DIFFERENTIAL COUPLING OF RAT D2 DOPAMINE-RECEPTOR ISOFORMS EXPRESSED IN SPODOPTERA-FRUGIPERDA INSECT CELLS

By using a baculovirus expression system, the two isoforms of the rat D2 dopamine receptor were expressed at densities ranging up to 15 pmol /mg of protein. D2(L) and D2(S) dopamine receptors expressed in aline of Spodoptera frugiperda (Sf9) insect cells Sf9cells, displayed high a ffinity for the antagonists spiroperidol and (+)-butaclamol and the ag onist N-propylnorapomorphine. Antisera raised against the D2 receptor immunoprecipitated binding sites for a radiolabeled D2 antagonist from solubilized extracts of infected Sf9cells. In immunoblots of Sf9cells infected with recombinant D2 baculovirus, these antisera recognized a major species of protein of approximately 46 kDa. Photoaffinity-label ing of infected Sf9cells using N-(p-azido-m-[ (125)l]iodophenethyl)spi perone also identified a protein of this size, suggesting that D2 rece ptors expressed in Sf9cells are largely unglycosylated. In cells expre ssing receptors at a density greater than 1 pmol/mg, GTP-sensitive, hi gh-affinity binding of agonists was not detected in studies of the inh ibition of the binding of a radiolabeled D2 antagonist. When expressio n levels were under 1 pmol/mg, the binding of agonists was sensitive t o the addition of guanine nucleotides, indicating that D2 receptors we re coupled to endogenous G proteins. Endogenous G proteins enable both isoforms of D2 receptors to couple to the inhibition of adenylyl cycl ase activity. The high-affinity state of the D2 receptor was directly measured using a radiolabeled agonist. Although the density of recepto rs increased with longer times after infection, the density of high-af finity sites reached a maximum of approximately 40 fmol/mg 30 to 36 hr after infection. Coexpression of D2 receptors and G protein subunits in Sf9cells dramatically increased the density of high-affinity sites, whereas the total density of receptors was unchanged, confirming that D2 receptors in Sf9 cells can exist in the high-affinity-couple state , but that appropriate G proteins are expressed at relatively low leve ls. The density of D2(S) receptors converted to a coupled, agonist-pre ferring state when coexpressed with G proteins subunits (alpha(l1), be ta(1), and gamma(2)) was 5 times greater than that of D2(L) receptors expressed under the same conditions, consistent with the hypothesis th at D2 dopamine receptor isoforms differentially couple to alpha(l1).