PURIFICATION AND PARTIAL AMINO-ACID-SEQUENCE OF A MU-OPIOID RECEPTOR FROM RAT-BRAIN

A rat brain opioid receptor protein was isolated by binding [epsilon-b iotinyl-Lys32]beta-endorphin to membranes, solubilizing the receptor-l igand (R.L) complex with deoxycholate-lysophosphatidylcholine and puri fying on immobilized streptavidin and wheat germ agglutinin. The purif ied glycoprotein had a molecular mass of 60-70 kDa. Recovery of this p rotein was blocked by the nonselective opioid antagonist naloxone and the highly mu-selective agonist [D-Ala2,N-methyl-Phe4,Glyol5]enkephali n but not by the highly delta-selective agonist [D-Pen2,4'-Cl-Phe4 D-P en5]enkephalin when these compounds were added as competitors at the b inding step. The 60-70-kDa receptor protein co-purified through the st reptavidin column with 40-kDa protein recognized by anti-G(ialpha) ant ibodies. GTP and Na+ influenced dissociation of the solubilized R.I-12 5-L complex and elution of the receptor and G protein from streptavidi n in fashions consistent with the pharmacology of mu-opioid receptors. A 23-amino acid residue sequence from the purified receptor differs a t 4 positions from a similar sequence in the murine delta-opioid recep tor and is encoded within a novel rat brain cDNA isolated by polymeras e chain reaction with oligonucleotide primers related to the murine de lta-opioid receptor gene.