Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9

Abstract

G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four G_s-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β₂ adrenergic receptors (β₂ARs) and β₁ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V₂ and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β₂AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein-deleted cells to GPCR behavior in native systems.

Fig 1.. Variable effects of CRISPR/Cas9 gene editing on β₂AR-stimulated activation of ERK1/2 in three independently derived βArr1/2 CRISPR KO HEK293 cell lines.

(A to D) Top: Parental and corresponding CRISPR βArr1/2 KO HEK293 cell lines expressing endogenous β₂ARs (A to C) or overexpressed FLAG-β₂AR (D) were analyzed by Western blotting (IB) with antibody against βArr1/2. β-Actin was used as a loading control. The abundance of endogenous β₂AR determined by [¹²⁵I](–)-iodocyanopindolol binding was 15 ± 5 fmol/mg protein in all parental and CRISPR βArr1/2 KO lines, and the abundance of FLAG-β₂AR in the stably transfected AI-parental and CRISPR βArr1/2 KO lines was 2.3 ± 0.01 and 4.7 ± 0.6 pmol/mg protein, respectively. Middle: Serum-deprived cells were stimulated with 100 nM isoproterenol (Iso) for the indicated times and cell lysates were analyzed by Western blotting sequentially for pERK1/2 (pERK) and total ERK1/2 (ERK). Western blots for each parental-CRISPR pair are representative of four (B to D) or five (A) experiments. Bottom: For each parental-CRISPR βArr1/2 KO pair, Iso-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the parental line and data are expressed a percentage of the maximum control response (% max control). Data are means ± SEM of four or five biological replicates. Statistical significance was determined by two-way ANOVA and Sidak’s multiple comparison test. * P < 0.05 compared with parental cells; # P < 0.05 compared with parental cells at the corresponding time point; ns, not significant.

Fig 2.. Consistent effects of siRNA-mediated knockdown of βArr1/2 on β2AR-stimulated ERK1/2 activation in three parental HEK-293 cell lines.

(A to H) Top: Parental HEK293 cell lines expressing endogenous β₂AR (A to C) and (E to G) or overexpressed FLAG-β₂AR (D and H) and transfected with siRNAs targeting no mRNA (CTL), βArr2 (A to D), or both βArr1 and βArr2 (E to H) were analyzed by Western blotting (IB) with antibody against βArr1/2. β-Actin was used as a loading control. Middle: Serum-deprived cells were stimulated with 100 nM isoproterenol (Iso) for the indicated times and cell lysates were analyzed by Western blotting sequentially for pERK1/2 (pERK) and total ERK1/2 (ERK). Western blots for each pair of CTL siRNA– and βArr siRNA–treated cells are representative of three (H), four (B to C and E to G), or five (A and B) experiments. Bottom: For each CTL siRNA– (black symbols) and βArr siRNA–treated (red symbols) pair, Iso-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the CTL siRNA–treated cells and data expressed a percentage of the maximum control response (% max control). Data are means ± SEM of four or five biological replicates. Statistical significance was determined by two-way ANOVA and Sidak’s multiple comparison test. * P < 0.05 compared with parental group (control); # P < 0.05 compared with control at the corresponding time point; ns, not significant.

Fig 3.. Variable effects of restoring βArr1/2 on β2AR-stimulated ERK1/2 activation in three CRISPR βArr1/2 KO cell lines.

(A to F) Top: CRISPR βArr1/2 KO cell lines were transiently transfected with plasmid encoding FLAG-β₂AR together with either empty vector or plasmids encoding HA-βArr2 (A to C) or both HA-βArr1 and HA-βArr2 (D to F). Parental HEK293 cells, vector-transfected CRIPSR cells, and CRISPR cells expressing HA-βArr1, HA-βArr2, or HA-βArr1/2 were analyzed by Western blotting (IB) with antibody against βArr1/2. β-Actin was used as a loading control. Middle: Serum-deprived cells were stimulated with 100 nM isoproterenol (Iso) for the indicated times and cell lysates were analyzed by Western blotting sequentially for pERK1/2 (pERK) and total ERK1/2 (ERK). Western blots for each pair of vector-transfected and HA-βArr–expressing CRISPR cells are representative of three (B to C and E to F) or four (A and D) experiments. Bottom: For each vector-transfected (black symbols) and HA-βArr–expressing (green symbols) CRISPR cell pair, Iso-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the vector-transfected cells and data are expressed a percentage of the maximum control response (% max control). Data are means ± SEM of three or four biological replicates. Statistical significance was determined by two-way ANOVA and Sidak’s multiple comparison test. # P < 0.05 compared with control at the corresponding time point; ns, not significant.

