independent experiments. E and F, phosphorylation of ERK1/2 in CHO cells stably expressing the rat apelin receptor after stimulation by K17F (E) or K16P (F) in presence of PTX (25 ng/ml). Phosphorylation was quantified using the Alphascreen Surefire ERK1/2 assay kit. The values are expressed as the area under the curve (AUC) of ERK1/2 phosphorylation obtained from a time course of 20 min of stimulation. The data represent the means ± S.E. from at least four independent experiments. Statistical differences were assessed using Student's t test. *, p < 0.05; n.s., nonsignificant.

FIGURE 5.

Apelin receptor-mediated ERK1/2 activation induced by K17F or K16P in absence or presence of β-arrestin-2-K296A. A–D, phosphorylation of ERK1/2 in CHO cells stably expressing the rat apelin receptor transiently transfected with DsRed2 mono (empty vector) (A and C) or DsRed2 mono-β-arr2-K296A (B and D) stimulated with 10⁻⁶ m of K17F (A and B) or K16P (C and D) for 5–20 min without or with PTX (25 ng/ml). The values in graphics are expressed as area under the curve (AUC) values of ERK1/2 phosphorylation obtained for a time course of 20 min of stimulation. ERK1/2 phosphorylation was quantified using the Alphascreen Surefire ERK1/2 assay kit. The data correspond to the means ± S.E. from at least four independent experiments. Statistical differences were assessed using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., nonsignificant.

FIGURE 6.

Gα_i protein-independent activation of MAPK cascade could be correlated to K17F-induced apelin receptor internalization. A–D, phosphorylation of endogenous ERK1/2 in CHO cells stably expressing wild-type apelin receptor-EGFP (A and C) or mutated F255A apelin receptor-EGFP (B and D) stimulated with 10⁻⁶ m of K17F (A and B) or K16P (C and D) for 5–20 min without (left two columns) or with (right two columns) PTX (25 ng/ml). The values in graphs are expressed as area under the curve (AUC) of ERK1/2 phosphorylation obtained for a time course of 20 min of stimulation with each of both peptides. ERK1/2 phosphorylation was quantified with Alphascreen technology. The data are the means ± S.E. from at least four independent experiments. Statistical differences were assessed using Student's t test. *, p < 0.05; **, p < 0.01; n.s., nonsignificant.

FIGURE 7.

Effects of K17F and K16P on the afferent arteriole contractile response to Ang II. Arteriolar diameters were measured in the basal conditions, then 1.5 min after adding 10⁻⁹ M Ang II, and 1.5 min after addition of 10⁻⁷ m K17F or K16P on vasoconstricted arterioles (n = 5). **, p < 0.001; n.s., nonsignificant.