Asymmetric Recruitment of β-Arrestin1/2 by the Angiotensin II Type I and Prostaglandin F2α Receptor Dimer

Figures

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  Figure 1. Symmetric and asymmetric recruitment of β-arrestin 1/2 to the AT1R/FP dimer by Ang II and PGF2α, respectively. (A) Ang II (1 μM) induced a rapid and sustained symmetric recruitment of β-arrestin 1-RlucII (dashed lines; t1/2 < 2 min.) and β-arrestin 2-RlucII (full lines; t1/2 < 2 min) to AT1R-Venus alone (purple lines) and to a lesser extent, the AT1R-Venus/FP-WT dimer (blue lines), but not to the AT1R-WT/FP-Venus dimer (black lines). (B) No and very small responses were detected with Ang II by blocking AT1R-Venus with losartan (100 μM) (dashed and full blue lines), and when an AT1R[Δ325]-Venus mutant deficient for both β-arrestin 1/2-RlucII recruitments (dashed and full blue-green lines) were used in the context of the dimer, respectively. Responses were all specific to AT1R since no responses were detected for FP-Venus alone (dashed and full red lines) and β-arrestin 1/2-RlucII alone (dashed and full black lines). (C) PGF2α (1 μM) induced a slower, yet sustained asymmetric recruitment of βarr1-RlucII (dashed blue lines; t1/2 = 20 min) and β-arrestin 2-RlucII (full blue lines; t1/2 = 17 min) to the AT1R-Venus/FP-WT dimer only, but not to FP-Venus alone (dashed and full red lines)…

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  Figure 2. Specificity of the PGF2α-induced β-arrestin 2 recruitment to the AT1R/FP dimer. The recruitment of β-arrestin 2-RlucII to the AT1R-Venus (0.3 μg)/FP-WT (0.1 μg) dimer (fixed amount) induced by PGF2α (1 μM) was titrated by co-transfecting increasing amounts of AT1R-WT (0.0–0.7 μg). Increased expression of AT1R-WT competed with AT1R-Venus for dimerization with FP-WT, reducing the capacity of the AT1R-Venus/FP-WT dimer to recruit β-arrestin 2-RlucII. Data were normalized for AT1R-Venus expression and represent mean ± s.e.m. of independent experiments [biological replicates each performed in triplicate (n = 3–5)]. *p < 0.05, compared to AT1R-WT (0.0 μg).

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  Figure 3. Enhanced bystander BRET shows FP can stimulate AT1R internalization. HEK293 cells were transfected with the indicated enhanced BRET pairs. To monitor AT1R- or FP-mediated trafficking of AT1R-RlucII from the membrane (loss of Lyn-rGFP bystander BRET) or endosomal trafficking (as measured by increase in rGFP-FYVE bystander BRET), AT1R-RlucII were co-transfected with wildtype FP and the indicated bystander BRET partner. Cells were stimulated with (A) 1 μM Ang II or (B) 1 μM PGF2α and monitored over time. (C) FP-RlucII were co-transfected with wildtype AT1R and the indicated bystander BRET partner and stimulated with 1 μM PGF2α and monitored over time. BRET signals were measured as described in Materials and Methods. Data represent mean ± s.e.m. of independent experiments performed in triplicate (n = 3).

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  Figure 4. Biased allosteric regulation of the PGF2α- and cloprostenol-induced β-arrestin2 recruitment to the AT1R/FP dimer by AT1R antagonists. (A) Pretreatment (1 μM for 30 min) with telmisartan or azilsartan allosterically inhibited recruitment of β-arrestin 2-RlucII to the AT1R-Venus/FP-WT dimer in response to PGF2α (1 μM). Under the same experimental conditions, irbesartan, candersartan and losartan produced no allosteric effects. (B) Pretreatment (1 μM for 30 min) with telmisartan allosterically inhibited the recruitment of β-arrestin 2-RlucII to the AT1R-Venus/FP-WT dimer in response to cloprostenol (1 μM). Under the same experimental conditions, azilsartan, irbesartan, candersartan and losartan produced no allosteric effects. Data represent mean ± s.e.m. of independent experiments [biological replicates performed in triplicate (n = 3–6)]. *p < 0.05. N.S., non-significant.

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  Figure 5. Role of G proteins in Ang II- and PGF2α-induced β-arrestin 1/2 recruitment to the AT1R/FP dimer. (A,B) Compared to the HEK 293 parental cell line (black lines), Ang II (1 μM) induced a decaying and much lower, yet sustained recruitment of β-arrestin 1/2-RlucII to the AT1R-Venus/FP-WT dimer when evaluated in the HEK 293 ΔGαq/11/12/13 cell line (red lines). (C,D) PGF2α (1 μM) induced the recruitments of β-arrestin 1/2-RlucII to the AT1R-Venus/FP-WT dimer in the HEK 293 parental cell line (black lines) only, and virtually no responses were detected with the HEK 293 ΔGαq/11/12/13 cell line (red lines) (C), inset: The response in parental and ΔGαq/11/12/13 cell lines are shown alone for clarity. Data represent mean ± s.e.m. of [biological replicates performed in triplicate (n = 3–15)].

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  Figure 6. Effects of endocytosis inhibitors on Ang II- and PGF2α-induced β-arrestin 1/2 recruitment to the AT1R/FP dimer. (A,B) Sucrose (blue lines, 450 mM) and phenylarsine oxide (blue-green lines, 2.5 μM) pretreatment (30 min) potentiated and diminished the recruitment of β-arrestin1/2-RlucII to the AT1R-Venus/FP-WT dimer in response to Ang II (1 μM), respectively. To a much lesser extent, concanavalin A (purple lines, 0.4 g/L) pretreatment (30 min) and the dominant negative mutant K44A dynamic (salmon lines; 0.5 μg) also potentiated and diminished the responses, respectively. (C,D) Concanavalin A (purple lines, 0.4 g/L) and phenylarsine oxide (blue-green lines, 2.5 μM) pretreatment (30 min) potentiated and diminished the recruitments of β-arrestin 1/2-RlucII to the AT1R-Venus/FP-WT dimer in response to PGF2α (1 μM), respectively. To a lower extent, sucrose (blue lines, 450 mM) and the dominant negative mutant K44A of dynamin (salmon lines; 0.5 μg) both diminished the responses. Data represent mean ± s.e.m. of independent experiments performed in triplicate (n = 3–4).