Research ArticleGPCR SIGNALING

Genetic evidence that β-arrestins are dispensable for the initiation of β2-adrenergic receptor signaling to ERK

See allHide authors and affiliations

Science Signaling  20 Jun 2017:
Vol. 10, Issue 484, eaal3395
DOI: 10.1126/scisignal.aal3395
  • Fig. 1 Internalization of β2AR in HEK293, β-arr1 KO, and β-arr–less cells.

    (A) Schematic of FLAG-β2AR and FLAG-β2AR 3S internalization assays. Iso, isoproterenol. (B) Surface FLAG-β2AR or FLAG-β2AR 3S abundance in HEK293 cells before or after 15 min of isoproterenol (10 μM) stimulation compared with isotype-stained reference control and control stimulation at 4°C. (C) Internalization of FLAG-β2AR or FLAG-β2AR 3S after isoproterenol stimulation; mean of three independent experiments as in (B) ± SD. (D) Western blot of β-arr1 and β-arr2 and α-tubulin (loading control) in HEK293 cells transfected with β-arrestin or control siRNAs (C). Representative of four independent experiments. (E) Internalization of FLAG-β2AR after isoproterenol stimulation in HEK293 cells transfected with β-arrestin or control siRNAs; mean of three independent experiments ± SD. (F) Flow cytometry quantification of median fluorescence intensity (MFI) of 15-min transferrin-546 uptake in HEK293 FLAG-β2AR cells transfected with control siRNA (siCont) or indicated siRNAs; mean of three independent experiments ± SD. ns, not significant. (G) Schematic of the β-arr1 TALEN construct design targeting exon 6. (H and I) Western blot for β-arrestins in (H) HEK293 and β-arr1 KO cells and in (I) HEK293 and β-arr1 KO cells transfected with two different siRNAs (A, B) targeting β-arr1 or β-arr2 or siRNA pools (A + B) or control scrambled siRNA (C). (J) Internalization of FLAG-β2AR after isoproterenol stimulation in indicated cells; mean of three independent experiments ± SD.

  • Fig. 2 β2AR internalization into endocytic vesicles through clathrin-coated pits.

    (A) Schematic of SNAP-tagged β2AR used for immunofluorescence (IF) imaging. Immunofluorescence of SNAP-β2AR and EEA1 endocytic vesicles (B) and respective Pearson’s correlation (R) values (C). Mean of 12 individual cells imaged from two independent experiments ± SEM in the indicated cells upon 10-min isoproterenol stimulation. Statistical significance was determined by t test. Scale bars, 10 μm. (D) Surface FLAG-β2AR abundance in the indicated cells after isoproterenol stimulation, determined by TIRF microscopy using fluorescently labeled β-arr2 siRNA. Representative of three independent experiments. (E) TIRF imaging of FLAG-β2AR (green) and clathrin-coated pits (red) in indicated cells before and after 5-min isoproterenol stimulation. Graphs depict the overlap in fluorescence intensity between FLAG-β2AR and clathrin-coated pits across designated lines through the cells, as an indication of colocalization. Scale bars, 5 μm; inset scale bars, 500 nm. Representative of three independent experiments.

  • Fig. 3 ERK activation in HEK293 and β-arr–less cells.

    (A) In-Cell Western blot with quantification of ERK phosphorylation (pERK) induced by isoproterenol stimulation, performed in triplicate ± SEM; representative of two independent experiments shown. (B) Western blot and quantification of isoproterenol-induced pERK normalized to total ERK from three independent Western blots ± SEM. Statistical significance was determined by t test. Westerns are from the same blot and exposure; representative of three independent experiments. (C) Immunofluorescence of FLAG-β2AR and FLAG-β2AR 3S in HEK293 cells upon 5-min isoproterenol stimulation. Scale bars, 10 μm. Representative image from two independent experiments. (D) Western blot of pERK and indicated controls upon isoproterenol stimulation of HEK293 FLAG-β2AR or FLAG-β2AR 3S cells. (E) Quantification of pERK normalized to ERK from three independent Western blots ± SEM in the indicated cells after isoproterenol stimulation. Statistical significance was determined by t test. (F) pERK Western blots of the indicated cells after 3-min isoproterenol and epinephrine (Epi) stimulations (representative of three independent experiments) and the corresponding representative ELISA data quantifying amounts of pERK (representative of three independent experiments). Relative Emax and EC50 values are indicated. Nonlinear regression (least squares fit) analysis was used to fit curves and determine the EC50 and Emax values. Statistical significance (*) signifies nonoverlapping 95% confidence intervals. (G and H) Western blot for β-arr1 and β-arr2 in parental HEK293 cells and two separate CRISPR/Cas9 β-arr1/2 KO clones (asterisks in the β-arrestin 2 panel indicate nonspecific immunoreactive bands) (G) and pERK amounts upon isoproterenol stimulation of these cells (H). Representative of three independent experiments. (I) Western blot for pERK, total ERK, and α-tubulin in unstimulated and 5-min isoproterenol (10 μM) stimulated parental HEK293 and β-arr1/2 KO cell lines with endogenous levels of β2AR. Results represent three independent experiments.

