Purification and partial amino acid sequence of a μ opioid receptor from rat brain

A rat brain opioid receptor protein was isolated by binding [∈-biotinyl-Lys³²]β-endorphin to membranes, solubilizing the receptor-ligand (R·L) complex with deoxycholate-lysophosphatidylcholine and purifying on immobilized streptavidin and wheat germ agglutinin. The purified glycoprotein had a molecular mass of 60-70 kDa. Recovery of this protein was blocked by the non-selective opioid antagonist naloxone and the highly μ-selective agonist [D-Ala²,N-methyl-Phe⁴,Glyol⁵]-enkephalin but not by the highly δ-selective agonist [D-Pen²,4′-Cl-Phe⁴,D-Pen⁵]enkephalin when these compounds were added as competitors at the binding step. The 60-70-kDa receptor protein co-purified through the streptavidin column with 40-kDa protein recognized by anti-G_iα antibodies. GTP and Na⁺ influenced dissociation of the solubilized R·¹²⁵I-L complex and elution of the receptor and G protein from streptavidin in fashions consistent with the pharmacology of μ-opioid receptors. A 23-amino acid residue sequence from the purified receptor differs at 4 positions from a similar sequence in the murine δ-opioid receptor and is encoded within a novel rat brain cDNA isolated by polymerase chain reaction with oligonucleotide primers related to the murine δ-opioid receptor gene.