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  Characterization of the Human Gonadotropin-Releasing Hormone Receptor Heterologously Produced Using the Baculovirus/Insect Cell and the Semliki Forest Virus Systems

Marheineke, K., Lenhard, T., Haase, W., Beckers, T., Michel, H., & Reiländer, H. (1998). Characterization of the Human Gonadotropin-Releasing Hormone Receptor Heterologously Produced Using the Baculovirus/Insect Cell and the Semliki Forest Virus Systems. Cellular and Molecular Neurobiology, 18(5), 509-524. doi:10.1023/A:1026379326184.

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 Creators:
Marheineke, Kathrin1, Author           
Lenhard, Thomas1, Author           
Haase, Winfried1, Author           
Beckers, Thomas2, Author
Michel, Hartmut1, Author           
Reiländer, Helmut1, Author           
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              
2Abt. Tumorforschung, ASTA Medica AG, Weissmüllerstr. 45, D-60314, Frankfurt/M, Germany, ou_persistent22              

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Free keywords: human gonadotropin-releasing hormone receptor; baculovirus/insect cell; Semliki Forest virus.
 Abstract: 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 ≈2000 receptors per cell be detected. 3. Production of the GnRH receptor in BHK cells using the Semliki Forest virus system resulted in ≈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.

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Language(s): eng - English
 Dates: 1997-06-011997-11-191998-10
 Publication Status: Published in print
 Pages: 16
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1023/A:1026379326184
 Degree: -

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Title: Cellular and Molecular Neurobiology
  Other : Cell. Mol. Neurobiol.
Source Genre: Journal
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Affiliations:
Publ. Info: New York, NY : Kluwer Academic/Plenum Publishers
Pages: - Volume / Issue: 18 (5) Sequence Number: - Start / End Page: 509 - 524 Identifier: ISSN: 0272-4340
CoNE: https://pure.mpg.de/cone/journals/resource/954925503218