Establishment of immunological probes to study human gonadotropin-releasing hormone receptors

doi:10.1016/0303-7207(95)03641-J

Molecular and Cellular Endocrinology

Volume 114, Issues 1–2, 30 October 1995, Pages 51-56

https://doi.org/10.1016/0303-7207(95)03641-J Get rights and content

Abstract

Monoclonal antibodies were raised to a synthetic peptide corresponding to amino acids 1–29 of the human gonadotropin-releasing hormone (GnRH) receptor. One of the two antibodies was found to recognise GnRH receptors on human pituitary gonadotrophs as determined by immunohistochemistry and supported by Western blotting. The antibody also bound to T47D human breast carcinoma cell line as determined by flow cytometric analysis.

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Cited by (31)

Gonadotropin-releasing hormone (GnRH) receptors of cattle aggregate on the surface of gonadotrophs and are increased by elevated GnRH concentrations
2014, Animal Reproduction Science
Citation Excerpt :
First, this peptide has only 60.7% homology the corresponding region of guinea pig GnRHR (NP_001166428.1). Second, the corresponding 29 amino acids from the human GnRHR were successfully used to develop a mouse monoclonal antibody to human GnRHR (Karande et al., 1995). The anti-human GnRHR mouse antibody was not used because the corresponding region of human GnRHR (MANSASPEQNQNHCSAINNSIPLMQGNLP; NP_000397.1) has only 79% homology with the GnRHR region and it was necessary to use another mouse monoclonal antibody to detect LH as subsequently described.
The presence of gonadotropin-releasing hormone (GnRH) receptors (GnRHRs) on gonadotrophs in the anterior pituitary (AP) is an important factor for reproduction control. However, little is known regarding GnRHR gene expression in gonadotrophs of cattle owing to the lack of an appropriate anti-GnRHR antibody. Therefore, an anti-GnRHR antibody for immunohistochemistry, flow cytometry, and immunocytochemistry assays was developed to characterize GnRHR gene expression in gonadotrophs. The anti-GnRHR antibody could suppress GnRH-induced LH secretion from cultured AP cells of cattle. The GnRHR, luteinizing hormone (LH), and follicle stimulating hormone (FSH) in the AP tissue was analyzed by fluorescence immunohistochemistry. The GnRHRs were aggregated on a limited area of the cell surface of gonadotrophs, possibly localized to lipid rafts. The LH secretion was stimulated with increasing amounts of GnRH; however, excessive concentrations (>1 nM) resulted in a decrease in LH secretion. A novel method to purify gonadotrophs was developed using the anti-GnRHR antibody and fluorescence-activated cell sorting. Flow cytometric analysis using the anti-GnRHR antibody for cultured bovine AP cells, however, failed to support the hypothesis that GnRH induces GnRHR internalization and decreases GnRHR on the surface of GnRHR-positive AP cells. In contrast, immunocytochemistry using primary antibodies for cultured bovine AP cells showed that 10 nM (P < 0.05) and 100 nM (P < 0.01) GnRH, but not 0.01–1 nM GnRH, increased GnRHR in the cytoplasm of LH-positive cells. In conclusion, these data suggested that GnRHRs were aggregated on the surface of gonadotrophs and GnRHR inside gonadotrophs increased with elevated concentrations of GnRH.
Gonadotropin releasing hormone (GnRH) receptor
2007, xPharm: The Comprehensive Pharmacology Reference
Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) that mediates central control of reproduction by binding to GnRH receptors on pituitary gonadotropes and stimulating secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This peptide (also known as luteinizing hormone-releasing hormone, gonadoliberin, luliberin, gonadorelin) is also designated GnRH I, to distinguish it from GnRH II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Plo-Gly-NH₂, also known as chicken GnRH-II), GnRH III (pGlu-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH₂ also known as lamprey GnRH III), and related peptides. Receptors selective for all three of these ligands exist in amphibians, but only GnRH I and GnRH II and their cognate receptors (GnRH I receptors and GnRH II receptors) have been found in mammals …
Expression and function of gonadotropin-releasing hormone (GnRH) receptor in human olfactory GnRH-secreting neurons. An autocrine GnRH loop underlies neuronal migration
2004, Journal of Biological Chemistry
Citation Excerpt :
GnRH antagonist cetrorelix (Ac-d-Nal (2)1, d-Phe (4Cl)2, d-Pal (3)3, d-Cit6, d-Ala10) was purchased from ASTA Medica GmbH (Frankfurt, Germany). Antisera—The following monoclonal antibodies (MA) were used for immunocytochemical or immunoblot analysis: mouse MA to human pituitary GnRH-R (F1G4), kindly provided by Dr. A. A. Karande, Department of Biochemistry, Indian Institute of Science, Bangalore, India (27); mouse MA to human vimentin (Vm; clone V9); mouse MA to human GAP43 (clone NCL-GAP43); and MA to human neurofilament 200 (NF200) (clone N52) were from Sigma; mouse MA to human actin (C-2) sc-8432 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and mouse MA to human nestin (clone 10C2) was from Chemicon International Inc. (Temecula, CA). Other reagents were obtained at the highest grade available from commercial sources.
Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmann's syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time- and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dose-dependent increase in migration (up to 3–4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons.
The biology of gonadotropin hormone-releasing hormone: Role in the control of tumor growth and progression in humans
2003, Frontiers in Neuroendocrinology
It is now well known that different forms of GnRH coexist in the same vertebrate species. In humans, two forms of GnRH have been identified so far. The first form corresponds to the hypophysiotropic decapeptide, and is now called GnRH-I. The second form has been initially identified in the chicken brain, and it is referred to as GnRH-II. GnRH-I binds to and activates specific receptors, belonging to the 7 transmembrane (7TM) domain superfamily, present on pituitary gonadotropes. These receptors (type I GnRH receptors) are coupled to the Gq/11/PLC intracellular signalling pathway. A receptor specific for GnRH-II (type II GnRH receptor) has been identified in non-mammalian vertebrates as well as in primates, but not yet in humans. In the last 10–15 years experimental evidence has been accumulated indicating that GnRH-I is expressed, together with its receptors, in tumors of the reproductive tract (prostate, breast, ovary, and endometrium). In these hormone-related tumors, activation of type I GnRH receptors consistently decreases cell proliferation, mainly by interfering with the mitogenic activity of stimulatory growth factors (e.g., EGF, IGF). Recent data seem to suggest that GnRH-I might also reduce the migratory and invasive capacity of cancer cells, possibly by affecting the expression and/or activity of cell adhesion molecules and of enzymes involved in the remodelling of the extracellular matrix. These observations point to GnRH-I as an autocrine negative regulatory factor on tumor growth progression and metastatization. Extensive research has been performed to clarify the molecular mechanisms underlying the peculiar antitumor activity of GnRH-I. Type I GnRH receptors in hormone-related tumors correspond to those present at the pituitary level in terms of cDNA nucleotide sequence and protein molecular weight, but do not share the same pharmacological profile in terms of binding affinity for the different synthetic GnRH-I analogs. Moreover, the classical intracellular signalling pathway mediating the stimulatory activity of the decapeptide on gonadotropin synthesis and secretion is not involved in its inhibitory activity on hormone-related tumor growth. In these tumors, type I GnRH receptors are coupled to the Gi-cAMP, rather than the Gq/11-PLC, signal transduction pathway. Recently, we have reported that GnRH-I and type I GnRH receptors are expressed also in tumors not related to the reproductive system, such as melanoma. Also in melanoma cells, GnRH-I behaves as a negative regulator of tumor growth and progression. Interestingly, the biochemical and pharmacological profiles of type I GnRH receptors in melanoma seem to correspond to those of the receptors at pituitary level. The data so far reported on the expression and on the possible functions of GnRH-II in humans are still scanty. The decapeptide has been identified, together with a ‘putative’ type II GnRH receptor, both in the central nervous system and in peripheral structures, such as tissues of the reproductive tract (both normal and tumoral). The specific biological functions of GnRH-II in humans are presently under investigation.
Two distinct humanized monoclonal antibodies for immunotherapy of ovarian cancer
2014, Journal of Cancer Science and Therapy
Recombinant GnRH-p53 protein sensitizes breast cancer cells to 5-fluorouracil-induced apoptosis in vitro and in vivo
2013, Apoptosis

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Research articleEstablishment of immunological probes to study human gonadotropin-releasing hormone receptors

Abstract

Cancer Lett.

Placenta

Immunol. Methods

Mol. Cell. Endocrinology

Mol. Cell. Endocrinol.

Biochem. Biophys. Res. Commun.

Mol. Cell. Endocrinol.

J. Immunol. Methods

Mol. Cell. Endocrinol.

Methods Enzymol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Immunol. Methods

J. Immunol. Methods

J. Clin. Endocrinol. Metab.

J. Endocrinol.

Research article
Establishment of immunological probes to study human gonadotropin-releasing hormone receptors