Characterization of the human gonadotropin-releasing hormone receptor heterologously produced using the baculovirus/insect cell and the Semliki Forest virus systems.
1. Two eukaryotic viral systems, the baculovirus/insect cell and the Semliki Forest virus systems, were tested for heterologous expression of human gonadotropin-releasing hormone receptor (GnRHR) cDNA. 2. An unmodified as well as a c-myc epitope-tagged human GnRH receptor was produced in two insect cell lines (Spodoptera frugiperda, Trichoplusia ni) after infection with the respective recombinant baculoviruses. In both insect cell lines, the receptor was identified by immunoblot analysis as a triplet of bands between 35 and 40 kDa. After deglycosylation of the receptor the molecular mass decreased to 35 kDa. The GnRH receptor was localized in membrane compartments within the infected insect cells. However, only in membranes of infected Trichoplusia ni insect cells could approximately 2000 receptors per cell be detected. 3. Production of the GnRH receptor in BHK cells using the Semliki Forest virus system resulted in approximately 50,000 receptors per cell. A maximal yield of 0.42 pmol/mg membrane protein was obtained 24 hr after electroporation of BHK cells with in vitro synthesized RNA. Binding of the antagonist [125I]Cetrorelix was saturable with a KD of 1.3 nM. The receptor produced in the BHK cells was further characterized by ligand displacement studies. The rank order of agonist and antagonist affinities was Cetrorelix > Triptorelin > Antide > GnRH.
Max-Planck-Institut für Biophysik, Abteilung Mol. Membranbiologie, Frankfurt/M, Germany.
K
K
Marheineke
T
T
Lenhard
W
W
Haase
T
T
Beckers
H
H
Michel
H
H
Reiländer
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008647:Mesocricetus; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010902:Pituitary Gland / Q000737:chemistry; D011966:Receptors, LHRH / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics*; D018411:Spodoptera / Q000166:cytology
D018076:DNA, Complementary; D011966:Receptors, LHRH; D011993:Recombinant Fusion Proteins; D007987:Gonadotropin-Releasing Hormone; C062876:cetrorelix