Functional analysis of the human α2C-C4 adrenergic receptor in insect cells expressed by a luciferase-based baculovirus vector

Abstract

A click beetle luciferase-based baculovirus expression vector is described for functional analysis and high level expression of a human α2-adrenergic receptor (α2AR) in Sf9 insect cells. The resultant recombinant baculovirus construct, AcLucGR-α2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abundant expression of the α2C-C4 receptor protein in this lepidopteran insect cell line. A maximal expression of α2-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 α2-receptor antagonist, [ 3H]rauwolscine. The receptor agonists, noradrenaline and clonidine, displaced the [ 3H]rauwolscine binding with K i values 12.3 ± 1.54 μM and 1.23 ± 0.11 μM, 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 α2-receptor subtypes. In addition, co-expression of luciferase proved to be useful for screening and isolation of the recombinant baculovirus.