Functional analysis of the human alpha 2C-C4 adrenergic receptor in insect cells expressed by a luciferase-based baculovirus vector.
A click beetle luciferase-based baculovirus expression vector is described for functional analysis and high level expression of a human alpha 2-adrenergic receptor (alpha 2AR) in Sf9 insect cells. The resultant recombinant baculovirus construct, AcLucGR-alpha 2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abundant expression of the alpha 2C-C4 receptor protein in this lepidopteran insect cell line. A maximal expression of alpha 2-receptors at a level of 1.370 pmol/mg protein was obtained at 48 h after infection as determined by ligand-binding experiments using the alpha 2-receptor antagonist, [3H]rauwolscine. The receptor agonists, noradrenaline and clonidine, displaced the [3H]rauwolscine binding with Ki values 12.3 +/- 1.54 microM and 1.23 +/- 0.11 microM, respectively. The recombinant receptors were functionally intact since the agonists inhibited forskolin-stimulated cAMP production. Here, however, the maximal inhibition was obtained at 36 h after the infection. The results presented here, suggest that the baculovirus expression vector system (BEVS) provides a simple method for abundant expression of functional alpha 2-receptor subtypes. In addition, co-expression of luciferase proved to be useful for screening and isolation of the recombinant baculovirus.
Oker-Blom
C
C
Abo Akademi University, Department of Biochemistry and Pharmacy, Turku, Finland.
Jansson
C
C
Karp
M
M
Lindqvist
C
C
Savola
J M
JM
Vlak
J
J
Akerman
K
K
D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D003000:Clonidine / Q000494:pharmacology; D000242:Cyclic AMP / Q000032:analysis; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D009638:Norepinephrine / Q000494:pharmacology; D010957:Plasmids; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000235:genetics*; D011995:Recombination, Genetic
D000317:Adrenergic alpha-Antagonists; D011942:Receptors, Adrenergic, alpha; D000242:Cyclic AMP; D008156:Luciferases; D003000:Clonidine; D009638:Norepinephrine