Akademik Veri Yönetim Sistemi
EUROPEAN JOURNAL OF PHARMACOLOGY, cilt.425, sa.3, ss.181-188, 2001 (SCI-Expanded)
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(s alpha). (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(s alpha) 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. (C) 2001 Elsevier Science B.V. All rights reserved.