Receptor-mediated activation of G-proteins by κ opioid agonists in frog (Rana esculenta) brain membranes

This study delineates the heterotrimeric guanine nucleotide binding regulatory protein (G-protein) types in frog (Rana esculenta) brain membranes and their activation by kappa opioid agonists. Ethylketocyclazocine (EKC), trans-(+/-)-3,4-dichloro-N-methy-N-(2-[1-pyrrolidinyl]cyclohexyl)benzene-acetamide (U-50,488) and bremazocine displayed dose-dependent, norbinaltorphimine-reversible stimulation of guanosine-5'-O-(3-[S-35]thio)triphosphate ([S-35]GTP gamma S) binding in crude membrane preparations. G-proteins were identified by Western-blotting using previously characterized specific antisera that were generated against mammalian G-protein alpha-subunits and beta-subunits. A photoreactive guanosine 5'-triphosphate (GTP) analog, [alpha-P-32]GTP azidoanilide ([alpha-P-32]AA-GTP) irreversibly labeled four proteins in the molecular weight range of 39-43 kDa. Ethylketocyclazocine and U-50,488 stimulated photolabelling of these proteins among which the 39 kDa band comigrated with the protein specifically labelled with the alpha(i2) antibody and the 40 kDa band was identified as alpha(o1). The other two bands were also stained with the alpha(common) antibody, but were not further identified. These results suggest that the endogenously expressed kappa opioid receptors that are present in frog brain interact with multiple G-proteins in situ. Furthermore, the structure of most G-proteins seems to be well preserved during phylogenesis. (C) 1998 Elsevier Science Inc.