Purification and mass spectrometric analysis of the μ opioid receptor

Abstract

A mouse μ opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl-terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B max of 10 pmol/mg protein. 3[H]Bremazocine exhibited high affinity binding to the epitope-tagged μ opioid receptor with a K D of 1.0 nM. The agonists [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]enkephalin (DAMGO), morphine and [ d-Ala 2, d-Leu 5]enkephalin (DADL) competitively inhibited bremazocine binding to the tagged μ receptor with K I's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged μ receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The μ receptor was solubilized from HEK 293 cell membranes with n-dodecyl-β- d-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of wheat germ agglutinin (WGA)–agarose chromatography, Sephacryl S300 gel filtration chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) permitted purification of the receptor. The purified μ opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 80 kDa. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides derived from the μ opioid receptor following in-gel digestion with trypsin or chymotrypsin, and precursor-derived tandem mass spectrometry (ms/ms) confirmed the identity of several peptides derived from enzymatic digestion of the μ opioid receptor.