Purification and mass spectrometric analysis of the mu opioid receptor.
A mouse mu 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 mu opioid receptor with a KD 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 mu receptor with KI's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged mu receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The mu receptor was solubilized from HEK 293 cell membranes with n-dodecyl-beta-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 mu 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 mu 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 mu opioid receptor.
Christoffers
Keith H
KH
Department of Neuroscience, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103, USA.
Li
Hong
H
Keenan
Susan M
SM
Howells
Richard D
RD
D000700:Analgesics / Q000378:metabolism / Q000494:pharmacology; D001575:Benzomorphans / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D000939:Epitopes; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D009294:Narcotics / Q000378:metabolism / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D017450:Receptors, Opioid, mu / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D012995:Solubility
D000700:Analgesics; D001575:Benzomorphans; D000939:Epitopes; D008024:Ligands; D009294:Narcotics; D010455:Peptides; D017450:Receptors, Opioid, mu; C027248:bremazocine