  • Fig. 4 s, but not PKA, is critical for β2AR-mediated ERK phosphorylation.

    (A) Immunofluorescence showing GFP expression of FLAG-β2AR Gnas f/f MEFs transduced with control adenoviral (adeno)–GFP or adeno-Cre-GFP and Western blot of pERK upon 3-min isoproterenol stimulation of these cells (representative of three independent experiments). (B) Western blot for Gαs in HEK293 CRISPR/Cas9-edited Gαs KO cells. (C) Western blot of pERK upon 3-min isoproterenol or EGF (10 ng/ml) stimulation in the indicated cells. Representative of three independent experiments. (D and E) Relative amounts (mean ± SEM of three independent experiments) of cAMP (D) and CRE luciferase activity (E) in the indicated cells upon isoproterenol stimulation. (F) Western blot of pERK after isoproterenol stimulation in Gαs KO FLAG-β2AR cells transfected with vector control or Gαs. Representative of three independent experiments. (G and H) Western blot of pERK in HEK293 FLAG-β2AR cells stimulated with isoproterenol after pretreatment with dimethyl sulfoxide (DMSO) (control), H89 (10 μM) (G), or cAMPS-RP (100 μM) (H). Representative of three independent experiments for (G) and (H). (I) Relative CRE luciferase activity in isoproterenol- and forskolin (FSK; 5 mg/ml)–stimulated HEK293 FLAG-β2AR cells pretreated with H89, cAMPS-RP, or DMSO control; mean ± SEM of three experiments. (J) Immunofluorescence of GFP expression, relative CRE luciferase activity (mean ± SEM, three experiments), and Western blot of pERK in HEK293 FLAG-β2AR cells transfected with GFP-PKI or GFP-PKI-mutant (PKI-Mut) plasmids upon stimulation with isoproterenol (representative of three independent experiments).

  • Fig. 5 β2AR-mediates ERK activation through Gβγ signaling and activation of a signaling cascade that involves SRC, SHC, SOS, RAS, RAF, and MEK.

    (A and B) Western blot of pERK after isoproterenol or EGF stimulation (3 min) in HEK293 FLAG-β2AR cells pretreated with DMSO or U0126 (10 μM) (A), SB-590885 (SB; 10 μM) (B), or GDC-0879 (GDC; 10 μM) (B). Representative of three independent experiments for (A) and (B). (C) Western blot of pERK after isoproterenol stimulation in HEK293 FLAG-β2AR cells transfected with dominant-negative (DN) KRAS, dominant-negative HRAS, or control. Representative of four independent experiments. (D) Relative CRE luciferase activity in isoproterenol- and forskolin-stimulated HEK293 FLAG-β2AR cells transfected with dominant-negative KRAS or control plasmid; mean ± SEM of three experiments. (E) Western blot showing active RAS-GTP pull-down (IP) and input for RAS and α-tubulin after isoproterenol stimulation. Representative of three independent experiments. (F) Immunofluorescence showing GFP expression and pERK Western blot of isoproterenol-stimulated FLAG-β2AR–Rasless MEFs transduced with adeno-GFP or adeno-Cre-GFP virus (representative of three independent experiments). (G) Effects of an siRNA library screen of various RAS–guanine nucleotide exchange factors (GEFs) on isoproterenol-stimulated pERK amounts. Representative of three independent experiments. (H) Western blot of pERK in HEK293 FLAG-β2AR cells transfected with siRNA to SOS1 and SOS2 (SOS) or control in response to isoproterenol. Representative of three independent experiments. (I and J) Western blot of pSRC and pSHC in (I) response to isoproterenol stimulation and (J) with the SRC inhibitor PP1 (10 μM), SU6656 (SU; 10 μM), or DMSO. Representative of three independent experiments for (I) and (J). (K) Western blot of pERK in HEK293 FLAG-β2AR cells pretreated with SRC inhibitors upon isoproterenol stimulation. Representative of three independent experiments. (L) Western blot of pSRC in the indicated cells upon isoproterenol stimulation. Representative of three independent experiments. (M) Western blot of pERK after isoproterenol or EGF stimulation in HEK293 FLAG-β2AR cells transfected with control or hemagglutinin (HA)–tagged GRK2ct plasmids. Representative of three independent experiments. (N) Western blot of isoproterenol-stimulated pSRC and pERK in HEK293 FLAG-β2AR cells transfected with control or HA-tagged GRK2ct. Representative of three independent experiments.

  • Fig. 6 Schematic depicting β2AR-mediated ERK activation.

