Quasi-irreversible binding of agonist to beta-adrenoceptors and formation of non-dissociating receptor-G(s) complex in the absence of guanine nucleotides.
Here, we tested the hypothesis that receptor-G protein and agonist may form an irreversible complex in the absence of guanine nucleotides. We used the beta-adrenoceptor-G(s) system of guinea pig lung parenchymal membranes as a model. Two groups of membranes were used in the experiments: (1) washed with nucleotide-free buffer in the presence of isoproterenol (isoproterenol-treated), and (2) washed with buffer alone or with agonist+GDP (both were treated as control). Results were as follows: (1) the iodopindolol binding capacity of isoproterenol-treated membranes was reduced by about 30%. (2) No such reduction was observed in control membranes. (3) Addition of GDP to the isoproterenol-treated membranes completely restored the pindolol binding capacity. We interpreted this result as indicating irreversible agonist-receptor complex is formed when the receptor interacts with nucleotide-free G(salpha). (4) We observed a single peak of beta(2)-adrenoceptor activity in the control group by size-exclusion chromatography of the solubilized membranes. Inclusion of isoproterenol in the washing buffer led to an additional (heavier) peak of beta(2)-adrenoceptor activity. This peak disappeared when GDP was added to the detergent extract before high-pressure liquid chromatography (HPLC) analysis. Western blot analysis of these HPLC fractions showed that the agonist-induced heavier peak contained significantly more G(salpha) protein than did the other fractions. We interpreted this result as indicating that a practically irreversible complex of receptor and G protein is formed in the absence of GDP. We suggest that the tightly bound (nucleotide-free) receptor-G protein complex also contains the agonist, and that this complex can be reversed only by the addition of nucleotides. The implications of these results are also discussed.