GLYCOSYLATION, PALMITOYLATION, AND LOCALIZATION OF THE HUMAN D-2S RECEPTOR IN BACULOVIRUS-INFECTED INSECT CELLS

In order to evaluate the baculovirus expression system as a means for high-yield production of homogeneous D-2S receptor, we have expressed various D-2S receptor constructs in two Spodoptera frugiperda cell. li nes, a Trichoplusia ni and a Mammestra brassicae cell line. To improve expression yield, the environment of the polyhedrin gene translationa l initiation site was retained by fusing the first 12 codons of the po lyhedrin gene to the 5'-end of the D-2S receptor coding sequence. The pharmacological profile of the expressed D-2S receptor was similar to that reported for neuronal D-2 receptors. Sf9 and Tn cells were best s uited for overexpression, yielding about 2 x 10(6) and 4 x 10(6) recep tors/cell, respectively, corresponding to 6 pmol/mg of cell protein in Sf9 cells and 10 pmol/mg of cell protein in Tn cells. We have develop ed a D-2 receptor-specific anti-peptide antibody to study glycosylatio n, palmitoylation, and localization of the heterologously produced rec eptor. Immunoprecipitation of digitonin/cholate-solubilized receptor f rom control and tunicamycin-treated Sf9, Tn, and Mb cells revealed an apparent molecular mass of 47-48 kDa for the glycosylated receptor and of 39-40 kDa for the unglycosylated receptor. Although pulse-chase st udies showed that glycosylation occurred rapidly and efficiently, the glycosylated receptor only constituted a small fraction of the overall produced receptor protein, which was mainly located intracellularly. The glycosylation of the receptor was of the high-mannose-type in cont rast to the complex-type glycosylation found in native tissue. The gly cosylated D-2S receptor was palmitoylated. Glycosylation, however, was not a prerequisite for palmitoylation which was insensitive to tunica mycin, brefeldin A, and monensin. NH2-terminal addition of the signal sequence of prepromelittin to the D-2S receptor increased expression l evels 2-3-fold and significantly enhanced membrane insertion and proce ssing, resulting in increased targeting of the synthesized receptor to the plasma membrane.