help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Finch, A. R.
Right arrow Articles by McArdle, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Finch, A. R.
Right arrow Articles by McArdle, C. A.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Breast Cancer
The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 4 1823-1832
Copyright © 2004 by The Endocrine Society

Signaling and Antiproliferative Effects of Type I and II Gonadotropin-Releasing Hormone Receptors in Breast Cancer Cells

Ann R. Finch, Lisa Green, James N. Hislop, Eamonn Kelly and Craig A. McArdle

Wellcome Laboratories for Integrative Neuroscience and Endocrinology (A.R.F., L.G., C.A.M.) and Department of Pharmacology (E.K.), University of Bristol, Bristol, BS1 3NY, United Kingdom; and University of California at San Francisco (J.N.H.), San Francisco, California 94143

Address all correspondence and requests for reprints to: Dr. Craig A. McArdle, Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Whitson Street, Bristol, BS1 3NY United Kingdom. E-mail: craig.mcardle{at}bris.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH receptors (GnRH-Rs) mediate direct antiproliferative effects on hormone-dependent cancer cells. GnRH-Rs can be grouped according to ligand specificity (for GnRH-I and -II), and there is evidence that type II GnRH ligands and/or receptors can inhibit proliferation. Type I GnRH-Rs (e.g. human and sheep) lack the C-terminal tails found in other G protein-coupled receptors including type II GnRH-Rs (e.g. Xenopus; XGnRH-R). This underlies the remarkable resistance of type I GnRH-Rs to desensitization and may be important for chronic effects on proliferation. To test this, we have compared the antiproliferative effects of GnRH-Rs expressed in MCF7 breast cancer cells using recombinant adenovirus (Ad). Endogenous GnRH-Rs were not detected, but infection with Ad-expressing sheep GnRH-Rs (sGnRH-R) facilitated proliferation inhibition by Buserelin, and maximum inhibition required only 10,000–20,000 sGnRH-Rs. XGnRH-Rs were much less efficient at inhibiting proliferation and were internalized faster than sGnRH-Rs. Thus, the type II GnRH-R is less efficient at inhibiting proliferation, presumably because it is rapidly desensitized and/or internalized. Moreover, comparisons of human GnRH-R, sGnRH-R, and XGnRH-R, as well as chimeric receptors (type I GnRH-Rs with C-terminal tails from XGnRH-Rs), revealed that C-terminal tail addition increases receptor expression and thereby increases the efficiency with which the vector facilitates the antiproliferative effect.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GNRH REGULATES THE secretion of LH and FSH from the pituitary and thereby controls gametogenesis and steroidogenesis in the gonads (1). At the pituitary, GnRH binds G protein-coupled receptors (GPCRs), which act via Gq/11 to stimulate phospholipase C (PLC), thereby causing an inositol (1, 4, 5) trisphosphate-mediated mobilization of Ca2+ from intracellular stores. This Ca2+ mobilization and the entry of Ca2+ across the plasma membrane and the concomitant activation of protein kinase C are thought to mediate GnRH-stimulated gonadotrophin secretion (1, 2, 3). Activation of GPCRs typically causes rapid desensitization of the receptor as a consequence of receptor phosphorylation by second messenger-regulated kinases or by GPCR kinases. This phosphorylation most often occurs in the receptor’s C-terminal tail and can facilitate the binding of ß-arrestins that not only prevent G protein activation but also target the desensitized receptor for internalization. However, the cloning of the mouse GnRH-R revealed that it lacks a C-terminal tail, and subsequent functional studies revealed that it is only slowly internalized and is resistant to desensitization (4, 5, 6, 7). Since 1992, numerous GnRH-Rs have been cloned, and these can be divided into distinct groups. The type I GnRH-Rs that mediate hypothalamic control of mammalian reproduction are preferentially activated by GnRH. They lack C-terminal tails, internalize slowly, and resist desensitization (7, 8). In contrast, type II GnRH-Rs (which include all cloned nonmammalian GnRH-Rs) are preferentially activated by GnRH-II [originally termed chicken GnRH-II (cGnRH-II)], do have C-terminal tails, and where investigated, have been found to bind ß-arrestin and to internalize and desensitize rapidly (7, 8).

GnRH-stimulated gonadotropin secretion can be blocked with antagonists or mimicked by agonists, but in the latter case, sustained stimulation causes desensitization. Thus, both treatments will ultimately reduce circulating levels of gonadotropins and gonadal steroids, and this medical castration underlies the use of GnRH analogs to treat sex hormone-dependent neoplasms such as those of the prostate, ovary, endometrium, or mammary (9, 10). Because the type I GnRH-Rs mediating gonadotrophin secretion from the pituitary do not rapidly desensitize, desensitization of gonadotrophin secretion likely reflects downstream adaptation (11, 12). In addition to expression in the pituitary, GnRH-Rs are found (often along with GnRH) in some mammary, prostate, endometrial, and ovarian cancers (13, 14, 15). Interest in these extrapituitary GnRH-Rs stems primarily from the fact that GnRH analogs (or their cytotoxic derivatives) can inhibit proliferation of cell lines derived from such cancers and that direct antiproliferative effects may, therefore, contribute to the therapeutic effects of GnRH analogs in cancer treatment (13, 14, 15, 16, 17, 18, 19, 20, 21, 22).

Although type I GnRH-R transcripts detected in breast and ovarian cancers are identical to those of the pituitary (17), the receptors may differ functionally. Pituitary GnRH-Rs have high affinity for agonists, such as Buserelin [nanomolar dissociation constant (Kd) values], whereas the vast majority of GnRH-Rs in extrapituitary sites are low affinity (micromolar Kd values), and there are also apparent differences in signaling. Whereas GnRH-Rs in gonadotropes are positively coupled to PLC and MAPK activation, those in ovarian and endometrial cell lines have little, if any, effect on PLC and, in the presence of epidermal growth factor, actually inhibit ERK phosphorylation (18). Similar inhibition was observed in prostatic cancer cell lines (19), and it has been suggested that a Gi-mediated activation of protein phosphatase activity underlies differences in signaling between peripheral tumor and anterior pituitary GnRH-Rs (20, 21). Moreover, the antiproliferative effects of GnRH-R agonists in some cancer cell models can be mimicked by analogs, such as cetrorelix, which are competitive antagonists at pituitary GnRH-Rs. This implies that the agonist/antagonist dichotomy established for pituitary GnRH-Rs does not apply in extrapituitary sites (13, 22).