Fig 4.. Effects of β-arrestin expression on Gαs activation, receptor internalization, and ERK1/2 activation by the β2AR.

(A) Top: Schematic representation of the BRET biosensors used to monitor Gα_s activation. Activation of Gα_s induces dissociation of RlucII-Gα_s from GFP10-Gγ1 resulting in a decrease in BRET signal (55). Bottom: Isoproterenol (Iso) concentration-response curves generated in each parental HEK293 cell line (black symbols; solid black line) together with those observed in the corresponding CRISPR βArr1/2 KO line in the absence (open symbols; dashed black line) or presence of exogenous βArr1 (open symbols; dashed blue line) or βArr2 (open symbols; dashed red line). Data are means ± SEM of four to 11 biological replicates as follows: SL parental (n = 11), SL CRISPR (n = 11), SL CRISPR + βarr1 (n = 5), SL CRISPR + βarr2 (n = 5), AI parental (n = 10), AI CRISPR (n = 10), AI CRISPR + βarr1 (n = 4), CRISPR + βarr2 (n = 4), HAR parental (n = 8), HAR CRISPR (n = 8), CRISPR + βarr1 (n = 4), CRISPR + βarr2 (n = 4). (B) Top: Schematic representation of the BRET biosensors used to monitor loss of β₂AR from the plasma membrane. Receptor internalization was measured by the decrease in BRET between β2AR-RlucII and rGFP-CAAX labeling the plasma membrane (56). Bottom: Iso concentration-response curves generated in each parental HEK293 cell line (black symbols; solid black line) together with those observed in the corresponding CRISPR βArr1/2 KO line in the absence (open symbols; dashed black line) or presence of exogenous βArr1 (open symbols; dashed blue line) or βArr2 (open symbols; dashed red line). Data are means ± SEM of four to six biological replicates as follows: SL parental (n = 6), SL CRISPR (n = 6), SL CRISPR + βarr1 (n = 5), SL CRISPR + βarr2 (n = 5), AI parental (n = 5), AI CRISPR (n = 5), AI CRISPR + βarr1 (n = 4), AI CRISPR + βarr2 (n = 4), HAR parental (n = 5), HAR CRISPR (n = 5), HAR CRISPR + βarr1 (n = 4), HAR CRISPR + βarr2 (n = 4). (C) Top: Schematic representation of the FRET-based assay used to monitor ERK1/2 phosphorylation. The AlphaLISA SureFire Ultra system is a sandwich ELISA in which bridging of donor and acceptor beads by the activated pThr²⁰²/pTyr²⁰⁴ ERK1/2 analyte produces an increase in fluorescence emission (59). Middle: The effect of pretreatment with PTX, the PKA inhibitor 6–22, or both, on 1 µM Iso-stimulated ERK1/2 phosphorylation in SL-parental HEK293 cells. Bottom: Results of identical experiments performed using the corresponding SL-CRISPR βArr1/2 KO line. In each graph, responses are expressed as a percentage of the maximal Iso-stimulated response in the absence of inhibitor (control). Data are means ± SEM of three to six biological replicates as follows: SL parental: control (n = 6), PTX (n = 5), 6–22 (n = 6), PTX + 6–22 (n = 3). SL CRISPR: control (n = 6), PTX (n = 5), 6–22 (n = 6), PTX + 6–22 (n = 3). Significance was assessed by two-way ANOVA. * P < 0.05; ** P < 0.01 for the indicated comparisons.

Fig 5.. Contrast between the consistent effects of siRNA-mediated knockdown of βArr1/2 in parental HEK293 cells and the variable effects of βArr1/2 reconstitution in CRISPR βArr1/2 KO cells on ERK1/2 activation by the β₁AR.

