Determination of the amino acid residue involved in [3H]beta-funaltrexamine covalent binding in the cloned rat mu-opioid receptor.
J Biol Chem
; 271(35): 21422-9, 1996 Aug 30.
Article
em En
| MEDLINE
| ID: mdl-8702924
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Receptores Opioides mu
/
Lisina
/
Naltrexona
Limite:
Animals
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
1996
Tipo de documento:
Article
País de afiliação:
Estados Unidos