The data above could mean that GnRH-R signaling is context dependent, and we have recently addressed this issue using recombinant adenovirus (Ad) to express type I GnRH-Rs in human mammary (MCF7) and prostate (PC3) cancer-derived cell lines. In each case, PLC-coupled receptors were expressed. No evidence was obtained for context-dependent receptor function, but type I GnRH-R expression did mediate potent and pronounced antiproliferative effects of receptor agonists (23, 24). An alternative possibility is that GnRH analog effects in extrapituitary sites are actually mediated by type II GnRH-Rs. This possibility is supported by the recent cloning of type II GnRH-Rs in primates (25, 26, 27, 28), as well as the demonstration that hormone-dependent cancers can express type II GnRH-R ligands and transcripts. Moreover, these ligands can directly inhibit proliferation of ovarian and endometrial cancer cell lines (29, 30). Although the expression of functional human type II GnRH-R mRNA and protein remains controversial (see Discussion), these observations raise the question of whether type II GnRH-Rs can mediate signaling and antiproliferative effects in hormone-dependent cancer cells. This issue is of particular interest in light of recent work suggesting that vectors encoding GPCRs may be used therapeutically. For example, administration of recombinant Ad-expressing ß2-adrenergic receptors (AR) can be beneficial in animal models of cardiac insufficiency due to ß2-AR loss (31). Similarly, Ad-mediated expression of type II somatostatin receptors in pancreatic cancers can inhibit cancer growth and metastasis in vivo and can increase the effect of receptor-targeted therapy (32). A logical extension of this idea is that the vector may be engineered to incorporate desirable pharmacological characteristics, and a desensitization-resistant ß2-AR has recently been engineered for such applications (33). In this context, we are interested in the possibility that the antiproliferative effects mediated by type I GnRH-Rs might actually be dependent upon the unique resistance of these wild-type receptors to desensitization. Accordingly, we have now used recombinant Ad to express type I (sheep) and type II (Xenopus) GnRH-Rs (and chimeras thereof) in MCF7 cells. Using this model, we have addressed the specific question of whether a type II GnRH-R can mediate inhibition of proliferation, as well as the broader question of the relevance of GPCR desensitization and internalization as determinants of the efficiency with which proliferation is inhibited.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and cell culture

GnRH and cGnRH-II were purchased from Peninsula Laboratories Europe Ltd. (Merseyside, UK) or from Sigma (Poole, UK). Buserelin and [125I]Buserelin (2000 Ci/mmol) were provided by Prof. J. Sandow (Aventis Pharma GmbH, Frankfurt, Germany). [125I]cGnRH-II (~3400 Ci/mmol, determined by self-displacement) was prepared using chloramine-T and purified by G25 Sephadex column chromatography. [2-3H]Inositol (14–16 Ci/mmol) was from Amersham International PLC (Little Chalfont, UK). Culture media was from Sigma, sera was from First Link (Brierly Hill, UK), and plasticware was from Life Technologies, Inc. (Paisley, UK) or Falcon (Becton Dickinson & Co., Oxford, UK). All other reagents were from standard commercial suppliers. MCF7 cells were purchased from the European Collection of Cell Cultures (Salisbury, UK) and routinely cultured in DMEM supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin. Cultures were maintained at 37 C in humidified air-CO2 (19:1) and passaged weekly. For experiments, they were harvested by trypsinization and then incubated for 1–8 d in flasks or culture plates as described in the legends to the figures. Cells were infected on the second day, and after 6 h, the medium was replaced with fresh 1% FCS-supplemented media. Recombinant, early response gene 1 (E1)-deleted, Ad-expressing sheep GnRH-R (Ad sGnRH-R), human GnRH-R (Ad hGnRH-R), or Xenopus laevis type I GnRH-R (Ad XGnRH-R) or chimeric receptors consisting of the entire sGnRH-R or hGnRH-R with an added XGnRH-R C-terminal tail (Ad s.XGnRH-R and Ad h.XGnRH-R) were generated using standard techniques (34, 35) as previously described (35, 36). Adenoviral stocks were bulked up by growth in HEK-293. After extraction and CsCl2 gradient purification, they were stored at –80 C. Viral titer was determined using a standard plaque assay and is given in plaque-forming units per milliliter or plaque-forming units per infected cell (equivalent to multiplicity of infection).

Accumulation of [3H]inositol phosphates ([3H]IPx)

[3H]IPx accumulation was used as a measure of PLC activity as described (11, 23), using cells labeled by preincubation with [3H]inositol and stimulated in the presence of lithium chloride (LiCl). Cells were cultured in 24-well plates in 1 ml of media, and 2 µCi 2-[3H]inositol (14–16 Ci/mmol) was added to each well for the final 16 h of incubation. After two washes in physiological salt solution [PSS; 127 mM NaCl, 1.8 mM CaCl2, 5 mM KCl, 2 mM MgCl2, 0.5 mM NaH2PO4, 5 mM NaHCO3, 10 mM glucose, 0.1% (wt/vol) BSA, and 10 mM HEPES, pH 7.4], each well was stimulated for the period indicated in the figure legends with 200–250 µl of PSS containing 10 mM LiCl and the indicated concentration of stimulatory peptide. The stimuli were terminated by adding 1 ml of water at 95 C. The cells were lysed by freezing and thawing, and [3H]IPx was separated from free [3H]inositol using anion exchange chromatography in formate form Dowex-1 columns (Sigma-Aldrich), and the amount of 3H eluted in each fraction was determined by liquid scintillation spectroscopy (11, 23).

Radioligand binding and internalization assays

GnRH-R expression was assessed by whole-cell binding assays using cells in suspension or growing in culture plates. For the suspension binding assays, approximately 50,000 cells were incubated for 30 min at 21 C in 100 µl PSS containing 1 mg/ml bacitracin, with approximately 0.1 nM [125I]Buserelin (a high affinity GnRH-R agonist for type I GnRH-Rs) or approximately 0.25 nM [125I]cGnRH-II (a type II GnRH-R-specific ligand) and none or 10–11 to 10–5 M of the unlabelled competitor peptide. Free and bound peptide were then separated by centrifugation through oil as described (11, 23, 35), and nonlinear regression (Graphpad Prism; GraphPad Software Inc., San Diego, CA) was used to determine Kd and binding capacity values, assuming that the tracer and competitor bind with identical affinity to a single class of receptor. Cell counts were performed in parallel, enabling the number of receptors per cell to be calculated. For flat-plate binding assays, cells were seeded at 50,000 cells/well (grown in 24-well plates). For receptor internalization studies, plated cells were incubated for 5–120 min at 37 C with radioligand, and internalized ligand was determined using an acid wash procedure to remove cell surface bound ligand, as described (36).

[3H]Thymidine incorporation

Incorporation of [3H]thymidine into newly synthesized DNA was used as a index of cell proliferation (23, 37). Cells were seeded in 96-well plates in 250 µl DMEM with 10% FCS at a density of 10,000 cells/ml. After 24 h, they were transferred to DMEM containing 1% FCS and incubated with the required amount of Ad GnRH-R. After 6 h, the cells were transferred to fresh DMEM (1% FCS) and incubated overnight before addition of test peptides. After a further 5–7 d, 10 µl (0.5 µCi) [3H]thymidine was added to each well and left to incorporate for 6 h. Media was removed, and the cells were then incubated in 100 µl/well of trypsin/EDTA at 37 C for 30 min, after which the plates were stored at –20 C. The cells were then thawed, and incorporated [3H]thymidine was collected on filter papers and quantified as described (23).

