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Molecular Endocrinology, doi:10.1210/me.2006-0537
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Molecular Endocrinology 22 (7): 1711-1722
Copyright © 2008 by The Endocrine Society

Gonadotropin-Releasing Hormone Analog Structural Determinants of Selectivity for Inhibition of Cell Growth: Support for the Concept of Ligand-Induced Selective Signaling

Rakel López de Maturana, Adam J. Pawson, Zhi-Liang Lu, Lindsay Davidson, Stuart Maudsley, Kevin Morgan, Simon P. Langdon and Robert P. Millar

Ardana Bioscience (R.L.d.M., R.P.M.), Edinburgh EH3 7HA, United Kingdom; Medical Research Council Human Reproductive Sciences Unit (R.L.d.M., A.J.P., Z.-L.L., L.D., S.M., K.M., R.P.M.), The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom; and Cancer Research Center (S.P.L.), University of Edinburgh, Edinburgh EH4 2XR, United Kingdom

Address all correspondence and requests for reprints to: Prof. R. P. Millar, MRC Human Reproductive Sciences Unit, The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom. E-mail: r.millar{at}hrsu.mrc.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH and its receptor are expressed in human reproductive tract cancers, and direct antiproliferative effects of GnRH analogs have been demonstrated in cancer cell lines. The intracellular signaling responsible for this effect differs from that mediating pituitary gonadotropin secretion. The GnRH structure-activity relationship is different for the two effects. Here we report a structure-activity relationship study of GnRH agonist antiproliferative action in model cell systems of rat and human GnRH receptors stably expressed in HEK293 cells. GnRH II was more potent than GnRH I in inhibiting cell growth in the cell lines. In contrast, GnRH I was more potent than GnRH II in stimulating inositol phosphate production, the signaling pathway in gonadotropes. The different residues in GnRH II (His5, Trp7, Tyr8) were introduced singly or in pairs into GnRH I. Tyr5 replacement by His5 produced the highest increase in the antiproliferative potency of GnRH I. Tyr8 substitution of Arg8 produced the most selective analog, with very poor inositol phosphate generation but high antiproliferative potency. In nude mice bearing tumors of the HEK293 cell line, GnRH II and an antagonist administration was ineffective in inhibiting tumor growth, but D-amino acid stabilized analogs (D-Lys6 and D-Arg6) ablated tumor growth. Docking of GnRH I and GnRH II to the human GnRH receptor molecular model revealed that Arg8 of GnRH I makes contact with Asp302, whereas Tyr8 of GnRH II appears to make different contacts, suggesting these residues stabilize different receptor conformations mediating differential intracellular signaling and effects on gonadotropin and cell growth. These findings provide the basis for the development of selective GnRH analog cancer therapeutics that directly target tumor cells or inhibit pituitary gonadotropins or do both.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH I IS THE PRIMARY regulator of the reproductive system through stimulation of pituitary gonadotropin secretion and consequent stimulation of gametogenesis and steroidogenesis (1, 2). Androgen and estrogen are end products of this cascade and stimulate accessory reproductive tissues, such as the prostate and breast in both the normal and neoplastic states. The pituitary GnRH receptor (type I) is a G protein-coupled receptor that couples preferentially to G{alpha}q to stimulate Ca2+, protein kinase C, and MAPK signaling pathways (1, 3, 4). Prolonged GnRH receptor stimulation with agonists results in gonadotrope desensitization and a decrease in circulating biologically active gonadotropin, which culminates in reduced steroid hormone production. GnRH agonist analogs have therefore found wide and extensive therapeutic application in hormone-dependent diseases including endometriosis, fibroids, and breast and prostatic cancers (5, 6, 7, 8, 9, 10, 11).

