The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line is coupled to adenylyl cyclase through a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein.
The R1.1 mouse thymoma cell line expresses a high-affinity kappa opioid binding site. Opioid binding to this site is inhibited by guanine nucleotides, suggesting that the receptor is coupled to a guanine nucleotide-binding protein. Here, we present evidence that the kappa opioid binding site on R1.1 cell membranes is negatively coupled to adenylyl cyclase. The kappa-selective agonists (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide methane-sulfonate hydrate [(-)-U50,488], (5 alpha,7 alpha, 8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxas- piro(4,5)dec-8-yl)benzeneacetamide (U69,593) and several dynorphin peptides inhibited basal and forskolin-stimulated cyclic AMP production by up to 40% in R1.1 cell membranes. The order of potency for the inhibition of adenylyl cyclase activity by opioid agonists correlated with their Ki values for the inhibition of [3H]U69,593 binding. Opioid-mediated inhibition of adenylyl cyclase activity was stereoselective, as (-)-U50,488 was more potent than the (+) isomer, and the inhibition was blocked by the kappa-selective antagonist nor-binaltorphimine. The opioid-mediated inhibition of adenylyl cyclase activity was also completely blocked by incubating R1.1 cells with Bordetella pertussis toxin (PTX). Incubation of R1.1 cell membranes with PTX and [adenylate-32P]NAD+ resulted in the exclusive labeling of a 41-kDa protein, as determined by separating the membrane proteins under reducing conditions on a SDS polyacrylamide gel, followed by autoradiography. These results suggest that a PTX-sensitive inhibitory guanine nucleotide-binding protein mediates the link between the thymoma kappa opioid receptor and adenylyl cyclase.
Lawrence
D M
DM
Department of Pharmacology, University of Rochester, School of Medicine and Dentistry, New York.
Bidlack
J M
JM
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism / Q000502:physiology*; D000700:Analgesics / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D000242:Cyclic AMP / Q000096:biosynthesis; D004399:Dynorphins / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D037342:Pertussis Toxin; D011759:Pyrrolidines / Q000494:pharmacology; D017473:Receptors, Opioid, kappa / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D012680:Sensitivity and Specificity; D013945:Thymoma / Q000201:enzymology / Q000648:ultrastructure*; D013953:Thymus Neoplasms / Q000201:enzymology / Q000648:ultrastructure*; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000700:Analgesics; D011759:Pyrrolidines; D017473:Receptors, Opioid, kappa; D010566:Virulence Factors, Bordetella; C051844:norbinaltorphimine; D009271:Naltrexone; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D004399:Dynorphins; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases