Transient overexpression of kappa and mu opioid receptors using recombinant adenovirus vectors.
In order to study the trafficking and signal transduction mechanisms of the multiple opioid receptors, these receptors are expressed either transiently or stably in cell lines. Often, it is difficult to express receptors at a sufficiently high density to obtain reproducible results. To achieve a high density of receptors, replication-defective adenovirus (rAd5) vectors encoding the mu (MOR) and kappa (KOR) opioid receptors, both in their native form and as fusion proteins bearing the green fluorescent protein (GFP) at their C-terminus, were constructed. These vectors efficiently and reproducibly infected Chinese hamster ovary (CHO) cells that stably express the human coxsackie-adenovirus receptor (hCAR), with up to 90% of cells becoming infected at a low multiplicity of infection (MOI). Saturation receptor binding studies using mu- and kappa-selective agonists, [3H][D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) and [3H](5alpha7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzeneacetamide (U69,593), respectively, and a nonselective antagonist, [3H]diprenorphine, revealed that rAd5-transduced cells expressed MOR and KOR for at least 3 days, at levels which exceeded those present on widely-used CHO sublines that stably express MOR or KOR. Expression levels were highest for the vectors encoding native MOR or KOR, and slightly reduced for the GFP fusion proteins. These findings demonstrate the feasibility of using rAd5 vectors to express opioid receptors at high densities, which may facilitate opioid receptor studies.
Zhen
Zhu
Z
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Bradel-Tretheway
Birgit G
BG
Dewhurst
Stephen
S
Bidlack
Jean M
JM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D005786:Gene Expression Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D009292:Narcotic Antagonists / Q000494:pharmacology; D009294:Narcotics / Q000819:agonists; D011869:Radioligand Assay; D011940:Receptor Aggregation / Q000187:drug effects / Q000235:genetics*; D017473:Receptors, Opioid, kappa / Q000819:agonists / Q000096:biosynthesis / Q000235:genetics*; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins; D009292:Narcotic Antagonists; D009294:Narcotics; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins