Rapid Binding of Guanosine 5'-o-(3-thiotriphosphate) To An Apparent Complex of ß-adrenergic Receptor and The Gtp-binding Regulatory Protein G_s

When reconstituted phospholipid vesicles that contain purified ß-adrenergic receptors and the GTP-binding regulatory protein G_swere preincubated with agonist before the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP7S), the typical receptor-stimulated GTP7S binding reaction was preceded by an even more rapid burst of GTP7S binding. This burst was studied in detail at 0 °C. The rate of the burst was second order in nucleotide and G_s[k_assoc~ 2 X 10⁷(M-min)^-1], consistent with diffusion-controlled binding. The magnitude of the burst was always less than the number of receptors present and was roughly linear with receptor number when similarly prepared vesicles were compared. There was no obvious quantitative correlation between the burst and the amount of G_s. The species that gave rise to the burst formed with t_1/2~15 min at 0 °C in the presence of agonist and decayed by ~3 min upon addition of antagonist or detergent. Formation and decay of this species was much faster at 30 °C. The data suggest that a complex of agonist, receptor, and G_sthat is primed for the rapid binding of guanine nucleotide can form and be analyzed in reconstituted vesicles.