N-homocysteinylation of K296 within β-arrestin2 suppresses ligand-induced β-arrestin2 recruitment to GPCRs and receptor internalization and biases signal transduction toward G proteins. (A) Sequence alignment of rat, murine, and human β-arrestin2. (B-C) There were no significant differences between WT, K34R, and K313R β-arrestin2 recruited to P2Y1 (B) and P2Y12 (C) after ADP (5 μM) was activated under any conditions (with or without HTL). The data are from 6 independent experiments (n = 6). (D-G) BRET assay results demonstrated that the Hcy/HTL-dependent suppression of the interaction between β-arrestin2 and P2Y1 (D), P2Y12 (E), TP (F), and PAR1 (G) was rescued by mutation of K295 within human β-arrestin2. The data are from 6 independent experiments (n = 6). (H-K) The BRET assay results demonstrated that the Hcy/HTL-dependent decrease in the internalization of P2Y1 (H), P2Y12 (I), TP (J), and PAR1 (K) was abrogated by the mutation of K295 within human β-arrestin2. The data are from 6 independent experiments (n = 6). (L) Confocal microscopy revealed that the ADP-induced (5 μM) internalization of P2Y12 in HEK293A cells that expressed WT but not K295R human β-arrestin2 was largely prevented by pretreatment with HTL (30 μM) and Hcy (100 μM). The arrows indicate the colocalization of P2Y12 (YFP) and β-arrestin2 (RFP). The 0-, 5-, 10-, 20-minute markers indicate to the time points at which ADP acted on the HEK293A cells. Scale bars, 10 μm. (M-N) GloSensor cAMP inhibition assays confirmed that (M) P2Y12- and (N) PAR1-coupled Gi activity in HEK293A cells that expressed WT but not K295R human β-arrestin2 was significantly amplified by pretreatment with HTL (30 μM) and Hcy (100 μM). The data are from 4 independent experiments (n = 4). (O-Q) A TGFA shedding assay confirmed that (O) P2Y1-, (P) TP-, and (Q) PAR1-coupled Gq activity in HEK293A cells that expressed WT but not K295R hs-β-arrestin2 was significantly amplified by pretreatment with HTL (30 μM) and Hcy (100 μM). The data are from 5 independent experiments (n = 5). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The bars represent means ± SEMs. All the data were analyzed using the 1-way ANOVA. β-arr2, β-arrestin2.

N-homocysteinylation of K296 within β-arrestin2 suppresses ligand-induced β-arrestin2 recruitment to GPCRs and receptor internalization and biases signal transduction toward G proteins. (A) Sequence alignment of rat, murine, and human β-arrestin2. (B-C) There were no significant differences between WT, K34R, and K313R β-arrestin2 recruited to P2Y1 (B) and P2Y12 (C) after ADP (5 μM) was activated under any conditions (with or without HTL). The data are from 6 independent experiments (n = 6). (D-G) BRET assay results demonstrated that the Hcy/HTL-dependent suppression of the interaction between β-arrestin2 and P2Y1 (D), P2Y12 (E), TP (F), and PAR1 (G) was rescued by mutation of K295 within human β-arrestin2. The data are from 6 independent experiments (n = 6). (H-K) The BRET assay results demonstrated that the Hcy/HTL-dependent decrease in the internalization of P2Y1 (H), P2Y12 (I), TP (J), and PAR1 (K) was abrogated by the mutation of K295 within human β-arrestin2. The data are from 6 independent experiments (n = 6). (L) Confocal microscopy revealed that the ADP-induced (5 μM) internalization of P2Y12 in HEK293A cells that expressed WT but not K295R human β-arrestin2 was largely prevented by pretreatment with HTL (30 μM) and Hcy (100 μM). The arrows indicate the colocalization of P2Y12 (YFP) and β-arrestin2 (RFP). The 0-, 5-, 10-, 20-minute markers indicate to the time points at which ADP acted on the HEK293A cells. Scale bars, 10 μm. (M-N) GloSensor cAMP inhibition assays confirmed that (M) P2Y12- and (N) PAR1-coupled Gi activity in HEK293A cells that expressed WT but not K295R human β-arrestin2 was significantly amplified by pretreatment with HTL (30 μM) and Hcy (100 μM). The data are from 4 independent experiments (n = 4). (O-Q) A TGFA shedding assay confirmed that (O) P2Y1-, (P) TP-, and (Q) PAR1-coupled Gq activity in HEK293A cells that expressed WT but not K295R hs-β-arrestin2 was significantly amplified by pretreatment with HTL (30 μM) and Hcy (100 μM). The data are from 5 independent experiments (n = 5). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The bars represent means ± SEMs. All the data were analyzed using the 1-way ANOVA. β-arr2, β-arrestin2.

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