FIGURE 4.

Cholesterol depletion induces β₂AR phosphorylation, which in turn is responsible for loss of high affinity binding state. A, nontransfected cells (Mock) as well as cells stably expressing 1–3 pmol.mg^-1 of either β₂AR_WTorβ₂AR_PKA- were treated (CD) or not (-) with 2% CD for 1 h at 37 °C. Total membranes were solubilized, and the receptor phosphorylation state was assessed by Western blot analysis (IB) on 5 μg of protein using an antibody directed against phosphorylated Ser²⁶² amino acid of the receptor. Upper panel, immunoblot using the anti-phospho-Ser²⁶² antibody. Lower panel, immunoblot using an anti-β₂AR antibody. Data are representative of five independent experiments. B, cells stably expressing 1–3 pmol.mg^-1 of either β₂AR_WT (leftmost and rightmost panels) or β₂AR_PKA- (middle panel) were treated (CD, □) or not (vehicle, ▪) with 2% CD for 1 h at 37 °C to promote cholesterol depletion. Inhibition of PKA with 5 μm KT5720 was performed 2 h prior to depletion with CD (rightmost panel, ▪). [¹²⁵I]CYP binding on total membranes was competed with increasing isoproterenol concentrations. Data represent the mean ± S.E. of three independent experiments carried out in triplicate. C, cells stably expressing equivalent amount (700 fmol.mg^-1) of either β₂AR_WT (▪/□) or β₂AR_PKA- (•/○) were cholesterol-depleted (empty symbols) or not (filled symbols) with 2% CD for 1 h at 37 °C. An adenylyl cyclase assay was then performed in the presence of increasing concentrations of isoproterenol. D, cAMP production was measured in the presence of Gpp(NH)p (0.5 mm). The data shown in C and D represent the mean ± S.E. of three independent experiments carried out in triplicate.