File S1 - Broad-Scale Phosphoprotein Profiling of Beta Adrenergic Receptor (β-AR) Signaling Reveals Novel Phosphorylation and Dephosphorylation Events

Figures S1-S6. Figure S1. Competition binding study in MEFs with ICI 118,551. Membranes were prepared from MEFs and were used in binding reactions. For each reaction, 60 μg of membranes was incubated with 10 nM [3H]dihydroalprenolol ([3H]DHA) and varying concentrations of the β2-AR selective antagonist ICI 188,551. Non-specific binding was determined in the presence of 1 μM alprenolol. Results show mean ± SEM values from three experiments. Competition data were best fit with a two-site model that included a high-affinity binding site (74.7%) and low-affinity binding site (25.3%) for ICI 118,551. The high-affinity site corresponds to β2-AR, whereas low-affinity sites are β1-AR or β3-AR. Figure S2. Phosphoprotein signaling in MEFs after stimulation with epinephrine. A heat map representation of phosphoprotein changes over time. MEFs were grown to confluence and then were stimulated with different doses of epinephrine (Epi) (1 μM, 10 μM, or 100 μM) for various times (0, 5, 10, 20, 30, or 60 minutes). Stimulations were performed in the presence or absence of the alpha 1 adrenergic receptor antagonist prazosin (Praz) (1 μM). Using data from the lysate microarrays, a heat map was constructed which revealed distinct clusters of phosphorylation (yellow) and dephosphorylation (blue) events after epinephrine stimulation. The color scale shows fold change as compared with unstimulated MEFs. Data are representative of two independent experiments. Figure S3. Phosphoprotein signaling in MEFs after stimulation with norepinephrine. A heat map representation of phosphoprotein changes over time. MEFs were grown to confluence and then were stimulated with different doses of norepinephrine (Norepi) (1 μM, 10 μM, or 100 μM) for various times (0, 5, 10, 20, 30, or 60 minutes). Stimulations were performed in the presence or absence of the alpha 1 adrenergic receptor antagonist prazosin (Praz) (1 μM). Using data from the lysate microarrays, a heat map was constructed which revealed distinct clusters of phosphorylation (yellow) and dephosphorylation (blue) events after norepinephrine stimulation. The color scale shows fold change as compared with unstimulated MEFs. Data are representative of two independent experiments. Figure S4. Western blotting confirmatory studies on MEF lysates used for lysate microarrays. Figures show phospho (denoted by “p-“) and total levels of proteins as detected by western blotting. Each blot represents an isoproterenol time course study with 0, 5, 10, 20, 30 and 60 minute stimulations. Dephosphorylated proteins are shown at the top of the figure and phosphorylated proteins are shown at the bottom of the figure. Figure S5. Isoproterenol induced phosphoprotein signaling in the presence of pertussis toxin. MEFs were pretreated with pertussis toxin (PTX) (1 μg/ml) or vehicle control for 30 minutes and then stimulated with isoproterenol (Iso) (1 μM) for various times (0, 5, 10, 20, 30, or 60 minutes). A heat map shows phosphorylation (yellow) and dephosphorylation (blue) events after isoproterenol stimulation. The isoproterenol time course study on the left was performed in the presence of PTX and the time course study on the right was performed in the presence of vehicle. The color scale shows fold change as compared with unstimulated MEFs. Data are means of three independent stimulation experiments. Figure S6. Epac activation as assessed by the Rap GAP assay. MEFs were stimulated with vehicle control (C), forskolin (FSK) (50 μM), or the Epac-specific analog 8-pCPT-2'-O-Me-cAMP (Epac) (250 μM) for 5 or 20 minutes. Following stimulation, cells were lysed and incubated with Ral GDS-Rap Binding Domain bound to glutathione-agarose beads to specifically pull down Rap1-GTP (see Materials and Methods). Western blotting was then performed to detect Rap1. Positive (+) and negative (-) controls are included in the figure. Data depicted are representative of two independent stimulation experiments.
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