Statistical analysis and data presentation

The figures show data from a single representative experiment or the mean ± SEM of data pooled from n independent experiments (data normalized as described in the figure legends). Data are typically reported in the text as mean ± SEM, and statistical analysis was performed using the Student’s t test, accepting P < 0.05 as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In the first experiments, radioligand binding was used to assess expression of type II ligand-specific binding sites in control and Ad XGnRH-R infected MCF7 cells. We were unable to detect specific binding of [125I]GnRH-II in control (uninfected) MCF7 cells, but 1 d after infection with Ad XGnRH-R at varied viral titer (10, 30, 100, and 300 pfu/cell), there was a clear titer-dependent increase in specific binding (Fig. 1Go). This binding was competed for in a concentration-dependent manner by unlabeled GnRH-II, and curve fitting (nonlinear regression assuming a single class of binding site) revealed binding to high-affinity sites with Kd values of 1.4 ± 0.3 nM (n = 3). The Kd was not dependent upon viral titer, whereas increasing Ad XGnRH-R titer from 10–300 pfu/cell increased the number of binding sites from 2,100 ± 700 to 36,300 ± 10,300 sites/cell (Fig. 1Go). To assess whether these sites were indeed functional receptors, activation of PLC was assessed using an [3H]IPx accumulation assay. As shown (Fig. 1Go, inset), GnRH-II failed to activate PLC in control MCF7 cells, and infection with Ad XGnRH-R did not increase basal [3H]IPx accumulation in the absence of ligand. However, infection with Ad XGnRH-R did facilitate a clear concentration- and titer-dependent stimulation of [3H]IPx accumulation by GnRH-II.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 1. Titer dependence of receptor expression and signaling in MCF7 cells infected with Ad XGnRH-R. Main panel, MCF7 cells cultured in 60-mm Petri dishes were infected with Ad XGnRH at 0, 30, 100, or 300 pfu/cell as indicated and then cultured for 1 d before being scraped from the culture vessels and used for suspension binding assays using approximately 0.25 nM [125I]GnRH-II and the indicated concentration of unlabelled GnRH-II. The pooled Kd value was 1.4 nM, and the values shown are means ± SEM (n = 3) normalized as a percentage of the binding seen without competitor in cells infected at 300 pfu/cell. Inset, MCF7 cells cultured in 24-well plates were infected with Ad XGnRH-R at the indicated titer then cultured for 1 d. [3H]Inositol was added to the medium for the final 16 h of culture, after which the cells were washed and stimulated for 30 min with none or 10–7 M GnRH-II (as indicated) in the presence of 10 mM LiCl. Data shown are the means ± SEM (n = 2–4) from repeated experiments, each having triplicate determinations. For data pooling, the radioactivity in the [3H]IPx fraction (e.g. the third fraction eluted from the Dowex column) was normalized as a percentage of the total eluted radioactivity to provide an internal control for labeling in each well.

 
Ligand specificity was next examined in cells infected with Ad XGnRH-R (107 pfu/ml) by constructing competition binding curves with [125I]GnRH-II and increasing concentrations of Buserelin, GnRH, or GnRH-II. For comparison, similar experiments were performed in cells infected with Ad sGnRH-R or with Ad s.XGnRH-R. As shown (Fig. 2Go), GnRH-II and Buserelin both inhibited binding of [125I]GnRH-II to the XGnRH-R, whereas GnRH failed to do so. The observed rank order of potency (GnRH-II > Buserelin > GnRH) is characteristic for type II GnRH-Rs (2, 7, 32) and was identical to that seen when [3H]IPx accumulation was stimulated in Ad XGnRH-R-infected cells (data not shown). In cells infected with Ad sGnRH-R or Ad s.XGnRH-R, the binding of [125I]Buserelin was inhibited by the same three peptides but with the rank order of potency (Buserelin > GnRH > GnRH-II) expected for a type I GnRH-R (2, 7, 32). The same rank order of potency was also observed when [3H]IPx accumulation was stimulated in Ad sGnRH-R- or Ad s.XGnRH-R-infected cells (data not shown) (20).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 2. Ligand specificity in competition binding assays. MCF7 cells were infected with Ad sGnRH-R, Ad XGnRH-R, or Ad s.XGnRH-R and then cultured for 1–2 d before being scraped from the culture vessels and used for suspension binding assays using approximately 0.1 nM [125I]Buserelin (sGnRH-R and s.XGnRH-R) or 0.25 nM [125I]GnRH-II and the indicated concentration of unlabelled GnRH, Buserelin, or GnRH-II (as indicated), as described in Materials and Methods. Data shown are the means ± SEM (n = 2–7) from seven experiments, each having duplicate or triplicate determinations. Where error bars are not visible, they are smaller than the symbols.

 
When the relationship between viral titer and receptor number was explored (Fig. 3Go), increasing titer of Ad sGnRH-R, Ad XGnRH-R, or Ad s.XGnRH-R from 0.3–20 x 106 pfu/ml caused a clear increase in ligand binding, but numbers of chimeric receptors were considerably higher than for either of the wild-type receptors (e.g. expression of over 300,000 s.XGnRH-R/cell at the highest titer, compared with 25,000–50,000 sites/cell for the wild-type receptors). When the kinetics of binding were assessed at 37 C, rates of association were comparable for each receptor (data not shown), but internalization rates differed dramatically. The XGnRH-R was internalized much faster than the sGnRH-R, whereas internalization was slowed by addition of the Xenopus tail to the sGnRH-R (Fig. 4Go).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 3. Relationship between adenoviral titer and receptor expression. MCF7 cells were infected with Ad sGnRH-R, Ad XGnRH-R, or Ad s.XGnRH-R at the indicated titer and then used in a single point flat plate binding assay using approximately 0.1 nM [125I]Buserelin (sGnRH-R and s.XGnRH-R) or 0.25 nM [125I]GnRH-II with or without 10–6 M unlabelled homologous competitor peptide, as described in Materials and Methods. Assuming binding to a single class of receptor (Kd values of 1.4 nM for type I and II GnRH-Rs, each binding their cognate ligand) and a Hill coefficient of 1, we calculate that the tracers occupy 9 and 15%, respectively, of the cell surface receptors at equilibrium. Specific binding (femtomoles per plated cell) was determined by subtraction of nonspecific from total binding, and this was used along with the percentage of occupancy and cell counts to calculate receptor number per cell. The data shown are pooled from 10 separate experiments, each having duplicate or triplicate observations (mean ± SEM, n = 3–10).

 


View larger version (18K):
[in this window]
[in a new window]
  		FIG. 4. Internalization of wild-type and chimeric GnRH-Rs in MCF7 cells. Cells were infected with Ad sGnRH-R, Ad XGnRH-R, or Ad s.XGnRH-R at 107 pfu/ml and then used to assess receptor internalization by incubation at 37 C in medium with 0.1 nM [125I]Buserelin (sGnRH-R and s.XGnRH-R) or 0.25 nM [125I]GnRH-II (XGnRH-R) with or without 10–6 M unlabelled homologous competitor. After the indicated incubation time, internalization was stopped by the addition of medium at 4 C. Cell surface and internalized receptors were distinguished by acid washing, and at each time point, specific acid-resistant binding was expressed as a percentage of total (cell surface plus internalized) specific binding. The time courses for total specific binding were comparable for all three receptors (data not shown). The data shown are pooled from three separate experiments, each having triplicate observations (mean ± SEM, n = 3).

