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

Overview

Journal Cell Mol Neurobiol

Publisher Springer

Specialties Cell Biology
Molecular Biology
Neurology

Date 1998 Oct 20

PMID 9777251

Citations 2

Authors

K Marheineke

T Lenhard

W Haase

T Beckers

H Michel

H Reilander

Affiliations

Soon will be listed here.

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 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.

Citing Articles

Probing the GnRH receptor agonist binding site identifies methylated triptorelin as a new anti-proliferative agent.

Morgan K, Leighton S, Millar R J Mol Biochem. 2014; 1(2):86-98.

PMID: 24490142 PMC: 3906704.

Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System.

Blasey H, Brethon B, Hovius R, Vogel H H, Tairi A, Lundstrom K Cytotechnology. 2008; 32(3):199-208.

PMID: 19002981 PMC: 3449892. DOI: 10.1023/A:1008192709549.

References

Beckers T, Marheineke K, Reilander H, Hilgard P . Selection and characterization of mammalian cell lines with stable over-expression of human pituitary receptors for gonadoliberin. Eur J Biochem. 1995; 231(3):535-43. View

Illing N, Jacobs G, Becker I, Flanagan C, Davidson J, Eales A . Comparative sequence analysis and functional characterization of the cloned sheep gonadotropin-releasing hormone receptor reveal differences in primary structure and ligand specificity among mammalian receptors. Biochem Biophys Res Commun. 1993; 196(2):745-51. DOI: 10.1006/bbrc.1993.2312. View

Lenhard T, MAUL G, Haase W, Reilander . A new set of versatile vectors for the heterologous expression of foreign genes using the baculovirus system. Gene. 1996; 169(2):187-90. DOI: 10.1016/0378-1119(95)00831-4. View

Kakar S, Musgrove L, Devor D, Sellers J, Neill J . Cloning, sequencing, and expression of human gonadotropin releasing hormone (GnRH) receptor. Biochem Biophys Res Commun. 1992; 189(1):289-95. DOI: 10.1016/0006-291x(92)91556-6. View

Tsutsumi M, Zhou W, Millar R, Mellon P, Roberts J, Flanagan C . Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor. Mol Endocrinol. 1992; 6(7):1163-9. DOI: 10.1210/mend.6.7.1324422. View

Kakar S, Rahe C, Neill J . Molecular cloning, sequencing, and characterizing the bovine receptor for gonadotropin releasing hormone (GnRH). Domest Anim Endocrinol. 1993; 10(4):335-42. DOI: 10.1016/0739-7240(93)90037-c. View

Liljestrom P, Garoff H . A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y). 1991; 9(12):1356-61. DOI: 10.1038/nbt1291-1356. View

Reinhart J, Mertz L, Catt K . Molecular cloning and expression of cDNA encoding the murine gonadotropin-releasing hormone receptor. J Biol Chem. 1992; 267(30):21281-4. View

Towbin H, Staehelin T, Gordon J . Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979; 76(9):4350-4. PMC: 411572. DOI: 10.1073/pnas.76.9.4350. View

10.

Clayton R, Catt K . Gonadotropin-releasing hormone receptors: characterization, physiological regulation, and relationship to reproductive function. Endocr Rev. 1981; 2(2):186-209. DOI: 10.1210/edrv-2-2-186. View

11.

Hazum E, Garritsen A, Keinan D . Role of lipids in gonadotropin releasing hormone agonists and antagonist binding to rat pituitary. Biochem Biophys Res Commun. 1982; 105(1):8-13. DOI: 10.1016/s0006-291x(82)80003-x. View

12.

Grisshammer R, Tate C . Overexpression of integral membrane proteins for structural studies. Q Rev Biophys. 1995; 28(3):315-422. DOI: 10.1017/s0033583500003504. View

13.

Conn P, Crowley Jr W . Gonadotropin-releasing hormone and its analogs. Annu Rev Med. 1994; 45:391-405. DOI: 10.1146/annurev.med.45.1.391. View

14.

Sherwood N, Lovejoy D, Coe I . Origin of mammalian gonadotropin-releasing hormones. Endocr Rev. 1993; 14(2):241-54. DOI: 10.1210/edrv-14-2-241. View

15.

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

16.

Vasudevan S, Hulme E, Bach M, Haase W, Pavia J, Reilander H . Characterization of the rat m3 muscarinic acetylcholine receptor produced in insect cells infected with recombinant baculovirus. Eur J Biochem. 1995; 227(1-2):466-75. DOI: 10.1111/j.1432-1033.1995.tb20411.x. View

17.

Bigelow D, Thomas D . Rotational dynamics of lipid and the Ca-ATPase in sarcoplasmic reticulum. The molecular basis of activation by diethyl ether. J Biol Chem. 1987; 262(28):13449-56. View

18.

Marheineke K, Bach M, Haase W, Reilander H . High level production and localization of bovine rod cGMP-gated cation channel subunit 1 in baculovirus-infected insect cells and Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1995; 215(3):961-7. DOI: 10.1006/bbrc.1995.2557. View

19.

Kaiser U, Zhao D, Cardona G, Chin W . Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor. Biochem Biophys Res Commun. 1992; 189(3):1645-52. DOI: 10.1016/0006-291x(92)90266-n. View

20.

Tate C, Blakely R . The effect of N-linked glycosylation on activity of the Na(+)- and Cl(-)-dependent serotonin transporter expressed using recombinant baculovirus in insect cells. J Biol Chem. 1994; 269(42):26303-10. View