Characterization of irreversible binding of beta-funaltrexamine to the cloned rat mu opioid receptor.
Binding of beta-funaltrexamine (beta-FNA) to the cloned rat mu opioid receptor expressed in COS-1 cells or Chinese hamster ovary cells was examined. beta-FNA bound to the mu receptor with high affinity. Irreversible binding of [3H]beta-FNA was defined as the binding that could not be dissociated by trichloroacetic acid. Na+ greatly enhanced the specific irreversible binding of [3H]beta-FNA to the mu receptor, which was concentration- and time-dependent. Specific irreversible binding of [3H]beta-FNA was potently inhibited by CTAP (a mu ligand), but not by ICI174,864 (a delta ligand) or U50,488H (a kappa ligand). These results indicate that [3H]beta-FNA binds irreversibly to the cloned mu opioid receptor. SDS-polyacrylamide gel electrophoresis and fluorography showed that [3H]beta-FNA-labeled receptors migrated as one broad and diffuse band with a mass of 80 kDa in Chinese hamster ovary or COS cells and as one band with a mass of 67 kDa in the rat brain preparation. Upon removal of N-linked carbohydrates, labeled receptors became a sharper band with a mass of approximately 40 kDa. [3H]beta-FNA did not bind irreversibly to the cloned rat kappa receptor. [3H]beta-FNA binding to four chimeric mu/kappa receptors was examined. The region from the middle of the third intracellular loop to the C terminus of the mu receptor is necessary for irreversible binding of beta-FNA.
Chen
C
C
Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Xue
J C
JC
Zhu
J
J
Chen
Y W
YW
Kunapuli
S
S
Kim de Riel
J
J
Yu
L
L
Liu-Chen
L Y
LY
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004174:Diprenorphine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D006031:Glycosylation; D008969:Molecular Sequence Data; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011485:Protein Binding; D011759:Pyrrolidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017465:Receptors, Opioid, delta / Q000187:drug effects; D017473:Receptors, Opioid, kappa / Q000037:antagonists & inhibitors / Q000187:drug effects; D017450:Receptors, Opioid, mu / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D014316:Tritium
D009842:Oligopeptides; D011759:Pyrrolidines; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; C089773:phenylalanyl--cysteinyl-tyrosyl-tryptophyl-arginyl-penicillaminyl-threoninamide; D014316:Tritium; D004174:Diprenorphine; D004743:Enkephalin, Leucine; D009271:Naltrexone; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; C024524:beta-funaltrexamine; C040643:N,N-diallyl-tyrosyl-alpha-aminoisobutyric acid-phenylalanyl-leucine; D012964:Sodium