Positive responses of breast cancer to GnRH analogs in postmenopausal women (12) suggested that GnRH analogs may also have direct antiproliferative effects that are independent of their actions in decreasing sex steroid hormones. The presence of GnRH receptors in breast cancer tissue (13) and the demonstration of antiproliferative actions of GnRH analogs in breast cancer cell lines (14, 15) supported this suggestion. GnRHRs and antiproliferative effects of GnRH analogs have also been shown in a number of cell lines of reproductive tract tumors, including prostate, uterine, and ovarian cancers, and also in non-reproductive-tract tumors (7, 8, 9, 16, 17, 18). In contrast to GnRH actions at the pituitary, which are mediated through the G{alpha}q protein, these antiproliferative and apoptotic effects on tumor cells are thought to be mediated via the G{alpha}i protein, focal adhesion complexes involving c-Src, and the p38 and c-Jun N-terminal kinase stress-activated kinases (17, 18, 19, 20, 21, 22).

Additional intracellular mechanisms have been implicated in the antiproliferative effects, including the down-regulation of growth factor actions (by decreased expression of growth factors and their receptors and activation of phosphotyrosine phosphatase) and the inhibition of Akt and the 60S acidic ribosomal phosphoproteins (restraining cell survival and protein synthesis, respectively) (8, 21, 22, 23, 24, 25, 26, 27, 28). We have recently demonstrated that the pharmacology of GnRH analog effects on antiproliferation and apoptosis is distinctly different from their effects on gonadotropin secretion and attributed this to the preferential activation of different signaling cascades by certain analogs (21). We have dubbed the phenomenon ligand-induced selective signaling (LiSS) (1, 21, 29). The extrahypothalamic form of GnRH (GnRH II) (30, 31) is less potent than GnRH I in stimulating gonadotropin synthesis but more potent in inhibiting cell growth (32). The most persuasive demonstration of the phenomenon is manifest in certain antagonists of pituitary GnRH receptors that have similar activity to GnRH agonists in inducing inhibition of tumor cell lines (14, 21, 32, 33, 34).

Until now, research on the structure-activity relationship (SAR) of GnRH analogs has focused on the ability of ligands to modulate G{alpha}q activation, which stimulates gonadotropin secretion. Because antiproliferative and apoptotic effects are mediated by different signaling pathways from those stimulating gonadotropins, we have undertaken a SAR study on the inhibition of cell number by GnRH analogs using HEK293 cells expressing either the rat or the human GnRH receptor. Because GnRH II has been previously shown to be more potent than GnRH I, the three different amino acids in GnRH II were systematically incorporated in GnRH I and the relative antiproliferative and inositol phosphate (IP) production compared. The findings show that [His5] GnRH I is the most potent at inhibiting cell number and indicate that [Tyr8] GnRH I has the highest selectivity for this effect.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of GnRH I and GnRH II on DNA Synthesis and Apoptosis
We investigated whether the growth suppression caused by GnRH on HEK293 cells expressing the rat receptor (HEK293/rGnRHR cell line) was due to an inhibition of cell proliferation and/or an induction of apoptosis. Radioactive thymidine incorporation into DNA was used to monitor cell proliferation over a period of 96 h. Both GnRH I and GnRH II (100 nM each) decreased thymidine incorporation, GnRH II being more effective (Fig. 1AGo). Apoptosis was followed by monitoring cleavage of poly(ADP-ribose)polymerase (PARP), a substrate of caspase 3. Using this output, GnRH II (100 nM) induction of apoptosis was apparent by 24 h after commencing treatment, whereas GnRH I effects only occurred by 48 h (Fig. 1BGo). The increase of cleaved PARP in the cytoplasm was proportional to the increase of cleaved PARP in the nucleus (data not shown). Thus, the well-described inhibition of cell number by GnRH analogs is the product of inhibition of proliferation and induction of apoptosis.


Figure 1
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  		Fig. 1. Effects of GnRH I and GnRH II on DNA Synthesis and Apoptosis in HEK293 Cells Stably Expressing the Rat GnRH Receptor

A, Time course of thymidine incorporation into HEK293/rGnRHR cells exposed to 100 nM GnRH I or GnRH II. Control cells were untreated. The graph shown is one of two separate experiments that yielded similar results. Each point represents the mean ± SEM (n = 3). B, Time course of PARP cleavage in HEK293/rGnRHR cells exposed to 100 nM GnRH I or GnRH II. The strips show the signals obtained in crude cytoplasmic protein extracts in a representative Western blot using specific anti-cleaved PARP antibody. The data shown are representative of two separate experiments that produced similar results.

