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Plasma membrane expression of gonadotropin-releasing hormone receptors: regulation by peptide and nonpeptide antagonists.

ABSTRACT: Gonadotropin-releasing hormone acts via cell surface receptors but most human (h) GnRH receptors (GnRHRs) are intracellular. A membrane-permeant nonpeptide antagonist [(2S)-2-[5-[2-(2-axabicyclo[2.2.2]oct-2-yl)-1,1-dimethy-2-oxoethyl]-2-(3,5-dimethylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl)propan-1-amine (IN3)] increases hGnRHR expression at the surface, apparently by facilitating its exit from the endoplasmic reticulum. Here we have quantified GnRHR by automated imaging in HeLa cells transduced with adenovirus expressing hemagglutinin-tagged GnRHR. Consistent with an intracellular site of action, IN3 increases cell surface hGnRHR, and this effect is not blocked or mimicked by membrane-impermeant peptide antagonists [Ac-D2Nal-D4Cpa-D3Pal-Ser-Tyr-d-Cit-Leu-Arg-Pro-d-Ala-NH(2) (cetrorelix) and antide]. However, when the C-terminal tail of a Xenopus (X) GnRHR was added (h.XGnRHR) to increase expression, both peptides further increased cell surface GnRHR. Cetrorelix also synergized with IN3 to increase expression of hGnRHR and a G-protein coupling-deficient mutant (A261K-hGnRHR). Cetrorelix also increased cell surface expression of hGnRHR, h.XGnRHR, and mouse GnRHR in gonadotrope-lineage LbetaT2 cells, and in HeLa cells it slowed h.XGnRHR internalization (measured by receptor-mediated antihemagglutinin uptake). Thus cetrorelix has effects other than GnRHR blockade; it acts as an inverse agonist in internalization assays, supporting the potential importance of ligand-biased efficacy at GnRHR. We also developed an imaging assay for GnRH function based on Ca(2+)-dependent nuclear translocation of a nuclear factor of activated T cells reporter. Using this in HeLa and LbetaT2 cells, IN3 and cetrorelix behaved as competitive antagonists when coincubated with GnRH, and long-term pretreatment (16 h) with IN3 reduced its effectiveness as an inhibitor whereas pretreatment with cetrorelix increased its inhibitory effect. This distinction between peptide and nonpeptide antagonists may prove important for therapeutic applications of GnRH antagonists.

SUBMITTER: Finch AR

PROVIDER: S-EPMC2830153 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Plasma membrane expression of gonadotropin-releasing hormone receptors: regulation by peptide and nonpeptide antagonists.

Finch Ann R AR Caunt Christopher J CJ Armstrong Stephen P SP McArdle Craig A CA

Molecular endocrinology (Baltimore, Md.) 20091215 2

Gonadotropin-releasing hormone acts via cell surface receptors but most human (h) GnRH receptors (GnRHRs) are intracellular. A membrane-permeant nonpeptide antagonist [(2S)-2-[5-[2-(2-axabicyclo[2.2.2]oct-2-yl)-1,1-dimethy-2-oxoethyl]-2-(3,5-dimethylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl)propan-1-amine (IN3)] increases hGnRHR expression at the surface, apparently by facilitating its exit from the endoplasmic reticulum. Here we have quantified GnRHR by automated imaging in HeLa cells trans ...[more]

PMID: 20009083

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Project description:Androgen deprivation therapy (ADT) is the main treatment approach in advanced prostate cancer and in recent years has primarily involved the use of gonadotropin-releasing hormone (GnRH) agonists. However, despite their efficacy, GnRH agonists have several drawbacks associated with their mode of action. These include an initial testosterone surge and testosterone microsurges on repeat administration. GnRH antagonists provide an alternative approach to ADT with a more direct mode of action that involves immediate blockade of GnRH receptors. Antagonists produce a more rapid suppression of testosterone (and prostate-specific antigen [PSA]) without a testosterone surge or microsurges and appear to offer an effective and well tolerated option for the hormonal treatment of prostate cancer. Comparisons with GnRH agonists have shown GnRH antagonists to be at least as effective in achieving and maintaining castrate testosterone levels in patients with prostate cancer. Furthermore, with antagonists, the lack of an initial testosterone surge (which may cause clinical flare) may allow more rapid relief of symptoms related to prostate cancer, avoid the need for concomitant antiandrogens to prevent clinical flare (so avoiding any antiandrogen-associated adverse events) and allow GnRH antagonist use in patients with high tumour burden and/or acute problems such as spinal cord compression. Although several antagonists have been investigated, only degarelix and abarelix are currently available for clinical use in prostate cancer. Currently, degarelix is the most extensively studied and widely available agent in this class. Degarelix is one of a newer generation of antagonists which, in a comprehensive and ongoing clinical development programme, has been shown to provide rapid, profound and sustained testosterone suppression without the systemic allergic reactions associated with earlier antagonists. This review examines the currently available data on GnRH antagonists in prostate cancer.

Project description:Gonadotropin-releasing hormones (GnRHs) play pivotal roles in reproductive functions via the hypothalamus, pituitary, and gonad axis, namely, HPG axis in vertebrates. GnRHs and their receptors (GnRHRs) are likely to be conserved in invertebrate deuterostomes and lophotrochozoans. All vertebrate and urochordate GnRHs are composed of 10 amino acids, whereas protostome, echinoderm, and amphioxus GnRH-like peptides are 11- or 12-residue peptide containing two amino acids after an N-terminal pyro-Glu. In urochordates, Halocynthia roretzi GnRH gene encodes two GnRH peptide sequences, whereas two GnRH genes encode three different GnRH peptides in Ciona intestinalis. These findings indicate the species-specific diversification of GnRHs. Intriguingly, the major signaling pathway for GnRHRs is intracellular Ca2+ mobilization in chordates, echinoderms, and protostomes, whereas Ciona GnRHRs (Ci-GnRHRs) are endowed with multiple GnRHergic cAMP production pathways in a ligand-selective manner. Moreover, the ligand-specific modulation of signal transduction via heterodimerization among Ci-GnRHR paralogs suggests the species-specific development of fine-tuning of gonadal functions in ascidians. Echinoderm GnRH-like peptides show high sequence differences compared to those of protostome counterparts, leading to the difficulty in classification of peptides and receptors. These findings also show both the diversity and conservation of GnRH signaling systems in invertebrates. The lack of the HPG axis in invertebrates indicates that biological functions of GnRHs are not release of gonadotropins in current invertebrates and common ancestors of vertebrates and invertebrates. To date, authentic or putative GnRHRs have been characterized from various echinoderms and protostomes as well as chordates and the mRNAs have been found to be distributed not only reproductive organs but also other tissues. Collectively, these findings further support the notion that invertebrate GnRHs have biological roles other than the regulation of reproductive functions. Moreover, recent molecular phylogenetic analysis suggests that adipokinetic hormone (AKH), corazonin (CRZ), and AKH/CRZ-related peptide (ACP) belong to the GnRH superfamily but has led to the different classifications of these peptides and receptors using different datasets including the number of sequences and structural domains. In this review, we provide current knowledge of, and perspectives in, molecular basis and evolutionary aspects of the GnRH, AKH, CRZ, and ACP.

Dataset Information

Plasma membrane expression of gonadotropin-releasing hormone receptors: regulation by peptide and nonpeptide antagonists.

Publications

Plasma membrane expression of gonadotropin-releasing hormone receptors: regulation by peptide and nonpeptide antagonists.

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