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Evidence for receptor‐mediated inhibition of intrinsic activity of GTP‐binding protein, G i 1 and G i 2, but not G 0 in reconstitution experiments
Author(s) -
Ueda Hiroshi,
Uno Shouichi,
Harada Jun,
Kobayashi Ichiro,
Katada Toshiaki,
Ui Michio,
Satoh Masamichi
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)81534-u
Subject(s) - g protein , gtp' , pertussis toxin , intrinsic activity , receptor , chemistry , agonist , gtp binding protein regulators , biophysics , heterotrimeric g protein , chaps , biochemistry , biology , membrane , enzyme
The receptor‐mediated inhibition of intrinsic activities of GTP‐binding proteins (G‐proteins) was studied. Pertussis toxin (IAP)‐substrate G‐protein, G i 1, G i 2 or G 0 , was prelabeled with [α‐ 32 P]GDP and reconstituted with synaptic membranes of the guinea pig cerebellum in the presence of 0.02% of Chaps. Intrinsic activities of G‐proteins were evaluated by the release of [α‐ 32 P]GDP in exchange for added GppNHp or GDP in reconstituted preparations. U‐50,488H (1 nM‐10 μM), a specific ϰ‐subtype of opioid receptor agonist, inhibited the [α‐ 32 P]GDP release in exchange for added 1 μM GppNHp in G i 1‐reconstituted preparations in a concentration‐dependent manner. On the other hand, the ϰ‐opioid agonist at 10 μM increases the K m values of GppNHp, but not GDP in exchange for [α‐ 32 P]GDP release in preparations reconstituted with G i 1 or G i 2, but not with G 0 . These findings indicate that ϰ‐opioid receptor is coupled to inhibition of intrinsic activities of G i 1 and G i 2, but not G 0 , in guinea pig cerebellar membranes. In addition, it was revealed that the mode of action is mediated by a decrease in affinity of GTP (or its analog) for G‐proteins, but not by a change in affinity of GDP.