(A to C) Top: Parental HEK293 cell lines co-transfected with plasmid encoding FLAG-β₁AR and siRNAs targeting no mRNA (CTL) or βArr1/2 were analyzed by Western blotting (IB) with antibody against βArr1/2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Middle: Serum-deprived cells were stimulated with 10 µM isoproterenol (Iso) for the indicated times and cell lysates were analyzed by Western blotting sequentially for pERK1/2 (pERK) and total ERK1/2 (ERK). Western blots for each pair of CTL siRNA– and βArr1/2 siRNA–treated cells are representative of four experiments. Bottom: For each CTL siRNA– (black symbols) and βArr1/2 siRNA–treated (red symbols) pair, Iso-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the CTL siRNA–treated cells and data are expressed a percentage of the maximum control response (% max control). Data are means ± SEM of four biological replicates. (D to F) Top: CRISPR βArr1/2 KO cell lines transiently transfected with plasmid encoding FLAG-β1AR together with either empty vector or plasmids encoding HA-βArr1 and HA-βArr2 were analyzed by Western blotting (IB) with antibody against βArr1/2. GAPDH was used as a loading control. Middle: Serum-deprived cells were stimulated with 10 µM Iso for the indicated times and cell lysates were analyzed by Western blotting sequentially for pERK1/2 (pERK) and total ERK1/2 (ERK). Western blots for each pair of vector-transfected and HA-βArr1/2–expressing CRISPR cells are representative of three experiments. Bottom: For each vector-transfected (black symbols) and HA-βArr1/2– expressing (green symbols) CRISPR cell pair, Iso-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the vector-transfected cells and the data are expressed a percentage of the maximum control response (% max control). Data are means ± SEM of three biological replicates. In all panels, statistical significance was determined by two-way ANOVA and Sidak’s multiple comparison test. # P < 0.05 compared with control at the corresponding time point; ns, not significant.

Fig 6.. Reciprocal effects of βArr1/2 knockdown in parental HEK293 cells and βArr1/2 reconstitution in CRISPR βArr1/2 KO cells on ERK1/2 activation by the V2R.

(A to C) Top: Parental HEK293 cell lines co-transfected with plasmid encoding HA-V₂R and siRNAs targeting no mRNA (CTL) or βArr1/2 were analyzed by Western blotting (IB) with antibody against βArr1/2 (top row). Total ERK1/2 was used as a loading control. Serum-deprived cells were stimulated with 1 µM arginine-vasopressin (AVP) for the indicated times and cell lysates were analyzed by Western blotting sequentially for pERK1/2 (pERK) (middle row) and total ERK1/2 (ERK) (bottom row). Western blots for each pair of CTL siRNA– and βArr1/2 siRNA–treated cells are representative of three experiments. Bottom: For each CTL siRNA– (black symbols) and βArr1/2 siRNA–treated (red symbols) pair, AVP-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the CTL siRNA–treated cells and the data are expressed a percentage of the maximum control response (% max control). Data are means ± SEM of three biological replicates. (D to F) Top: CRISPR βArr1/2 KO cell lines transiently transfected with plasmid encoding HA-V₂R together with either empty vector or plasmids encoding FLAG-βArr1 or FLAG-βArr2 were analyzed by Western blotting (IB) with antibody against βArr1/2 (top rows). Total ERK1/2 was used as a loading control. Serum-deprived cells were stimulated with 1 µM AVP for the indicated times and cell lysates were analyzed by Western blotting sequentially for pERK1/2 (pERK) (middle rows) and total ERK1/2 (ERK) (bottom rows). Western blots for each pair of vector-transfected and FLAG-βArr– expressing CRISPR cells are representative of three experiments. Bottom: For vector-transfected (black symbols), FLAG-βArr1–expressing (purple symbols), and FLAG-βArr2–expressing (green symbols) cell comparisons, AVP-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the vector-transfected cells and the data are expressed as a percentage of the maximum control response (% max control). Data are means ± SEM of three biological replicates. In all panels, statistical significance was determined by two-way ANOVA and Sidak’s multiple comparison test. & P < 0.05 for βArr1 reconstitution compared with control at the corresponding time point; # P < 0.05 for βArr2 reconstitution compared with control at the corresponding time point; ns, not significant.

Fig 7.. Reciprocal effects of βArr1/2 knockdown in parental HEK293 cells and βArr1/2 reconstitution in CRISPR βArr1/2 KO cells on ERK1/2 activation by the FSHR.