 
To assess possible antiproliferative effects, MCF7 cells were infected with Ad sGnRH-R, Ad XGnRH-R, or with no Ad (control cells) and then incubated with various concentrations of Buserelin or GnRH-II before estimation of DNA synthesis ([3H]thymidine incorporation) on the last day of culture. Neither of these peptides had any measurable effect in control cells (data not shown) (20), but after infection with Ad sGnRH-R, Buserelin caused the expected pronounced and potent inhibition of DNA synthesis (Fig. 5Go). This effect was mimicked by GnRH-II, but the dose-response curve was right shifted, as anticipated from the lower affinity of GnRH-II for type I GnRH-Rs (Fig. 2Go). In contrast, GnRH-II (and Buserelin) failed to inhibit proliferation in control cells and in cells infected with Ad XGnRH-R (Fig. 5Go and data not shown).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 5. Effects of Buserelin and GnRH-II on [3H]thymidine incorporation into MCF7 cells infected with Ad sGnRH-R or Ad XGnRH-R. Cells plated at low density in 96-well plates were infected with Ad sGnRH-R (• and {blacktriangleup}, 107 pfu/ml) or with Ad XGnRH-R ({triangleup}) and then maintained for 5–6 d in the presence of the indicated concentration of Buserelin ({circ} and •) or GnRH-II ({triangleup}) before assessment of [3H]thymidine incorporation on the final day of culture. The figure shows data pooled from five separate experiments (mean ± SEM, n = 3–5), each of which had four to six replicate observations. Pooling was achieved by normalizing the data as a percentage of [3H]thymidine incorporation seen in control cells without peptide, and the three control values did not differ significantly from each other. Neither of the peptides had any measurable effect on [3H]thymidine incorporation in control cells receiving no Ad or receiving a control (empty) Ad (data not shown).

 
To explore the importance of receptor expression levels, experiments were performed in which receptor number, [3H]IPx accumulation, and inhibition of [3H]thymidine incorporation were all assessed in cells infected with Ad sGnRH-R, Ad XGnRH-R, or Ad s.XGnRH-R at varied titers. This approach is illustrated for sGnRH-R in Fig. 6Go. As shown earlier, increasing Ad titer from 0.6–20 x 106 pfu/ml increased receptor number to approximately 50,000 sites/cell (Fig. 3Go), and this was associated with an increase in receptor-mediated [3H]IPx accumulation and inhibition of [3H]thymidine incorporation (Fig. 6Go). To pool data and compare receptors, the functional responses were binned according to receptor number (2,000–20,000 sites/cell and 20,000–80,000 sites/cell). These categories were chosen because they approximate subphysiological and physiological levels for type I GnRH-Rs in pituitary gonadotrophs (35, 38). This analysis revealed that increasing sGnRH-R number into the physiological range increased receptor-mediated [3H]IPx accumulation but did not increase the antiproliferative effect (Fig. 7Go, upper panels). At comparable receptor numbers, XGnRH-Rs mediated comparable stimulation of [3H]IPx accumulation, but inhibition of proliferation was much lower than that with the sGnRH-R. The same approach was used to compare Ad sGnRH-R- and Ad s.XGnRH-R-infected cells (Fig. 7Go, lower panels), and this revealed that the chimeric receptor was no more effective than the sGnRH-R at inhibiting proliferation at matched receptor number (20,000–80,000 sites/cell).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 6. Relationship between receptor number and receptor-mediated effects in Ad sGnRH-R-infected MCF7. Cells were infected with Ad sGnRH-R (0.6–20 x 106 pfu/ml) and used for estimation of receptor expression as described in legend of Fig. 3Go. They were also used for assessment of receptor-mediated [3H]IPx accumulation and for inhibition of [3H]thymidine incorporation (as described in the legends of Figs. 1Go and 5Go), which enabled these two responses to be plotted against receptor number. The figures show data pooled from 11 separate experiments, each with two to four replicate observations at each adenoviral titer (mean ± SEM, n = 2–11).

 


View larger version (27K):
[in this window]
[in a new window]
  		FIG. 7. Comparison of responses to sGnRH-R, XGnRH-R, and s.XGnRH-R activation using data binned according to receptor number. Experiments identical to those described for Ad sGnRH-R-infected cells (Fig. 6Go) were performed using Ad XGnRH-R and Ad s.XGnRH-R, and the functional responses were binned according to receptor number (2,000–20,000; 20,000–80,000; or >80,000 sites/cell, as indicated). The upper panels show a comparison of sGnRH-Rs and XGnRH-Rs using data pooled from 10–20 separate experiments, each with two to four replicate observations (mean ± SEM, n = 10–20). For each receptor number bin, numbers of receptors expressed and [3H]IPx responses were indistinguishable, whereas XGnRH-R-mediated effects on [3H]thymidine incorporation were lower than those mediated by sGnRH-R (P < 0.01). The lower panels show a comparison of sGnRH-Rs and s.XGnRH-Rs using data pooled from five separate experiments, each with two to four replicate observations (mean ± SEM, n = 6–14). Wild-type sGnRH-R expression never exceeded 80,000 sites/cell.

 
In addition to comparing antiproliferative effects in cells binned according to receptor number, we have compared effects of these receptors after infection at a fixed adenoviral titer. When cells were infected with Ad sGnRH-R, Ad XGnRH-R, or Ad s.XGnRH-R (each at 5 x 106 pfu/ml), comparable [3H]IPx responses were observed, although receptor expression was approximately 10-fold higher with the chimeric receptor (Fig. 8Go). The Ad sGnRH-R and Ad s.XGnRH-R mediated comparable inhibition of [3H]thymidine incorporation, despite the higher receptor number with the chimera, and the XGnRH-R mediated no significant inhibition of [3H]thymidine incorporation, despite comparable receptor number with the sGnRH-R (Fig. 8Go). The lack of any additional antiproliferative effect of the chimeric receptor could simply reflect the fact that maximal inhibition is achieved with relatively low numbers of wild-type receptors (Fig. 6Go), in which case, the chimeric receptor vector would only be expected to be more effective where receptor number is limiting. To test this, we performed similar studies with Ad expressing an hGnRH-R or a full-length hGnRH-R with an additional XGnRH-R C-terminal tail. The rationale for these experiments is that we, and others, have found levels of hGnRH-R to be exceedingly low in a number of expression systems, including MCF7 cells (35, 39), and these h.XGnRH-R were indistinguishable from the s.XGnRH in preliminary pharmacological studies (data not shown). When MCF7 cells were infected with Ad hGnRH-R at 2 x 107 pfu/ml (the highest concentration used for the other receptors), receptor expression was too low for measurement using this single point plated cell binding assay (Fig. 8Go, lower panels). Consistent with this, receptor-mediated [3H]IPx accumulation and proliferation inhibition were much less marked than with sGnRH-Rs (Fig. 8Go, compare upper and lower panels). However, when cells were infected with Ad h.XGnRH-R at the same titer, receptor expression levels were increased into the physiological range, and a corresponding increase in receptor-mediated [3H]IPx accumulation and inhibition of [3H]thymidine incorporation was observed (Fig. 8Go, lower panel).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 8. Comparison of responses to wild-type and chimeric receptor activation after infection with Ad at fixed titer. Experiments identical to those described in Fig. 6Go were performed using Ad XGnRH-R, Ad sGnRH-R, and Ad s.XGnRH-R and a fixed Ad titer of 5 x 106 pfu/ml (upper panels) or using Ad XGnRH-R, Ad hGnRH-R, and Ad h.XGnRH-R at a fixed Ad titer of 20 x 106 pfu/ml (lower panels). The figures show receptor number (estimated using the flat plate binding assay) as well as receptor-mediated stimulation of [3H]IPx accumulation and inhibition of [3H]thymidine incorporation. Receptor internalization data (from the 30-min point in Fig. 4Go) are included for comparison in the upper panels but are absent from the lower panels because too few hGnRH-Rs were expressed for assessment of internalization. The upper panels each show data pooled from four or five separate experiments, each with two to four replicate observations (mean ± SEM, n = 3–5). The tendencies for inhibition of proliferation with Ad hGnRH-R- and Ad XGnRH-R-infected cells did not attain statistical significance (P > 0.05), whereas the effect mediated by the sGnRH-R, the s.XGnRH-R, and the h.XGnRH-R were all significant (P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Within the pituitary, GnRH acts via type I GnRH-Rs to cause a Gq/11-mediated activation of PLC and consequent acute stimulation of gonadotropin secretion as well as chronic regulation of gonadotropin and GnRH-R synthesis. The therapeutic effects of GnRH analogs are primarily (if not entirely) dependent upon activation, desensitization, or blockade of this system. There is also abundant evidence for expression of GnRH-Rs in extrapituitary sites, most notably in reproductive tissues and the hormone-dependent cancers or cancer cell lines derived from such tissues. Because GnRH analogs can inhibit proliferation, stimulate apoptosis, and slow invasion in various cancer models, there is immense interest in the possibility that the direct effects of GnRH analogs (or their cytotoxic derivatives) may also be exploited therapeutically. However, many aspects of GnRH analog action in hormone-dependent cancers remain controversial. Thus, although GnRH agonists are antiproliferative and/or proapoptotic in some cells (13, 14, 20, 21, 22, 23, 24, 40, 41, 42, 43), they appear to stimulate proliferation and/or inhibit apoptosis in others (44, 45). Similarly, although GnRH-Rs appear to couple faithfully to Gq/11 and thereby stimulate MAPK in some cells (44, 45), there is evidence that effects on cancer cell proliferation are mediated by Gi activation and consequent inhibition of MAPK signaling (20, 24). In several models, peptides that act as antagonists at pituitary GnRH-R actually mimic effects of agonists, an effect that could imply that the established agonist/antagonist dichotomy is not applicable to all cells or simply that agonist and antagonist are both reducing GnRH-R activation by down-regulation and blockade, respectively.

