| A high-titer antisera directed against amino acids 23--36 in the N-terminal of the GnRH receptor was generated and Western Blot analysis resulted in immunological detection in samples known to harbor GnRH receptors. Analysis of pituitary tissue from several species revealed the presence of a single 28 kD immunopositive band. Analysis of extra-pituitary tissues from the ovine, bovine, murine and lapine also resulted in consistent immunological detection of a 28 kD band in the adrenal, brain, endometrium, kidney, liver, myometrium and ovary. Heart and lung were devoid of product.; The product was smaller than either the predicted size of the peptide backbone (37,800) or GnRH receptors identified by photo-affinity labeling (32,000--62,000). In vitro transcription and translation of the cDNA resulted in a radiolabeled band with a Mr of 28,000. Western analysis of this product resulted in an immunopositive band of the same size. Therefore, the smaller is most likely due to aberrant migration of the peptide backbone in the polyacrylamide gel.; Southern blot analysis of reverse transcribed RNA was conducted and brain contained 3.7%, endometrium 1.9%, and myometrium 1.2%, respectively, of the pituitary. Thus, levels of GnRH receptor in these extra-pituitary tissues are extremely low when compared to the levels found in the pituitary.; Receptor assays were performed on partially purified membranes of ovine, rat and murine adrenal, brain, endometrium, kidney, liver and pituitary and rat testis. Ligand binding was demonstrated in the pituitary of all species and in the rat testis.; Larger sized receptors, like those reported from PAL studies, were not detected. This may be due to the location of two potential glycosylation signals close to the epitope our antibody recognizes (a.a. 23--36). If glycosylation occurs at either or both of these amino acids, the carbohydrate moieties may cause steric hindrance and inhibit the antibody from binding. Our results do not preclude the possibility that other forms of the receptor exist in any of the tissues surveyed. They may be present but not detected by our antibody in sufficient quantity to produce a positive signal. |
