Research ArticleGPCR SIGNALING

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

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Science Signaling  25 Sep 2018:
Vol. 11, Issue 549, eaat7650
DOI: 10.1126/scisignal.aat7650

Figures

  • 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 (o/e) 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, isoproterenol-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the parental line, and data are expressed as 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 analysis of variance (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 HEK293 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 [control (CTL)], βArr2 (A to D), or both βArr1 and βArr2 (E to H) were analyzed by Western blotting with antibody against βArr1/2. β-Actin was used as a loading control. Middle: Serum-deprived cells were stimulated with 100 nM isoproterenol 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 β-arrestin siRNA–treated cells are representative of three (H), four (B, C, and E to G), or five (A and D) experiments. Bottom: For each CTL siRNA–treated (open black symbols) and β-arrestin siRNA–treated (red symbols) pair, isoproterenol-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the CTL siRNA–treated cells, and data are expressed as a percentage of the maximum control response (% max control). Data are means ± SEM of three to 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.

  • 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 hemagglutinin (HA)–βArr2 (A to C) or both HA-βArr1 and HA-βArr2 (D to F). Parental HEK293 cells (P), vector-transfected CRIPSR cells, and CRISPR cells expressing HA-βArr1, HA-βArr2, or HA-βArr1/2 were analyzed by Western blotting with antibody against βArr1/2. β-Actin was used as a loading control. Middle: Serum-deprived cells were stimulated with 100 nM isoproterenol for the indicated times, and cell lysates were analyzed by Western blotting sequentially for pERK1/2 and total ERK1/2. Western blots for each pair of vector-transfected and HA–β-arrestin–expressing CRISPR cells are representative of three (B, C, E, and F) or four (A and D) experiments. Bottom: For each vector-transfected (black symbols) and HA–β-arrestin–expressing (green symbols) CRISPR cell pair, isoproterenol-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the vector-transfected cells, and data are expressed as a percentage of the maximum control response. 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.

  • 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 Gαs activation. Activation of Gαs induces dissociation of Rluc-II–Gαs from GFP10-Gγ1, resulting in a decrease in BRET signal (55). GDP, guanosine diphosphate; GTP, guanosine 5′-triphosphate. Bottom: Isoproterenol 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 4 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), and 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–Rluc-II and rGFP-CAAX labeling the plasma membrane (56). Bottom: Isoproterenol 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), and 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 enzyme-linked immunosorbent assay (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 isoproterenol-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 isoproterenol-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), and PTX + 6-22 (n = 3); SL-CRISPR: control (n = 6), PTX (n = 5), 6-22 (n = 6), and PTX + 6-22 (n = 3). Statistical 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 cotransfected with plasmid encoding FLAG-β1AR and siRNAs targeting no mRNA (CTL) or βArr1/2 were analyzed by Western blotting 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 for the indicated times, and cell lysates were analyzed by Western blotting sequentially for pERK1/2 and total ERK1/2. Western blots for each pair of CTL siRNA– and βArr1/2 siRNA–treated cells are representative of four experiments. Bottom: For each CTL siRNA–treated (black symbols) and βArr1/2 siRNA–treated (red symbols) pair, isoproterenol-stimulated pERK/ERK ratios were normalized to the maximum signal observed in the CTL siRNA–treated cells, and data are expressed as a percentage of the maximum control response. 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 with antibody against βArr1/2. GAPDH was used as a loading control. Middle: Serum-deprived cells were stimulated with 10 μM isoproterenol for the indicated times, and cell lysates were analyzed by Western blotting sequentially for pERK1/2 and total ERK1/2. 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, isoproterenol-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. Data are means ± SEM of three biological replicates. Statistical significance was determined by two-way ANOVA and Sidak’s multiple comparison test. *P < 0.05 compared with each corresponding control group; #P < 0.05 compared with control at the corresponding time point.

  • 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 cotransfected with plasmid encoding HA-V2R and siRNAs targeting no mRNA (CTL) or βArr1/2 were analyzed by Western blotting with antibody against βArr1/2. 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 and total ERK1/2. Western blots for each pair of CTL siRNA– and βArr1/2 siRNA–treated cells are representative of three experiments. Bottom: For each CTL siRNA–treated (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 as a percentage of the maximum control response. 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 with antibody against βArr1/2. 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 and total ERK1/2. Western blots for each pair of vector-transfected and FLAG–β-arrestin–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. Data are means ± SEM of three biological replicates. Statistical significance was determined by two-way ANOVA and Sidak’s multiple comparison test. *P < 0.05 compared with each corresponding control group; &P < 0.05 for βArr1 reconstitution compared with control at the corresponding time point; #P < 0.05 for βArr2 knockdown or βArr2 reconstitution compared with control at the corresponding time point.

  • 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 cotransfected with plasmid encoding FLAG-FSHR and siRNAs targeting no mRNA (CTL) or βArr1/2 were analyzed by Western blotting with antibody against βArr1/2. Total ERK1/2 was used as a loading control. Middle: Serum-deprived cells were stimulated with recombinant human FSH (100 ng/ml) for the indicated times, and cell lysates were analyzed by Western blotting sequentially for pERK1/2 and total ERK1/2. Western blots for each pair of CTL siRNA– and βArr1/2 siRNA–treated cells are representative of five experiments. Bottom: For each CTL siRNA–treated (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. 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 with antibody against βArr1/2. Total ERK1/2 was used as a loading control. Middle: Serum-deprived cells were stimulated with FSH (100 ng/ml) for the indicated times, and cell lysates were analyzed by Western blotting sequentially for pERK1/2 and total ERK1/2. 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. 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 each corresponding control group; #P < 0.05 for βArr1/2 knockdown or βArr1/2 reconstitution compared with control at the corresponding time point.

  • 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 cotransfected with plasmid encoding FLAG-β2AR and siRNAs targeting no mRNA (CTL) or βArr1/2 were analyzed by Western blotting 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 were analyzed by Western blotting sequentially for pERK1/2 and total ERK1/2. Western blots of CTL siRNA– and βArr1/2 siRNA–treated cells are representative of three experiments. Bottom: For CTL siRNA–treated (black symbols) and βArr1/2 siRNA–treated (red symbols) cells, carvedilol-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. 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 with antibody against βArr1/2. β-Actin was used as a loading control. Middle: Serum-deprived cells were stimulated with 10 μM carvedilol for the indicated times, and cell lysates were analyzed by Western blotting sequentially for pERK1/2 and total ERK1/2. 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, carvedilol-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. 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 HAR-parental HEK293 and HAR-CRISPR βArr1/2 KO cells expressing the β1AR. Western blots and densitometry data represent three (D) or four (C) biological replicates. Statistical significance was determined by two-way ANOVA and Sidak’s multiple comparison test. *P < 0.05 compared with each corresponding control group; #P < 0.05 for βArr1/2 knockdown or reconstitution compared with control at the corresponding time point.

  • 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 (βArr)–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 have little or no G protein efficacy rely predominantly on β-arrestin scaffolds to support ERK1/2 activation.

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