    β2AR-mediated ERK activation occurs through Gβγ induction of a signaling cascade that involves SRC, SHC, SOS, RAS, RAF, and MEK. β-arr2 mediates endocytosis of β2AR through clathrin-coated pits (CCPs), and Gα activates adenylate cyclase (ADCY) to generate cAMP, thereby activating PKA. Although PKA is dispensable for the activation of the SRC/SHC/SOS/RAS/RAF/MEK/ERK pathway by β2AR, it will require further investigation to determine whether PKA redundantly feeds into this pathway.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/484/eaal3395/DC1

    Fig. S1.Transferrin uptake experiments and genomic DNA sequencing of the β-arr1 KO cell line.

    Fig. S2. Clathrin is important for isoproterenol-mediated β2AR internalization.

    Fig. S3. Depletion of both β-arr1 and β-arr2 increases β2AR-mediated cAMP production.

    Fig. S4. Sequence of β-arr1/2 double-mutant cells.

    Fig. S5. Endogenous β2AR in parental HEK293 and β-arr1/2 KO and β2AR and V2R internalization in these cells.

    Fig. S6. β-Arrestin is not required for ERK phosphorylation in arginine vasopressin receptor 2 (V2R)–expressing cells.

    Fig. S7. Stable expression of FLAG-β2AR on Gnas f/f MEFs and Gαs KO cells and effect of EPAC inhibition on isoproterenol-mediated ERK phosphorylation and RAP1A activation.

    Fig. S8. EGFR, PI3K, and Gαi signaling are dispensable for activation of ERK by β2AR, but Gβγ signaling is important.

    Movie S1. Live-cell confocal imaging of β2AR internalization in HEK293 cells.

    Movie S2. Live-cell confocal imaging of β2AR internalization in β-arr1 KO cells.

    Movie S3. Live-cell confocal imaging of β2AR internalization in β-arr–less cells.

    Movie S4. Live-cell TIRF imaging of β2AR internalization into clathrin-coated pits in HEK293 cells.

    Movie S5. Live-cell TIRF imaging of β2AR internalization into clathrin-coated pits in β-arr1 KO cells.

    Movie S6. Live-cell TIRF imaging of β2AR internalization into clathrin-coated pits in β-arr–less cells.

  • Supplementary Materials for:

    Genetic evidence that β-arrestins are dispensable for the initiation of β2-adrenergic receptor signaling to ERK

    Morgan O'Hayre, Kelsie Eichel, Silvia Avino, Xuefeng Zhao, Dana J. Steffen, Xiaodong Feng, Kouki Kawakami, Junken Aoki, Karen Messer, Roger Sunahara, Asuka Inoue, Mark von Zastrow, J. Silvio Gutkind*

    *Corresponding author. Email: sgutkind{at}ucsd.edu

    This PDF file includes:

    • Fig. S1.Transferrin uptake experiments and genomic DNA sequencing of the β-arr1 KO cell line.
    • Fig. S2. Clathrin is important for isoproterenol-mediated β2AR internalization.
    • Fig. S3. Depletion of both β-arr1 and β-arr2 increases β2AR-mediated cAMP production.
    • Fig. S4. Sequence of β-arr1/2 double-mutant cells.
    • Fig. S5. Endogenous β2AR in parental HEK293 and β-arr1/2 KO and β2AR and V2R internalization in these cells.
    • Fig. S6. β-Arrestin is not required for ERK phosphorylation in arginine vasopressin receptor 2 (V2R)–expressing cells.
    • Fig. S7. Stable expression of FLAG-β2AR on Gnas f/f MEFs and Gαs KO cells and effect of EPAC inhibition on isoproterenol-mediated ERK phosphorylation and RAP1A activation.
    • Fig. S8. EGFR, PI3K, and Gαi signaling are dispensable for activation of ERK by β2AR, but Gβγ signaling is important.
    • Legends for movies S1 to S6

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.60 MB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Live-cell confocal imaging of β2AR internalization in HEK293 cells.
    • Movie S2 (.mp4 format). Live-cell confocal imaging of β2AR internalization in β-arr1 KO cells.
    • Movie S3 (.mp4 format). Live-cell confocal imaging of β2AR internalization in β-arr–less cells.
    • Movie S4 (.mp4 format). Live-cell TIRF imaging of β2AR internalization into clathrin-coated pits in HEK293 cells.
    • Movie S5 (.mp4 format). Live-cell TIRF imaging of β2AR internalization into clathrin-coated pits in β-arr1 KO cells.
    • Movie S6 (.mp4 format). Live-cell TIRF imaging of β2AR internalization into clathrin-coated pits in β-arr–less cells.

    Citation: M. O'Hayre, K. Eichel, S. Avino, X. Zhao, D. J. Steffen, X. Feng, K. Kawakami, J. Aoki, K. Messer, R. Sunahara, A. Inoue, M. von Zastrow, J. S. Gutkind, Genetic evidence that b-arrestins are dispensable for the initiation of β2-adrenergic receptor signaling to ERK. Sci. Signal. 10, eaal3395 (2017).

    © 2017 American Association for the Advancement of Science

Navigate This Article