 
Determination of SAR of GnRH Analogs for Inhibition of Cell Number
Because GnRH II appeared to be more effective in inhibiting proliferation and stimulation of apoptosis than GnRH I, we explored the structural basis of the difference. The decapeptides GnRH I and GnRH II differ in three amino acids. The three differing amino acids, His5, Trp7, and Tyr8 of GnRH II, were incorporated individually or in pairs into the GnRH I sequence, resulting in six different analogs structurally intermediate between GnRH I and GnRH II. The net effect of inhibition of proliferation and stimulation of apoptosis was monitored by determining cell number after 5 d of continuous exposure to the peptides (replenished every 12 h) in two HEK293 cell lines, one expressing the rat GnRH receptor and the other expressing the human GnRH receptor (HEK293/rGnRHR and HEK293/hGnRHR, respectively). The cell line expressing the human GnRH receptor was included in the study because of its clinical relevance and low receptor expression, which better reflects human cancer tissue and many cancer cell lines that respond to GnRH.

Comparative Inhibition of Cell Number, Receptor Binding, and IP Production
The affinities of all analogs correlated with their potency in stimulating IP accumulation but not with their potency to inhibit cell number (Tables 1Go and 2Go and Figs. 2–4GoGoGo). To illustrate this, at the rat receptor, GnRH I was 4.7-fold more potent in generating IP than GnRH II, but GnRH II was 13.0-fold more potent in inhibiting cell number (Table 1Go and Figs. 3AGo and 4AGo). Similarly, at the human receptor, GnRH I was 6.2-fold more potent in generating IP than GnRH II, but GnRH II was 6.7-fold more potent in inhibiting cell number (Table 2Go and Figs. 3CGo and 4CGo). Substitution of Tyr5 by and His5 resulted in increased affinity (Fig. 2BGo), which correlated with higher potencies for both of the responses studied (Tables 1Go and 2Go and Figs. 3Go, B–D, and 4Go, B–D). Although [His5] GnRH had a higher affinity than GnRH I, the difference did not achieve statistical significance (P = 0.06). We therefore repeated the binding study at the human receptor. The IC50 of [His5] GnRH (0.86 ± 0.25 nM) was significantly lower (P < 0.005) than that of GnRH I (2.75 ± 0.21 nM). The introduction of Trp7 into GnRH I did not significantly modify the affinity and IP production (Tables 1Go and 2Go and Figs. 2BGo and 3Go, B–D). However, [Trp7] GnRH I is 9.1-fold and 19.3-fold more potent than GnRH I in inhibiting cell number via the rat and human receptors, respectively (Tables 1Go and 2Go and Fig. 4Go, B–D). The substitution of Arg8 in GnRH I by Tyr8 was the single change that resulted in the most selective inhibitor of cell number. At the rat receptor, [Tyr8] GnRH I was 27.3-fold less potent in IP generation but 4.2-fold more potent in inhibiting cell number, compared with GnRH I (Table 1Go and Figs. 3BGo and 4BGo). Analogously, at the human receptor, this analog was 23.7-fold less potent in IP generation but 10.7-fold more potent in inhibiting cell number, again relative to the native peptide (Table 2Go and Figs. 3DGo and 4DGo). Notably, although this analog shows a very low affinity (Tables 1Go and 2Go and Fig. 2BGo), it is still more potent than GnRH I in inhibiting cell number. It therefore appears to be the most selective analog for inhibiting cell number. The two double-substitution peptides that incorporate His at position 5, namely [His5,Trp7] GnRH I and [His5,Tyr8] GnRH I, display phenotypes approximating the product of the single substitutions. Thus, [His5,Trp7] GnRH I has similar affinity and potency in IP generation to that of GnRH I but improved potency in inhibition of cell number (Tables 1Go and 2Go and Figs. 2BGo, 3Go, B–D, and 4Go, B–D). [His5,Tyr8] GnRH I shows features intermediate between GnRH I and [Tyr8] GnRH I (Tables 1Go and 2Go and Figs. 2BGo, 3Go, B–D, and 4Go, B–D). The loss of affinity due to the Tyr8 substitution and consequent decreased potency in IP generation seems to be partly rescued by the introduction of His5, making [His8,Tyr8] GnRH I more potent in inhibiting cell number than GnRH I (and [Tyr8] GnRH I). Finally, the features of [Trp7,Tyr8] GnRH I are almost identical to those of [Tyr8] GnRH I (Tables 1Go and 2Go and Figs. 2BGo, 3Go, B–D, and 4Go, B–D).


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  		Table 1. Receptor Binding (IC50), IP Accumulation, and Inhibition of Cell Number by GnRHs and Analogs in HEK293 Cells Stably Expressing the Rat GnRH Receptor

 

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  		Table 2. IP Accumulation and Inhibition of Cell Number by GnRHs and Analogs in HEK293 Cells Stably Expressing the Human GnRH Receptor

 

Figure 2
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  		Fig. 2. Binding of GnRHs and Analogs to HEK293 Cells Stably Expressing the Rat GnRH Receptor

A, Competition binding curves for GnRH I and GnRH II in HEK293/rGnRHR cells. The curves represent one of at least three independent experiments in which each point represents the mean of triplicate values with SEM displayed as error bars. Counts were normalized to the maximal specific binding within each data set. Means of all studies are shown in Table 1Go. B, Competition binding curves for GnRH analogs in HEK293/rGnRHR cells. The curves represent one of three or more independent experiments in which each point represents the mean of triplicate values with SEM displayed as error bars. Counts were normalized to the maximal specific binding within each data set. Means of all studies are shown in Table 1Go.

 

Figure 3
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  		Fig. 3. Stimulation of IPs by GnRHs and Analogs in HEK293 Cells Stably Expressing the Rat and Human GnRH Receptors

A, Total IP accumulation curves for GnRH I and GnRH II stimulation of HEK293/rGnRHR cells. The curves represent one of three or more independent experiments in which each point represents the mean of triplicate values with SEM displayed as error bars. Counts were normalized to the maximal response within each data set. Mean data are shown in Tables 1Go and 2Go. B, Total IP accumulation curves for GnRH analog stimulation of HEK293/rGnRHR cells. The curves represent one of three or more independent experiments in which each point represents the mean of triplicate values with SEM displayed as error bars. Counts were normalized to the maximal response within each data set. Mean data are shown in Tables 1Go and 2Go. C, Total IPs accumulation curves for GnRH I and GnRH II stimulation of HEK293/hGnRHR cells. The curves represent one of three independent experiments in which each point represents the mean of sextuplicate values with SEM displayed as error bars. Counts were normalized to the maximal response within each data set. Mean data are shown in Tables 1Go and 2Go. D, Total IP accumulation curves for GnRH analogs in HEK293/hGnRHR cells. The curves represent one of three or more independent experiments in which each point represents the mean of sextuplicate values with SEM displayed as error bars. Counts were normalized to the maximal response within each data set. Mean data are shown in Tables 1Go and 2Go.

 

Figure 4
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  		Fig. 4. Effect of GnRHs and Analogs on the Inhibition of Cell Number in HEK293 Cells Stably Expressing the Rat and Human GnRH Receptors

