Figure 3.
Homocysteine suppresses the functions of β-arrestins through N-homocysteinylation. (A-B) BRET assay results demonstrating that ADP-induced (5 μM) internalization of P2Y1 (A) and P2Y12 (B) was strongly decreased by pretreatment with HTL (30 μM) and Hcy (100 μM), whereas the decrease in the Hcy concentration was reversed by AHT (2 mM) and NAC (2 mM). The data are from 6 independent experiments (n = 6). (C-D) The BRET assay results indicated that ADP-induced (5 μM) interaction between β-arrestin1 and P2Y1 (C) and P2Y12 (D) was strongly decreased by pretreatment with HTL (30 μM) and Hcy (100 μM), whereas the Hcy-dependent decrease in the BRET ratio was reversed by AHT (2 mM) and NAC (2 mM). The data are from 6 independent experiments (n = 6). (E-F) BRET assay results demonstrated that the ADP-induced (5 μM) interaction between β-arrestin2 and P2Y1 (E) and P2Y12 (F) was strongly decreased by pretreatment with HTL (30 μM) and Hcy (100 μM), whereas the Hcy-dependent decrease in the BRET ratio was reversed by AHT (2 mM) and NAC (2 mM). The data are from 6 independent experiments (n = 6). (G) Confocal microscopy revealed that the ADP-induced (5 μM) internalization of P2Y12 was largely prevented by pretreatment with HTL (30 μM) and Hcy (100 μM) but reversed by AHT (2 mM). The arrows indicate the colocalization of P2Y12 (YFP) and β-arrestin2 (RFP). The 0-, 5-, 10-, and 20-minute indicators refer to the time points at which ADP acts upon the HEK293A cells. Scale bars, 10 μm. (H) Heat map visualization of the interaction between β-arrestin2 and representative cardiovascular GPCRs induced by agonists as demonstrated by BRET assays. The top of the heat map represents the GPCR-ligand pairs used in the BRET assays, whereas the left side of the heat map indicates the experimental groups, namely the control group and the HTL incubation group. The depth of color in the heat map cells corresponds to the BRET signal values for the recruitment of β-arrestin2 by GPCRs mediated by the ligand under the respective experimental conditions. The higher the signal value, the greater the interaction between the GPCR and β-arrestin2. The heat map shows that the agonist-induced interaction between β-arrestin2 and GPCRs was greatly diminished by pretreatment with HTL (30 μM). The data are from 6 independent experiments (n = 6). The principle and protocol of the BRET assay are detailed in supplemental Figure 4A and “Methods.” Angiotensin II (Ang II) activates AT1R, and TRV120026 or TRV120027 are AT1R β-arrestin1/2-biased agonists; isoprenaline (ISO) activates β2AR; DCA and P395 activate TGR5; TUG891 activates GPR120; PGE2 activates EP4; 9-HODE activates GPR132; S1P activates S1PR2; BM8-22 activates MRGPRX1; and PAMP12 activates MRGPRX2. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The bars represent means ± SEMs. All the data were analyzed using 1-way ANOVA. AT1R, adenosine transporter 1r; β2-AR, β2-adrenergic receptor; β-arr1, β-arrestin1; β-arr2, β-arrestin2; EP4, prostaglandin E2 receptor EP4 subtype; GPR120, free fatty acid receptor 4; GPR132, G protein-coupled receptor 132; MRGPRX1/2, mas-related G protein-coupled receptor member X1/2; S1PR2, sphingosine 1-phosphate receptor 2; TGR5, G protein-coupled bile acid receptor 1.

Homocysteine suppresses the functions of β-arrestins through N-homocysteinylation. (A-B) BRET assay results demonstrating that ADP-induced (5 μM) internalization of P2Y1 (A) and P2Y12 (B) was strongly decreased by pretreatment with HTL (30 μM) and Hcy (100 μM), whereas the decrease in the Hcy concentration was reversed by AHT (2 mM) and NAC (2 mM). The data are from 6 independent experiments (n = 6). (C-D) The BRET assay results indicated that ADP-induced (5 μM) interaction between β-arrestin1 and P2Y1 (C) and P2Y12 (D) was strongly decreased by pretreatment with HTL (30 μM) and Hcy (100 μM), whereas the Hcy-dependent decrease in the BRET ratio was reversed by AHT (2 mM) and NAC (2 mM). The data are from 6 independent experiments (n = 6). (E-F) BRET assay results demonstrated that the ADP-induced (5 μM) interaction between β-arrestin2 and P2Y1 (E) and P2Y12 (F) was strongly decreased by pretreatment with HTL (30 μM) and Hcy (100 μM), whereas the Hcy-dependent decrease in the BRET ratio was reversed by AHT (2 mM) and NAC (2 mM). The data are from 6 independent experiments (n = 6). (G) Confocal microscopy revealed that the ADP-induced (5 μM) internalization of P2Y12 was largely prevented by pretreatment with HTL (30 μM) and Hcy (100 μM) but reversed by AHT (2 mM). The arrows indicate the colocalization of P2Y12 (YFP) and β-arrestin2 (RFP). The 0-, 5-, 10-, and 20-minute indicators refer to the time points at which ADP acts upon the HEK293A cells. Scale bars, 10 μm. (H) Heat map visualization of the interaction between β-arrestin2 and representative cardiovascular GPCRs induced by agonists as demonstrated by BRET assays. The top of the heat map represents the GPCR-ligand pairs used in the BRET assays, whereas the left side of the heat map indicates the experimental groups, namely the control group and the HTL incubation group. The depth of color in the heat map cells corresponds to the BRET signal values for the recruitment of β-arrestin2 by GPCRs mediated by the ligand under the respective experimental conditions. The higher the signal value, the greater the interaction between the GPCR and β-arrestin2. The heat map shows that the agonist-induced interaction between β-arrestin2 and GPCRs was greatly diminished by pretreatment with HTL (30 μM). The data are from 6 independent experiments (n = 6). The principle and protocol of the BRET assay are detailed in supplemental Figure 4A and “Methods.” Angiotensin II (Ang II) activates AT1R, and TRV120026 or TRV120027 are AT1R β-arrestin1/2-biased agonists; isoprenaline (ISO) activates β2AR; DCA and P395 activate TGR5; TUG891 activates GPR120; PGE2 activates EP4; 9-HODE activates GPR132; S1P activates S1PR2; BM8-22 activates MRGPRX1; and PAMP12 activates MRGPRX2. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The bars represent means ± SEMs. All the data were analyzed using 1-way ANOVA. AT1R, adenosine transporter 1r; β2-AR, β2-adrenergic receptor; β-arr1, β-arrestin1; β-arr2, β-arrestin2; EP4, prostaglandin E2 receptor EP4 subtype; GPR120, free fatty acid receptor 4; GPR132, G protein-coupled receptor 132; MRGPRX1/2, mas-related G protein-coupled receptor member X1/2; S1PR2, sphingosine 1-phosphate receptor 2; TGR5, G protein-coupled bile acid receptor 1.

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