Determination of the amino acid residue involved in [3H]beta-funaltrexamine covalent binding in the cloned rat mu-opioid receptor.
We previously demonstrated that [3H]beta-funaltrexamine ([3H]beta-FNA) labeled the rat mu opioid receptor expressed in Chinese hamster ovary cells with high specificity, and [3H]beta-FNA-labeled receptors migrated as one broad band with a mass of 80 kDa. In this study, we determined the region and then the amino acid residue of the mu receptor involved in the covalent binding of [3H]beta-FNA. [3H]beta-FNA-labeled receptors were solubilized and purified to approximately 10% purity by immunoaffinity chromatography with antibodies against a C-terminal domain peptide. The site of covalent bond formation was determined to be within Ala206-Met243 by CNBr cleavage of partially purified labeled mu receptors and determinations of sizes of labeled receptor fragments. The amino acid residue of beta-FNA covalent incorporation was then determined by site-directed mutagenesis studies within this region. Mutation of Lys233 to Ala, Arg, His, and Leu completely eliminated covalent binding of [3H]beta-FNA, although these mutants bound beta-FNA with high affinity. Mutations of other amino acid residues did not affect covalent binding of [3H]beta-FNA. These results indicate that [3H]beta-FNA binds covalently to Lys233. Since [3H]beta-FNA is a rigid molecule, the information will be very useful for molecular modeling of interaction between morphinans and the mu receptor.
Chen
C
C
Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Yin
J
J
Riel
J K
JK
DesJarlais
R L
RL
Raveglia
L F
LF
Zhu
J
J
Liu-Chen
L Y
LY
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D006868:Hydrolysis; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014316:Tritium
D017450:Receptors, Opioid, mu; D014316:Tritium; D009271:Naltrexone; C024524:beta-funaltrexamine; D008239:Lysine