Taken together, the data above imply that GnRH-R function is dependent upon cellular context in terms of immediate and distal effectors (e.g. the G-proteins activated and the pathways downstream of the G-proteins). We have recently addressed this possibility using recombinant Ad to express type I GnRH-Rs at varied density in human breast cancer (MCF7) and prostate cancer (PC3) cell lines (23, 24), but this strategy has not revealed context-dependent signaling. In each case, high-affinity receptors were expressed that were positively coupled to PLC activation and MAPK (ERK1/2) activation. These had no measurable effect on cAMP accumulation and showed comparable ligand specificity (including agonist/antagonist discrimination) to that seen in pituitary cells. This context-independent signaling lends credence to the alternative possibility that GnRH analog effects in extrapituitary sites are actually mediated by a distinct receptor. The type II GnRH-R is the obvious candidate in light of recent evidence that GnRH-II is expressed in human reproductive tissues and can inhibit the proliferation of cell lines derived from hormone-dependent cancers, as well as the recent cloning of type II GnRH-Rs in humans (25, 26, 27, 28). Indeed, Grundker et al. (30) have recently shown that GnRH-II can inhibit growth in a cell line lacking type I GnRH-R transcripts and expressing type II GnRH-R transcripts by PCR. Moreover, the observations that a GnRH-R I antagonist can exhibit agonist activity at a type II GnRH-R (46) and that downstream effectors for type I and II GnRH-Rs can differ (30) are compatible with the idea that the functional dichotomy between pituitary and extrapituitary GnRH effects reflects mediation by type I and II GnRH-Rs, respectively. The fundamental problem with this argument is that the cloned human type II GnRH-R has a 5' nucleotide deletion (compared with the marmoset GnRH-R), causing a frame-shift, as well as a single base change in exon 2 that introduces a premature stop codon (25, 27, 28). Accordingly, the transcripts characterized to date could not encode a full-length seven-transmembrane region GnRH-R (27, 28). Another obvious concern is that chronic antiproliferative effects are likely to require sustained receptor activation (we have shown that several hours to days of peptide exposure are required for inhibition of [3H]thymidine incorporation into PC3 cells expressing type I GnRH-Rs) (22) and are, therefore, likely to be limited by receptor desensitization, internalization, and/or down-regulation. Because comparative studies have consistently revealed that receptor desensitization and internalization are rapid for type II GnRH-Rs and absent or slow for type I GnRH-Rs (7), we hypothesized that type II GnRH-Rs expressed by cancer cells would be relatively inefficient at mediating inhibition of proliferation. Here we have tested this by expressing type I (sheep) and type II (Xenopus) GnRH-Rs in MCF7 cells. We show that these cells do not express endogenous type II GnRH-Rs (lack of binding or [3H]IPx response in control cells) and that infection with Ad XGnRH-R leads to expression of functional receptors that are positively coupled to PLC (Fig. 1Go), have high affinity for GnRH-II (Figs. 1Go and 3Go), and are selective for the type II ligand (Fig. 2Go). Increasing adenoviral titer from 10–300 pfu/ml increased receptor number from 2,000–36,000 sites/cell (Fig. 3Go). Because at least 80% of these cells are transfected at 10 pfu/ml (20), this reflects primarily an increase in receptors per cell, rather than an increase in the proportion of cells expressing the receptors. These data are similar to those previously obtained with Ad sGnRH-R, except that the sGnRH-R displayed affinity and specificity (Buserelin > GnRH > GnRH-II) characteristic for a type I GnRH-R (23). In addition to ligand specificity, these type I and II GnRH-Rs differed markedly in internalization rates (Fig. 4Go). The XGnRH-R was internalized much more rapidly than the sGnRH-R (Fig. 4Go), as anticipated from earlier comparisons of type I and II GnRH-Rs expressed in {alpha}T4 and HeLa cells by infection with recombinant Ad (35, 36). Other GnRH-R comparisons have revealed that the rapid internalization of type II GnRH-Rs is associated with rapid desensitization, and we have also found that the number of cell surface XGnRH-R is dramatically reduced after activation, whereas the number of type I GnRH-Rs is not (data not shown). Thus, it is likely that desensitization, internalization, and down-regulation are all more rapid for XGnRH-Rs than for sGnRH-Rs in these cells.

