EXPRESSION AND CHARACTERIZATION OF HUMAN DOPAMINE D-2 RECEPTOR IN BACULOVIRUS-INFECTED INSECT CELLS

Multiple types of dopamine D-2-like receptors (D-2, D-3, D-4) 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 D-2-like receptor. We engineered a recombinant baculovirus containing the human D-2 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 D-2 antagonist [H-3]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 D-2 agonists and antagonists. Sodium ion stimulated [H-3]YM-09151-2 binding to D-2 receptor in infected Sf9 cell membranes. This effect was fit by an allosteric model which predicted the apparent affinity of [H-3]YM-09151-2. The D-2 receptor expressed in Sf9 and TN-5 cells was photolabeled with N-(p-azido-m-[I-125]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.