Purification and mass spectrometric analysis of the δ opioid receptor

• Published in: Brain research. Molecular brain research. 2005-05, Vol.136 (1), p.54-64
• Main Authors: Christoffers, Keith H., Li, Hong, Howells, Richard D.
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Analgesics - pharmacokinetics; Benzomorphans - pharmacokinetics; Biological and medical sciences; Blotting, Western - methods; Cell Line; Chromatography, Affinity; Chromatography, Gel - methods; Enkephalin; Fundamental and applied biological sciences. Psychology; Humans; Mass spectrometric analysis; Mass Spectrometry; Models, Molecular; Molecular Weight; Morphine; Opioid; Purification; Radioligand Assay - methods; Receptors, Opioid, delta - analysis; Receptors, Opioid, delta - chemistry; Receptors, Opioid, delta - drug effects; Receptors, Opioid, delta - isolation & purification; Solubility; Transfection - methods; Tritium - pharmacokinetics; Trypsin - pharmacology; Vertebrates: nervous system and sense organs
• ISSN: 0169-328X; 1872-6941
• DOI: 10.1016/j.molbrainres.2005.01.016

A mouse δ 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 δ opioid receptor with a K D of 1.4 nM. The agonists DADL, morphine, and DAMGO competitively inhibited bremazocine binding to the tagged δ receptor with K I's of 0.9, 370, and 620 nM, respectively. Chronic treatment of cells expressing the epitope-tagged δ receptor with DADL resulted in downregulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The δ 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 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 δ 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 δ opioid receptor following in-gel digestion with trypsin, and precursor-derived ms/ms confirmed the identity of peptides derived from enzymatic digestion of the δ opioid receptor.