We have previously shown that infection with Ad sGnRH-R facilitates a pronounced and potent inhibition of [3H]thymidine incorporation by type I GnRH-R agonists (but not antagonists) in MCF7 cells. Because this effect occurred without measurable stimulation of cytotoxicity or apoptosis, it apparently reflects inhibition of proliferation (23). Using the same model to compare effects mediated by type I and II GnRH-Rs, we now show that GnRH-II (the type II GnRH-R ligand) can inhibit proliferation by cross-reactivity at the type I receptor, but it failed to do so in cells infected with Ad XGnRH-R (Fig. 5Go). In the same study, neither of these ligands inhibited proliferation in control cells (uninfected or infected with empty Ad), whereas Buserelin did not inhibit proliferation in cells infected with Ad XGnRH-R (data not shown). Although these data are indicative of a further functional distinction between these receptors, receptor number was not measured, and an additional series of experiments was therefore planned in which receptor number was controlled by varying adenoviral titer and in which receptor number, [3H]IPx signaling, and inhibition of [3H]thymidine incorporation were all measured. As anticipated, we found that increasing Ad sGnRH-R titer increased receptor number from unmeasurable (<2,000 sites/cell) to approximately 50,000 sites/cell and that this was associated with increasing receptor-mediated stimulation of [3H]IPx accumulation and increased inhibition of [3H]thymidine incorporation (Fig. 6Go).

Interestingly, maximum inhibition of proliferation was achieved with only 10,000–20,000 receptors/cell, which are relatively low values that are below the physiological range of type I GnRH-Rs in pituitary gonadotrophs (25,000–85,000 sites/cell). When similar experiments were performed using Ad XGnRH-R, increasing adenoviral titer increased receptor number over a similar range (from unmeasurable to ~30,000 sites/cell). When the data for both receptors were binned according to receptor number (2,000–20,000 or 20,000–80,000 sites/cell), it was evident that both receptors elicited comparable [3H]IPx responses at comparable receptor numbers. Although we have previously shown (34) that XGnRH-R-mediated [3H]IPx accumulation desensitizes more rapidly than the effect mediated by a type I GnRH-R (human), the short stimulation period used in the current study (15 min) most probably obviated any difference in response kinetics due to receptor desensitization. This lack of distinction between the two receptors in acute signaling stands in marked contrast to the clear difference in the chronic, antiproliferative response. At both receptor densities, the XGnRH-R mediated only a modest inhibition of [3H]thymidine incorporation compared with the 50% inhibition seen with the sGnRH-R (Fig. 7Go). Moreover, this distinction is not simply a characteristic of the cell line used because we have obtained similar data in PC3 human prostate cancer cells infected with Ad at titers selected for comparable receptor expression. As in MCF7 cells (Fig. 5Go), Buserelin and GnRH-II had no effect on of [3H]thymidine incorporation in control (uninfected) PC3 cells, and Buserelin caused a concentration-dependent inhibition in Ad sGnRH-R-infected cells, which was mimicked by GnRH-II (albeit with lower potency), whereas neither peptide inhibited proliferation in Ad XGnRH-R-infected cells (data not shown) (24).

Having demonstrated that a type II GnRH-R is less efficient than a type I GnRH-R at mediating proliferation inhibition, we have explored the relevance of the C-terminal tail for this dichotomy. To do so, we engineered a recombinant Ad expressing the full-length sGnRH-R or hGnRH-R with an added XGnRH-R C-terminal tail. Pharmacological characterization revealed that this chimeric receptor is positively coupled to PLC and is indistinguishable from the wild-type sGnRH-R in terms of binding affinity and specificity (data not shown). When MCF7 cells were infected with Ad s.XGnRH-R at varied titer, receptor expression levels were considerably higher than those achieved with Ad XGnRH-Rs at the same titers. This finding had been anticipated from earlier studies showing that expression levels are increased by addition of a catfish GnRH-R C-terminal tail to the full-length rat GnRH-R (47). We had also anticipated that addition of the XGnRH-R tail to the sGnRH-R would accelerate internalization because addition of the TRH-receptor tail to the rat GnRH-R increases its rate of internalization and because truncation of the C-terminal tail of the cGnRH-R slows its internalization (48, 49). However, this proved not to be the case. In fact, internalization of the sGnRH-R was slowed by addition of the XGnRH-R C-terminal tail (Fig. 4Go).

Although unexpected, the reduction in internalization rate caused by the addition of the C-terminal tail provides a simple means of expressing receptors that are only slowly internalized and at high density. To determine how this modification influences receptor function, receptor number, [3H]IPx accumulation, and [3H]thymidine incorporation were assessed in cells infected with Ad s.XGnRH-R, just as described earlier for Ad sGnRH-R and Ad XGnRH-R. The data were again binned according to receptor number, and this revealed that, at comparable receptor density (20,000–80,000 sites/cell), the s.XGnRH-R is no more efficient than the sGnRH-R at mediating the inhibition of proliferation and is, in fact, less efficient at mediating [3H]IPx accumulation. Comparison of these parameters at a single Ad titer of 107 pfu/ml revealed that the dramatic increase in receptor number caused by addition of the Xenopus C-terminal tail to the sGnRH-R was not paralleled by an increase in the antiproliferative effect. Suspecting that this was simply because the wild-type sGnRH-R expression was sufficient for maximal expression (e.g. 20,000 sites/cell in Fig. 8Go compared with maximal effects at 10,000–20,000 sites/cell in Fig. 6Go), similar experiments were performed with the hGnRH-R. In many heterologous systems, hGnRH-R expression levels are low relative to those of other GnRH-Rs (35, 39, 49, 50). We have used Ad hGnRH-R to express hGnRH-Rs at 25,000–50,000 sites/cell in {alpha}T4 and HeLa cells (35, 36), but when MCF7 cells were infected with the same Ad, expression levels were much lower. Indeed, specific binding sites were not measurable (e.g. <2000 sites/cell) in MCF7 cells infected with Ad hGnRH-R at 2 x 107 pfu/ml (Fig. 8Go). Interestingly, a modest inhibition of proliferation (~20%) was seen, again indicating that the antiproliferative effect can occur at very low receptor number in this model. More importantly, addition of the XGnRH-R C-terminal tail to the hGnRH-R not only increased receptor expression levels and [3H]IPx signaling, but it also increased the antiproliferative effect. Thus, it is possible to increase the efficiency of antiproliferative signaling by modifying the receptor to increase its expression, but this is only effective when receptor expression is limiting (e.g. for the hGnRH-R but not for the sGnRH-R in this model).

These experiments are of interest in light of ongoing work suggesting that it may be possible to develop gene therapy with adenoviral vectors encoding GPCRs to increase the effects of endogenous or administered ligands (31, 32, 33). In such applications, it may be possible to engineer the viral genome to influence viral characteristics (such as tropism, infectivity, and immunogenicity) and to engineer or select for desirable receptor characteristics (such as ligand specificity, desensitization, and internalization). In this context, comparison of type I or II GnRH-Rs provides a clear example of the importance of receptor selection. The type I GnRH-R is more efficient at mediating inhibition of proliferation, as revealed by comparisons at matched receptor density (Fig. 7Go). Consequently, the Ad XGnRH-R is less efficient than the Ad sGnRH-R at facilitating the antiproliferative effect of agonists, as revealed by comparisons at matched Ad titer (Figs. 5Go and 8Go). In contrast, addition of the Xenopus C terminus does not increase the efficiency of type I GnRH-Rs at mediating proliferation inhibition (again revealed by comparisons at matched receptor density in Fig. 7Go), but because it increases receptor expression, it does increase the efficiency of the vector. The latter effect is evident from the fact that Ad h.XGnRH-R facilitates a greater inhibition of proliferation than Ad hGnRH-R (both at 2 x 107 pfu/ml in Fig. 8Go). Moreover, although comparable antiproliferative effects were seen with 20,000–80,000 sGnRH-R and s.XGnRH-R (Fig. 7Go), this required infection with 9.5 ± 1.6 x 106 pfu/ml of Ad s.GnRH-R compared with only 0.9 ± 0.3 x 106 pfu/ml of Ad s.XGnRH-R. Thus, our data show that the efficiency with which the vector facilitates the inhibition of proliferation is dependent upon receptor expression as well as function (desensitization and internalization) and that the efficiency of the vector can therefore be increased by appropriate selection or modification of the receptor.


