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 descr ibed for functional analysis and high level expression of a human alph a2-adrenergic receptor (alpha2AR) in Sf9 insect cells. The resultant r ecombinant baculovirus construct, AcLucGR-alpha2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abu ndant expression of the alpha2C-C4 receptor protein in this lepidopter an insect cell line. A maximal expression of alpha2-receptors at a lev el of 1.370 pmol/mg protein was obtained at 48 h after infection as de termined by ligand-binding experiments using the alpha2-receptor antag onist, [H-3]rauwolscine. The receptor agonists, noradrenaline and clon idine, displaced the [H-3]rauwolscine binding with K(i) values 12.3 +/ - 1.54 muM and 1.23 +/- 0.11 muM, respectively. The recombinant recept ors were functionally intact since the agonists inhibited forskolin-st imulated cAMP production. Here, however, the maximal inhibition was ob tained at 36 h after the infection. The results presented here, sugges t that the baculovirus expression vector system (BEVS) provides a simp le method for abundant expression of functional alpha2-receptor subtyp es. In addition, co-expression of luciferase proved to be useful for s creening and isolation of the recombinant baculovirus.