A, HEK293/rGnRHR cells were continuously treated with different concentrations of GnRH I and GnRH II for 5 d (with peptide replenishment every 12 h) and then viable cells assayed after WST-1 incubation. The curves represent one of three or more independent experiments in which each point represents the mean of triplicate values with S.E.M displayed as error bars. Data are presented as a percentage of the number of viable cells in untreated controls. Mean data are shown in Tables 1Go and 2Go. B, HEK293/rGnRHR cells were continuously treated with GnRH analogs for 5 days and then viable cells assayed after WST-1 incubation. The curves represent one of three independent experiments in which each point represents the mean of triplicate values with SEM displayed as error bars. Data are presented as a percentage of the number of viable cells in untreated controls. Mean data are shown in Tables 1Go and 2Go.C, HEK293/hGnRHR cells were continuously treated with GnRH I and GnRH II for 5 d and then viable cells assayed after WST-1 incubation. The curves represent one of three or more independent experiments in which each point represents the mean of triplicate values with SEM displayed as error bars. Data are presented as a percentage of the number of viable cells in untreated controls. Mean data are shown in Tables 1Go and 2Go. D, HEK293/hGnRHR cells were continuously treated with GnRH analogs for 5 d and then viable cells assayed after WST-1 incubation. The curves represent one of three or more independent experiments in which each point represents the mean of triplicate values with SEM displayed as error bars. Values were normalized to the number of cells that had been left untreated. Data are presented as a percentage of the number of viable cells in untreated controls. Mean data are shown in Tables 1Go and 2Go.

 
Inhibition of Tumor Growth in Nude Mice
To determine whether the inhibitory effects of GnRH analogs translated to in vivo effects, tumors of the HEK293 cell line stably expressing the rat GnRH receptor were transplanted sc in nude mice that were then treated with GnRH analogs. We first tested GnRH II, but it was ineffective (Fig. 5Go). We reasoned that this might be due to rapid degradation and metabolic clearance of the native GnRH (35, 36). We therefore tested GnRH II analogs with Gly6 substituted with D-amino acids that are known to protect against degradation (35, 36). D-Arg6 GnRH II halted tumor growth, whereas D-Lys6 GnRH II actually caused regression of tumors (Fig. 5Go). A GnRH antagonist (antide) was ineffective (Fig. 5Go).


Figure 5
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  		Fig. 5. Effects of GnRH Analogs on Tumor Growth in Nude Mice

HEK293 cells stably expressing the rat GnRH receptor were used to generate tumor xenografts in female nude mice. At least five animals per group were then treated daily with vehicle or GnRH analog, and tumor growth was measured. Antide (A) or GnRH II (equivalent to [His5,Trp7,Tyr8] GnRH I) (B) did not affect tumor growth, whereas [His5,D-Arg6,Trp7,Tyr8] GnRH I (C) and [His5,D-Lys6,Trp7,Tyr8] GnRH I significantly halted tumor growth (C) and induced tumor size regression (D), respectively.

 
Interactions of GnRH I and GnRH II with the Human GnRH Receptor
A β II' turn conformation of GnRH I derived from the nuclear magnetic resonance (NMR) structure and GnRH II were successfully docked to the receptor model to accommodate the experimentally confirmed interactions (Fig. 6Go). Both peptides exhibited the same receptor interactions between pGlu1 with Asn212(5.39), His2 with Asp98(2.61)/Lys121(3.32), and Pro9-Gly10NH2 with Asn102(2.65). A distinct difference was the interaction of Arg8 of GnRH I with Asp320(7.32), whereas Tyr8 of GnRH II faces away from this residue and toward extracellular loop 2 where it could make contact with His182 and His199.


Figure 6
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  		Fig. 6. Molecular Docking of GnRH I (A) and GnRH II (B) into the Human GnRH Receptor Model