   Acknowledgments
 
We are grateful to Prof. Sandow (Aventis Pharma GmbH, Frankfurt, Germany) for providing the Buserelin and [125I]Buserelin and to Prof. R. Millar (Medical Research Council Human Reproductive Sciences Unit, Edinburgh, UK) for providing the human and Xenopus GnRH-R plasmids.


   Footnotes
 
This work was supported by the Wellcome Trust for project grant support (062918).

Abbreviations: [3H]IPx, [3H]Inositol phosphate; Ad, adenovirus; AR, adrenergic receptor; cGnRH-II, chicken GnRH-II; FCS, fetal calf serum; GnRH-R, GnRH receptor; GPCR, G protein-coupled receptors; hGnRH-R, human GnRH-R; h.XGnRH-R, hGnRH-R with an added XGnRH-R C-terminal tail; PLC, phospholipase C; PSS, physiological salt solution; sGnRH-R, sheep GnRH-R; s.XGnRH-R, sGnRH-R with an added XGnRH-R C-terminal tail; XGnRH-R, Xenopus GnRH-R.

Received May 2, 2003.

Accepted December 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Conn PM, Crowley WFJ 1994 Gonadotropin-releasing hormone and its analogs. Annu Rev Med 45:391–405:391–405[CrossRef][Medline]
  2. Sealfon SC, Weinstein H, Millar RP 1997 Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev 18:180–205[Abstract/Free Full Text]
  3. Stojilkovic SS, Catt KJ 2000 Expression and signal transduction pathways of gonadotropin-releasing hormone receptors. Recent Prog Horm Res 50:161–205
  4. Tsutsumi M, Zhou W, Millar RP, Mellon PL, Roberts JL, Flanagan CA, Dong K, Gillo B, Sealfon SC 1992 Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor. Mol Endocrinol 6:1163–1169[Abstract]
  5. Reinhart J, Mertz LM, Catt KJ 1992 Molecular cloning and expression of cDNA encoding the murine gonadotropin-releasing hormone receptor. J Biol Chem 267:21281–21284[Abstract/Free Full Text]
  6. Davidson JS, Wakefield IK, Millar RP 1994 Absence of rapid desensitization of the mouse gonadotropin-releasing hormone receptor. Biochem J 300: 299–302
  7. McArdle CA, Franklin J, Green L, Hislop JN 2002 Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors. J Endocrinol 173:1–11[Abstract]
  8. Heding A, Vrecl M, Bogerd J, McGregor A, Sellar R, Taylor PL, Eidne KA 1998 Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics. J Biol Chem 273:11472–11477[Abstract/Free Full Text]
  9. Barbieri RL 1992 Clinical applications of GnRH and its analogues. Trend Endocrinol Metab 3:30–34
  10. Schally AV 1999 LH-RH analogues: their impact on human reproduction and the control of tumorigenesis. Peptides 20:1247–1262[CrossRef][Medline]
  11. McArdle CA, Forrest-Owen W, Willars G, Davidson J, Poch A, Kratzmeier M 1995 Desensitization of gonadotropin-releasing hormone action in the gonadotrope-derived {alpha} T3–1 cell line. Endocrinology 136:4864–4871[Abstract]
  12. Willars GB, Royall JE, Nahorski SR, El Gehani F, Everest H, McArdle CA 2001 Rapid down-regulation of the type I inositol 1,4,5-trisphosphate receptor and desensitization of gonadotropin-releasing hormone-mediated Ca2+ responses in {alpha} T3–1 gonadotropes. J Biol Chem 276:3123–3129[Abstract/Free Full Text]
  13. Emons G, Schally AV 1994 The use of luteinizing hormone releasing hormone agonists and antagonists in gynaecological cancers. Hum Reprod 9:1364–1379[Abstract/Free Full Text]
  14. Eidne KA, Flanagan CA, Harris NS, Millar RP 1987 Gonadotropin-releasing hormone (GnRH)-binding sites in human breast cancer cell lines and inhibitory effects of GnRH antagonists. J Clin Endocrinol Metab 64:425–432[Abstract]
  15. Schally AV, Nagy A 1999 Cancer chemotherapy based on targeting of cytotoxic peptide conjugates to their receptors on tumors. Eur J Endocrinol 141:1–14[Abstract]
  16. Schlick J, Dulieu P, Desvoyes B, Adami P, Radom J, Jouvenot M 2000 Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines. FEBS Lett 472:241–246[CrossRef][Medline]
  17. Kakar SS, Grizzle WE, Neill JD 1994 The nucleotide sequences of human GnRH receptors in breast and ovarian tumors are identical with that found in pituitary. Mol Cell Endocrinol 106:145–149[CrossRef][Medline]
  18. Emons G, Muller V, Ortmann O, Schulz KD 1998 Effects of LHRH-analogues on mitogenic signal transduction in cancer cells. J Steroid Biochem Mol Biol 65:199–206[CrossRef][Medline]
  19. Moretti RM, Marelli MM, Dondi D, Poletti A, Martini L, Motta M, Limonta P 1996 Luteinizing hormone-releasing hormone agonists interfere with the stimulatory actions of epidermal growth factor in human prostatic cancer cell lines, LNCaP and DU 145. J Clin Endocrinol Metab 81:3930–3937[Abstract/Free Full Text]
  20. Limonta P, Marelli MM, Moretti RM 2001 LHRH analogues as anticancer agents: pituitary and extrapituitary sites of action. Expert Opin Investig Drugs 10:709–720[CrossRef][Medline]
  21. Imai A, Horibe S, Takagi A, Tamaya T 1997 Gi protein activation of gonadotropin-releasing hormone-mediated protein dephosphorylation in human endometrial carcinoma. Am J Obstet Gynecol 176:371–376[CrossRef][Medline]
  22. Moretti RM, Montagnani MM, Van Groeninghen JC, Limonta P 2002 Locally expressed LHRH receptors mediate the oncostatic and antimetastatic activity of LHRH agonists on melanoma cells. J Clin Endocrinol Metab 87:3791–3797[Abstract/Free Full Text]
  23. Everest HM, Hislop JN, Harding T, Uney JB, Flynn A, Millar RP, McArdle CA 2001 Signaling and antiproliferative effects mediated by GnRH receptors after expression in breast cancer cells using recombinant adenovirus. Endocrinology 142:4663–4672[Abstract/Free Full Text]
  24. Franklin J, Hislop J, Flynn A, McArdle CA 2003 Signalling and anti-proliferative effects mediated by gonadotrophin-releasing hormone receptors after expression in prostate cancer cells using recombinant adenovirus. J Endocrinol 176:275–284[Abstract]
  25. Millar R, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, Ott T, Millar M, Lincoln G, Sellar R, Faurholm B, Scobie G, Kuestner R, Terasawa E, Katz A 2001 A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci USA 98:9636–9641[Abstract/Free Full Text]
  26. Neill JD, Duck LW, Sellers JC, Musgrove LC 2001 A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem Biophys Res Commun 282:1012–1018[CrossRef][Medline]
