Supplementary Materials

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

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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