Expression and characterization of human dopamine D2 receptor in baculovirus-infected insect cells.
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.
Javitch
J A
JA
Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York.
Kaback
J
J
Li
X
X
Karlin
A
A
D000818:Animals; D001386:Azides / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001549:Benzamides / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D017448:Receptors, Dopamine D2 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012964:Sodium / Q000494:pharmacology; D013134:Spiperone / Q000031:analogs & derivatives / Q000378:metabolism
D001386:Azides; D001549:Benzamides; D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins; D013134:Spiperone; C044567:N-(4-azido-3-iodophenethyl)spiperone; D012964:Sodium; C030265:nemonapride