(A to C) Top: Parental HEK293 cell lines co-transfected with plasmid encoding FLAG-FSHR and siRNAs targeting no mRNA (CTL) or βArr1/2 were analyzed by Western blotting (IB) with antibody against βArr1/2. Total ERK1/2 was used as a loading control. Middle: Serum-deprived cells were stimulated with 100 ng/mL recombinant human FSH for the indicated times and cell lysates were analyzed by Western blotting sequentially for pERK1/2 (pERK) and total ERK1/2 (ERK). Western blots for each pair of CTL siRNA– and βArr1/2 siRNA–treated cells are representative of five experiments. Bottom: For each CTL siRNA– (black symbols) and βArr1/2 siRNA–treated (red symbols) pair, FSH-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the CTL siRNA–treated cells and the data are expressed as a percentage of the maximum control response (% max control). Data are means ± SEM of five biological replicates. (D to F) Top: CRISPR βArr1/2 KO cell lines transiently transfected with plasmid encoding FLAG-FSHR together with either empty vector or plasmids encoding HA-βArr1 and HA-βArr2 were analyzed by Western blotting (IB) with antibody against βArr1/2. Total ERK1/2 was used as a loading control. Middle: Serum-deprived cells were stimulated with 100 ng/mL FSH for the indicated times and cell lysates were analyzed by Western blotting sequentially for pERK1/2 (pERK) and total ERK1/2 (ERK). Western blots for each pair of vector-transfected and FLAG-βArr1/2–expressing CRISPR cells are representative of four (D and F) or five (E) experiments. Bottom: For each vector-transfected (black symbols) and HA-βArr1/2–expressing (green symbols) CRISPR cell pair, FSH-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the vector- transfected cells and the data are expressed as a percentage of the maximum control response (% max control). Data are means ± SEM of four or five biological replicates. In all panels, statistical significance was determined by two-way ANOVA and Sidak’s multiple comparison test. # P < 0.05 for βArr1/2 reconstitution compared with the control at the corresponding time point; ns, not significant.

Fig 8.. Reciprocal effects of βArr1/2 knockdown in parental HEK293 cells and βArr1/2 reconstitution in CRISPR βArr1/2 KO cells on ERK1/2 activation by the arrestin pathway–selective biased agonist carvedilol.

(A) Top: SL-Parental HEK293 cells co-transfected with plasmid encoding FLAG-β₂AR and siRNAs targeting no mRNA (CTL) or βArr1/2 were analyzed by Western blotting (IB) with antibody against βArr1/2. β-Actin was used as a loading control. Middle: Serum-deprived cells were stimulated with 10 µM carvedilol (Carv) for the indicated times and cell lysates analyzed by Western blotting sequentially for pERK1/2 (pERK) and total ERK1/2 (ERK). Western blots of CTL siRNA– and βArr1/2 siRNA–treated cells are representative of three experiments. Bottom: For CTL siRNA– (black symbols) and βArr1/2 siRNA–treated (red symbols) cells, Carv-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the CTL siRNA–treated cells and the data are expressed as a percentage of the maximum control response (% max control). Data are means ± SEM of three biological replicates. (B) Top: SL-CRISPR βArr1/2 KO cells transiently transfected with plasmid encoding FLAG-β₂AR together with either empty vector or plasmids encoding HA-βArr1 and HA-βArr2 were analyzed by Western blotting (IB) with antibody against βArr1/2. β-Actin was used as a loading control. Middle: Serum-deprived cells were stimulated with 10 µM Carv for the indicated times and cell lysates were analyzed by Western blotting sequentially for pERK1/2 (pERK) and total ERK1/2 (ERK). Western blots of vector-transfected and FLAG-βArr1/2–expressing CRISPR cells are representative of four experiments. Bottom: For vector-transfected (black symbols) and HA-βArr1/2–expressing (green symbols) CRISPR cells, Carv-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the vector-transfected cells and the data are expressed as a percentage of the maximum control response (% max control). Data are means ± SEM of four biological replicates. (C and D) Analogous set of experiments to those described in (A) and (B) were performed in AI-parental HEK293 and AI-CRISPR βArr1/2 KO cells expressing the β₁AR. Western blots and densitometry data represent three (D) or four (C) biological replicates. In all panels, statistical significance was determined by two-way ANOVA and Sidak’s multiple comparison test. # P < 0.05 for βArr1/2 reconstitution compared with the control at the corresponding time point; ns, not significant.

Fig 9.. Model depicting the dual roles of β-arrestins in the GPCR-dependent stimulation of ERK1/2.

(A) In native cells, ERK1/2 activation reflects a balance between G protein–dependent pathways that are attenuated by β-arrestin–dependent desensitization and β-arrestin–dependent pathways that are augmented by its scaffolding function. (B) In the CRISPR/Cas9 βArr1/2 KO background, G protein–dependent ERK1/2 activation is unrestrained and β-arrestin–dependent ERK1/2 activation is absent. The resulting increase in signal strength in the G protein pathways may or may not offset the loss of β-arrestin–mediated signaling, leading to a net increase, decrease, or no change in GPCR-stimulated ERK1/2 activity. (C) β-Arrestin–biased ligands that possess little or no G protein efficacy rely predominantly on β-arrestin scaffolds to support ERK1/2 activation.