  27. Morgan K, Conklin D, Pawson AJ, Sellar R, Ott TR, Millar RP 2003 A transcriptionally active human type II gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the antisense orientation on chromosome 1q.12. Endocrinology 144:423–436[Abstract/Free Full Text]
  28. Millar RP 2002 GnRH II and type II GnRH receptors. Trends Endocrinol Metab 14:35–43
  29. Choi KC, Auersperg N, Leung PC 2001 Expression and antiproliferative effect of a second form of gonadotropin-releasing hormone in normal and neoplastic ovarian surface epithelial cells. J Clin Endocrinol Metab 86:5075–5078[Abstract/Free Full Text]
  30. Grundker C, Gunthert AR, Millar RP, Emons G 2002 Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. J Clin Endocrinol Metab 87:1427–1430[Abstract/Free Full Text]
  31. Rockman HA, Koch WJ, Lefkowitz RJ 2002 Seven-transmembrane-spanning receptors and heart function. Nature 415:206–212[CrossRef][Medline]
  32. Benali N, Ferjoux G, Puente E, Buscail L, Susini C 2000 Somatostatin receptors. Digestion 62(Suppl 1):27–32
  33. Small KM, Brown KM, Forbes SL, Liggett SB 2001 Modification of the {alpha} 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy. J Biol Chem 276:31596–31601[Abstract/Free Full Text]
  34. Graham FL, Prevec L 1995 Methods for construction of adenovirus vectors. Mol Biotechnol 3:207–220[Medline]
  35. Hislop J, Madziva MT, Everest H, Harding TC, Uney JB, Willars G, Millar R, Troskie B, Davidson J, McArdle CA 2000 Desensitization and internalization of human and Xenopus gonadotropin-releasing hormone receptors expressed in aT4 pituitary cells using recombinant adenovirus. Endocrinology 141:4564–4575[Abstract/Free Full Text]
  36. Hislop JN, Everest HM, Flynn A, Harding T, Uney JB, Troskie BE, Millar RP, McArdle CA 2001 Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling. J Biol Chem 276:39685–39694[Abstract/Free Full Text]
  37. Williams B, Brooks AN, Aldridge TC, Pennie WD, Stephenson R, McArdle CA 2000 Jan Oestradiol is a potent mitogen and modulator of GnRH signalling in {alpha} T3–1 cells: are these effects causally related? J Endocrinol 164:31–43[Abstract]
  38. Clayton RN, Catt KJ 1981 Gonadotropin-releasing hormone receptors: characterization, physiological regulation, and relationship to reproductive function. Endocr Rev 2:186–209[Medline]
  39. Davidson J, Flanagan C, Myburgh D, Elario R, Forrest-Owen W, McArdle CA 1996 Addition of an extra glycosylation site enhances expression and coupling of the human gonadotropin-releasing hormone receptor. Endocrine 4:207–212
  40. Huang YT, Hwang JJ, Lee LT, Liebow C, Lee PP, Ke FC, Lo TB, Schally AV, Lee MT 2002 Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells. Int J Cancer 99:505–513[CrossRef][Medline]
  41. Wang Y, Matsuo H, Kurachi O, Maruo T 2002 Down-regulation of proliferation and up-regulation of apoptosis by gonadotropin-releasing hormone agonist in cultured uterine leiomyoma cells. Eur J Endocrinol 146:447–456[Abstract]
  42. Kang SK, Choi KC, Cheng KW, Nathwani PS, Auersperg N, Leung PC 2000 Role of gonadotropin-releasing hormone as an autocrine growth factor in human ovarian surface epithelium. Endocrinology 141:72–80[Abstract/Free Full Text]
  43. Imai A, Tamaya T 2000 GnRH receptor and apoptotic signaling. Vitam Horm 59:1–33[Medline]
  44. Arencibia JM, Schally AV 2000 Luteinizing hormone-releasing hormone as an autocrine growth factor in ES-2 ovarian cancer cell line. Int J Oncol 16:1009–1013[Medline]
  45. Grundker C, Schulz K, Gunthert AR, Emons G 2000 Luteinizing hormone-releasing hormone induces nuclear factor {kappa}B-activation and inhibits apoptosis in ovarian cancer cells. J Clin Endocrinol Metab 85:3815–3820[Abstract/Free Full Text]
  46. Jacobs GF, Flanagan CA, Roeske RW, Millar RP 1995 Agonist activity of mammalian gonadotropin-releasing antagonists in chicken gonadotropes reflects marked differences in vertebrate gonadotropin-releasing receptors. Mol Cell Endocrinol 108:107–113[CrossRef][Medline]
  47. Lin X, Janovick JA, Brothers S, Blomenrohr M, Bogerd J, Conn PM 1998 Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation. Mol Endocrinol 12:161–171[Abstract/Free Full Text]
  48. Blomenrohr M, Heding A, Sellar R, Leurs R, Bogerd J, Eidne KA, Willars GB 1999 Pivotal role for the cytoplasmic carboxyl-terminal tail of a nonmammalian gonadotropin-releasing hormone receptor in cell surface expression, ligand binding, and receptor phosphorylation and internalization. Mol Pharmacol 56:1229–1237[Abstract/Free Full Text]
  49. Pawson AJ, Katz A, Sun YM, Lopes J, Illing N, Millar RP, Davidson JS 1998 Contrasting internalization kinetics of human and chicken gonadotropin-releasing hormone receptors mediated by C-terminal tail. J Endocrinol 156: R9–R12
  50. Maya-Nunez G, Janovick JA, Conn PM 2000 Combined modification of intracellular and extracellular loci on human gonadotropin-releasing hormone receptor provides a mechanism for enhanced expression. Endocrine 13:401–407[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J EndocrinolHome page
A. R Finch, K. R Sedgley, C. J Caunt, and C. A McArdle
Plasma membrane expression of GnRH receptors: regulation by antagonists in breast, prostate, and gonadotrope cell lines
J. Endocrinol., February 1, 2008; 196(2): 353 - 367.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
K. R Sedgley, A. R Finch, C. J Caunt, and C. A McArdle
Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells
J. Endocrinol., December 1, 2006; 191(3): 625 - 636.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. J. Caunt, A. R. Finch, K. R. Sedgley, L. Oakley, L. M. Luttrell, and C. A. McArdle
Arrestin-mediated ERK Activation by Gonadotropin-releasing Hormone Receptors: RECEPTOR-SPECIFIC ACTIVATION MECHANISMS AND COMPARTMENTALIZATION
J. Biol. Chem., February 3, 2006; 281(5): 2701 - 2710.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
K. Wada, L. Hu, N. Mores, C. E. Navarro, H. Fuda, L. Z. Krsmanovic, and K. J. Catt
Serotonin (5-HT) Receptor Subtypes Mediate Specific Modes of 5-HT-Induced Signaling and Regulation of Neurosecretion in Gonadotropin-Releasing Hormone Neurons
Mol. Endocrinol., January 1, 2006; 20(1): 125 - 135.
[Abstract]