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Volume 270, Number 30, Issue of July 28, pp. 17866-17870, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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Characterization of Irreversible Binding of -Funaltrexamine to the Cloned Rat Opioid Receptor

(Received for publication, May 4, 1995)

From the  (1)Departments of Pharmacology, Microbiology and Immunology, and (2)Physiology, the (3)Fels Institute for Molecular Biology and Cancer Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, and the (4)Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana 46202

Binding of -funaltrexamine (-FNA) to the cloned rat µ opioid receptor expressed in COS-1 cells or Chinese hamster ovary cells was examined. -FNA bound to the µ receptor with high affinity. Irreversible binding of [H]-FNA was defined as the binding that could not be dissociated by trichloroacetic acid. Na greatly enhanced the specific irreversible binding of [H]-FNA to the µ receptor, which was concentration- and time-dependent. Specific irreversible binding of [H]-FNA was potently inhibited by CTAP (a µ ligand), but not by ICI174,864 (a ligand) or U50,488H (a ligand). These results indicate that [H]-FNA binds irreversibly to the cloned µ opioid receptor. SDS-polyacrylamide gel electrophoresis and fluorography showed that [H]-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 40 kDa. [H]-FNA did not bind irreversibly to the cloned rat receptor. [H]-FNA binding to four chimeric µ/ receptors was examined. The region from the middle of the third intracellular loop to the C terminus of the µ receptor is necessary for irreversible binding of -FNA.

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