FIGURE 5.

In vitro activation of Gα_s by the β-adrenergic receptor. A, Coomassie Blue staining shows the purified turkey β-adrenergic receptor. B, Coomassie Blue staining shows some examples of purified G proteins (after GST cleavage). C, activation of Gα_s by the β-adrenergic receptor in the presence of alprenolol (■) or isoproterenol (●). D, activation of Gα_s + Gβ₁γ₂ by the β-adrenergic receptor in the presence of alprenolol (■) or isoproterenol (●). E, activation of Gα_sE195 by the β-adrenergic receptor in the presence of alprenolol (■) or isoproterenol (●). F, activation of Gα_sE195 + Gβ₁γ₂ by the β-adrenergic receptor in the presence of alprenolol (■) or isoproterenol (●). G, in vitro binding of Gα_s and representative mutants to β-AR. Different concentrations (150, 300, and 500 nm) of Gα_s and mutant proteins were used. H, left two panels, measurement of the initial rate of GTPγS loading onto wild-type Gα_s (top panel) or Gα_sK219A mutant (bottom panel) in the presence of different concentrations of Gβ₁γ₂. Right two panels, measurement of the initial rate of GTPγS loading onto wild-type Gα_s in the presence of Gβ₁γ₂ (top panel) or the Gα_sK219A mutant (bottom panel) in the absence of Gβ₁γ₂. One representative experiment from three independent experiments is shown for A-G. In H, data are shown as mean ± S.E.