title: Human mu-opioid receptor overexpressed in baculovirus system and its pharmacological characterizations.
abstract: OBJECTIVE
  To overexpress human mu-opioid receptor (muOR) with characteristics similar to those of mammalian origin.
  
  
  METHODS
  Human muOR with a tag of 6 consecutive histidines at its carboxyl terminus was expressed in recombinant baculovirus infected Sf9 insect cells. Then the pharmacological characterizations of the product were studied by receptor binding assay and cAMP assay.
  
  
  RESULTS
  The maximal binding capacity for the [3H]diprenorphine and [3H]ohmefentanyl (Ohm) were 9.1 +/- 0.7 and 6.52 +/- 0.23 nmol/g protein, respectively. The [3H]diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by alpha-selective agonists [D-Ala2, N-methyl-Phe4, glyol5] enkephalin (DAGO), Ohm, and morphine, but neither by the delta-selective agonist [D-Pen2, D-Pen5] enkephalin (DPDPE) nor by the kappa-selective agonist ¿trans-(+/-)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]¿ benzacetamide (U50488). NaCl 100 mmol.L-1 and guanosine triphosphate (GTP) 50 mumol.L-1 could reduce mu agonists Ohm and etorphine affinity binding to the expressed muOR. DAGO and Ohm effectively inhibited forskolin-stimulated cAMP accumulation. This agonist-dependent effect was blocked by opioid antagonist naloxone.
  
  
  CONCLUSIONS
  The overexpression of human muOR with a tag of six consecutive histidines at its carboxyl terminus in Sf9 insect cells retained the characteristics of wild-type human muOR.
authors.affiliation: Shanghai Institute of Materia Medica, China.
authors.forename: Q
authors.initials: Q
authors.lastname: Wei
authors.forename: D H
authors.initials: DH
authors.lastname: Zhou
authors.forename: Q X
authors.initials: QX
authors.lastname: Shen
authors.forename: C H
authors.initials: CH
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: C L
authors.initials: CL
authors.lastname: Li
authors.forename: G
authors.initials: G
authors.lastname: Pei
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Chi
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004174:Diprenorphine / Q000378:metabolism; D005048:Etorphine / Q000494:pharmacology; D005283:Fentanyl / Q000031:analogs & derivatives / Q000494:pharmacology; D015870:Gene Expression; D007313:Insecta / Q000166:cytology* / Q000821:virology; D011869:Radioligand Assay; D017450:Receptors, Opioid, mu / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
chemical_list: D017450:Receptors, Opioid, mu; D004174:Diprenorphine; D005048:Etorphine; C037016:F 7302; D000242:Cyclic AMP; D005283:Fentanyl