Evidence for receptor-mediated inhibition of intrinsic activity of GTP-binding protein, Gi1 and Gi2, but not G0 in reconstitution experiments.
The receptor-mediated inhibition of intrinsic activities of GTP-binding proteins (G-proteins) was studied. Pertussis toxin (IAP)-substrate G-protein, Gi1, Gi2 or G0, was prelabeled with [alpha-32P]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 [alpha-32P]GDP in exchange for added GppNHp or GDP in reconstituted preparations. U-50,488H (1 nM-10 microM), a specific kappa-subtype of opioid receptor agonist, inhibited the [alpha-32P]GDP release in exchange for added 1 microM GppNHp in Gi1-reconstituted preparations in a concentration-dependent manner. On the other hand, the kappa-opioid agonist at 10 microM increases the Km values of GppNHp, but not GDP in exchange for [alpha-32P]GDP release in preparations reconstituted with Gi1 or Gi2, but not with G0. These findings indicate that kappa-opioid receptor is coupled to inhibition of intrinsic activities of Gi1 and Gi2, but not G0, 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.
Ueda
H
H
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Uno
S
S
Harada
J
J
Kobayashi
I
I
Katada
T
T
Ui
M
M
Satoh
M
M
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D037342:Pertussis Toxin; D011759:Pyrrolidines / Q000494:pharmacology; D011957:Receptors, Opioid / Q000187:drug effects / Q000502:physiology*; D017473:Receptors, Opioid, kappa; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D011759:Pyrrolidines; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins