CHARACTERIZATION OF THE HUMAN GONADOTROPIN-RELEASING-HORMONE RECEPTORHETEROLOGOUSLY PRODUCED USING THE BACULOVIRUS-INSECT CELL AND THE SEMLIKI-FOREST-VIRUS SYSTEMS

1. Two eukaryotic viral systems, the baculovirus/insect cell and the S emliki Forest virus systems, were tested for heterologous expression o f human gonadotropin-releasing hormone receptor (GnRHR) cDNA. 2. An un modified as well as a c-myc epitope-tagged human GnRH receptor was pro duced in two insect cell lines (Spodoptera frugiperda, Trichoplusia ni ) after infection with the respective recombinant baculoviruses. In bo th insect cell lines, the receptor was identified by immunoblot analys is as a triplet of bands between 35 and 40 kDa. After deglycosylation of the receptor the molecular mass decreased to 35 kDa. The GnRH recep tor was localized in membrane compartments within the infected insect cells. However, only in membranes of infected Trichoplusia ni insect c ells could approximate to 2000 receptors per cell be detected. 3. Prod uction of the GnRH receptor in BHK cells using the Semliki Forest viru s system resulted in approximate to 50,000 receptors per cell. A maxim al yield of 0.42 pmol/mg membrane protein was obtained 24 hr after ele ctroporation of BHK cells with in vitro synthesized RNA. Binding of th e antagonist [I-125]Cetrorelix was saturable with a K-D of 1.3 nM. The receptor produced in the BHK cells was further characterized by ligan d displacement studies. The rank order of agonist and antagonist affin ities was Cetrorelix > Triptorelin > Antide > GnRH.