Expression and Characterization of Human Dopamine D2Receptor in Baculovirus-Infected Insect Cells

Abstract

Multiple types of dopamine D₂–like receptors (D₂, D₃, D₄) have been identified. Differences in pharmacology among these receptors may have profound clinical ramifications for the treatment of psychosis. Analysis of the structure and function of their binding sites requires a source of large amounts of receptor, uncontaminated by the other types of D₂–like receptor. We engineered a recombinant baculovirus containing the human D₂ receptor cDNA (DRD2) to express this receptor in insect cells. Spodoptera frugiperda cells (Sf9 and Sf21) and Trichoplusia ni cells (TN-5) were infected with the recombinant baculovirus. Binding of the D₂ antagonist [³H]YM-09151–2 to membranes fractions of these cells peaked at a specific activity of 5–8 pmol/mg protein, approximately 40 times that of membranes from bovine striatum. The receptor expressed in Sf9 cells was similar to that of striatum in its affinities for D₂ agonists and antagonists. Sodium ion stimulated [³H]YM-09151–2 binding to D₂ receptor in infected Sf9 cell membranes. This effect was fit by an allosteric model which predicted the apparent affinity of [³H]YM-09151–2. The D₂ receptor expressed in Sf9 and TN-5 cells was photolabeled with N-(p-azido-m-[¹²⁵I]iodophenylethyl)spiperone. The specifically labeled component(s) ran as a broad band of apparent molecular weight between 54,000 and 60,000. Deglycosylation of the labeled component(s) reduced its molecular weight to 46,000.