Purification and mass spectrometric analysis of the delta opioid receptor.
A mouse delta 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 10.5 pmol/mg protein. [3H]Bremazocine exhibited high affinity binding to the epitope-tagged delta opioid receptor with a K(D) of 1.4 nM. The agonists DADL, morphine, and DAMGO competitively inhibited bremazocine binding to the tagged delta receptor with K(I)'s of 0.9, 370, and 620 nM, respectively. Chronic treatment of cells expressing the epitope-tagged delta receptor with DADL resulted in downregulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The delta 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 Sephacryl S300 gel filtration chromatography, wheat germ agglutinin (WGA)-agarose chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and SDS/PAGE permitted purification of the receptor. The purified delta opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 65 kDa. MALDI-TOF mass spectrometry was used to identify and characterize peptides derived from the delta opioid receptor following in-gel digestion with trypsin, and precursor-derived ms/ms confirmed the identity of peptides derived from enzymatic digestion of the delta opioid receptor.
Christoffers
Keith H
KH
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103, USA.
Li
Hong
H
Howells
Richard D
RD
D000700:Analgesics / Q000493:pharmacokinetics; D001575:Benzomorphans / Q000493:pharmacokinetics; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008970:Molecular Weight; D011869:Radioligand Assay / Q000379:methods; D017465:Receptors, Opioid, delta / Q000032:analysis* / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification; D012995:Solubility; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000493:pharmacokinetics; D014357:Trypsin / Q000494:pharmacology
D000700:Analgesics; D001575:Benzomorphans; D017465:Receptors, Opioid, delta; D014316:Tritium; D014357:Trypsin; C027248:bremazocine