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. Author manuscript; available in PMC: 2015 Jun 1.
Published in final edited form as: Nat Chem Biol. 2014 Nov 2;10(12):1061–1065. doi: 10.1038/nchembio.1665

Figure 1. Endocytosis of β2-AR is required for cAMP accumulation and transcriptional response.

Figure 1

(a–b) Real-time cAMP levels were measured using the enzyme-based biosensor pGLO-20F (Promega) in response to bath application of (a) 1 μM or (b) 10 nM isoproterenol in cells pre-treated for 15 min with vehicle (DMSO) (blue) or 30 μM Dyngo (red). Data = average from n = 2–3 experiments. (c–e) Measurement by oligonucleotide microarrays of global gene expression in response to activation of β2-AR. Cells were pre-treated with either DMSO or Dyngo for 15 min as indicated. (c) Heat map showing the median-centered expression of core isoproterenol-responsive target genes (indicated on the y-axis; Supplementary Table 1) across all microarray experiments. (d–e) Scatter plots comparing expression levels for the target genes upon treatment of cells with (d) 1 μM or (e) 10 nM isoproterenol. Data = averaged log2 ratios (Iso/No Drug) from n = 2 experiments. In grey – isoproterenol targets, in red – endocytosis-dependent genes (Supplementary Table 3), dotted line has a slope of 1 (y = x). Arrows indicate RNA levels for PCK1 and DACT2. (f) Confirmation by qRT-PCR of PCK1 and DACT2 expression in response to block of receptor endocytosis. (g) Clathrin knockdown effects on β2-AR signalling in cells transfected with CHC17 or control siRNAs. RNA levels of PCK1 were analyzed by qRT-PCR. Data = averaged log2 ratios (Iso/No Drug) from n = 2 experiments. ND = no drug; Iso = isoproterenol. ** p < 0.005, * p < 0.05, p-values by unpaired t-test; error bars = ± s.e.m.