GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs.
We have recently demonstrated that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. A functional receptor for human GnRH-II has not yet been identified. In this study, we have generated a polyclonal antiserum to the putative human GnRH-II receptor using a peptide (YSPTMLTEVPPC) corresponding to the third extracellular domain coupled to keyhole limpet haemocyanin via the Cys residue. A database search showed no identical peptide sequences in any other human gene. To avoid cross-reactions against two similar amino acid sequences the antiserum was pre-absorbed using these peptides. Immune histological sections of human placenta and human endometrial, ovarian and prostate cancers using rabbit anti-human GnRH-II receptor antiserum showed GnRH-II receptor-like staining. Western blot analysis of cell membrane preparations of human endometrial and ovarian cancer cell lines yielded a band at approximately 43 kDa whereas Western blot analysis of cell membrane preparations of ovaries obtained from the marmoset monkey (Callithrix jacchus) yielded a band at approximately 54 kDa. To identify the GnRH-II receptor-like antigen we used the photo-affinity labelling technique. Photochemical reaction of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II (10(-9) M) with cell membrane preparations of human endometrial and ovarian cancer cells yielded a band at approximately 43 kDa. In competition experiments, the GnRH-I agonist Triptorelin (10(-7) M) showed a weak decrease of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II binding to its binding site. The GnRH-I antagonist Cetrorelix (10(-7) M) showed a clearly stronger decrease, whereas GnRH-II agonist [d-Lys(6)]-GnRH-II (10(-7) M) was the most potent competitor. Western blot analysis of the same gel using rabbit anti-human GnRH-II receptor antiserum identified this band as GnRH-II receptor-like antigen.
Eicke
Nicola
N
Department of Gynecology and Obstetrics, Georg-August-University, Robert-Koch-Street 40, D-37075 Göttingen, Germany.
Günthert
Andreas R
AR
Viereck
Volker
V
Siebold
Doreen
D
Béhé
Martin
M
Becker
Tamara
T
Emons
Günter
G
Gründker
Carsten
C
D000818:Animals; D000941:Antigens / Q000378:metabolism*; D015153:Blotting, Western; D002144:Callithrix; D045744:Cell Line, Tumor; D005260:Female; D005833:Genital Neoplasms, Female / Q000378:metabolism*; D005834:Genital Neoplasms, Male / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008297:Male; D010053:Ovary / Q000378:metabolism; D010920:Placenta / Q000378:metabolism*; D011966:Receptors, LHRH / Q000378:metabolism*
D000941:Antigens; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; C047717:LHRH, His(5)-Trp(7)-Tyr(8)-