The human GnRH receptor model was built using the crystal structure of a photoactivated deprotonated intermediate of bovine rhodopsin (61 ) as a template. GnRH I and GnRH II were docked into the model as described previously (54 55 62 ). Arg8 of GnRH I makes intermolecular interactions with Asp302 (green) in the third extracellular loop, but Tyr8 (yellow) of GnRH II faces away toward the second extracellular loop and may make intermolecular interactions with His182 and His199 (yellow). Only certain experimentally identified receptor inter- and intramolecular interactions are shown for clarity. These include H-bonds between Lys121 and His2 and between Asn102 and Gly10NH2 and between Asp98 and Lys121 (green and orange).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
GnRH analogs are widely employed to treat hormone-dependent diseases through their inhibition of gonadotropin and sex steroid hormones. However, this therapy of reproductive tissue cancers frequently leads to recurrence of hormone-insensitive disease. Since the original observations of direct antiproliferative effects of GnRH analogs on cancer cell lines 20 yr ago (14, 15), a substantial supportive literature on many cancer cell lines has emerged (7, 8, 9, 17, 21, 28, 37). The pharmacology of these effects by GnRH analogs is distinctly different from that on gonadotropin secretion. Initially, this was thought to be due to the existence of a GnRH receptor isoform (type II GnRHR). However, the human type II receptor was shown to be silenced due to a nucleotide deletion and frame shift as well as a premature stop codon (38, 39). The conundrum was resolved by our demonstration that the abnormal (nongonadotrope) pharmacology for antiproliferative effects could be demonstrated in HEK293 cells in which the type I GnRH receptor was stably transfected (21). This led us to propose the concept of LiSS in which the single type I GnRH receptor can take up different active conformations associated with different ligand analog-binding pharmacology and differential intracellular signaling (29). Our current studies with GnRH I, GnRH II, and analogs support this concept.

The development of more potent and more selective GnRH analogs provides the potential for improved cancer therapy. Until now, research on the SAR in the GnRH system had focused on the ability of ligands to modulate G{alpha}q, which is the chief regulatory pathway of gonadotropin synthesis and secretion in the gonadotrope (1, 2, 3). Because this is apparently not the predominant effector pathway leading to antiproliferation and apoptosis in a number of cancer cell lines, it is necessary to monitor other outputs in the pursuit of ligands with potent and selective cell growth inhibition. In this report, we have therefore studied the binding affinity and potency of IP stimulation by a series of analogs in conjunction with the ligand structural requirements to inhibit cell numbers using our model system of HEK293 cells expressing either the rat or the human GnRH receptor. The demonstration that certain GnRH antagonists inhibit cell growth in these model cell lines with a similar pharmacology to that seen by us in choriocarcinoma and benign prostatic hyperplasia cell lines (21) and in other cell lines (28, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45) motivated us to use this more robust model system to study GnRH analog antiproliferative SAR.

We have shown that GnRH inhibition of cell number is contributed to by an inhibition of cell proliferation along with induction of apoptosis. This is in line with previous reports of GnRH action on these cells and in various cancer cell lines (7, 8, 9, 17, 21, 28, 37, 40, 41, 42). Notably, Miles et al. (40) studied the effect of a prolonged treatment of the same HEK293 cells stably expressing the rat and human receptors with GnRH I and found a clear agonist dose-dependent inhibition of thymidine incorporation, along with a modest induction of apoptosis and cell cycle arrest, as revealed by flow cytometry. Because the net effect of antiproliferative and apoptotic effects is a decrease in total cell number, we have used this as the measure of GnRH analog effects.

The affinity of all peptides in the current study accord with previous reports using rat, sheep, and human anterior pituitary membranes or COS-7 and HEK293 cells transiently expressing the rat, sheep, or human receptors (1, 40, 43, 44, 45). Compared with GnRH I, GnRH II displayed a lower potency to induce IP accumulation but a much higher potency in inhibiting cell number. Substitution of Tyr5 by His5 in GnRH I resulted in an increase in affinity at the rat receptor, which correlated with a higher potency in the two responses studied. This substitution is thus a novel change that increases binding affinity. Moreover, it is the first natural (L) amino acid substitution shown to enhance GnRH binding affinity. All previously described agonists with increased affinity incorporate substitution of Gly6 with D-amino acids and Gly10 NH2 with N-ethylamide. These amino acid substitutions increase binding affinity and form the basis of all the super-active analogs used clinically (35, 36, 46). We did not conduct binding studies on GnRH I and [His5] GnRH I in the HEK293 cells stably expressing the human GnRH receptor because expression was too low to obtain accurate figures. To confirm that the His5 substitution increases affinity as for the rat GnRH receptor, we conducted studies on binding in COS-1 cells transfected with the human GnRH receptor (47). [His5] GnRH I had an IC50 of 0.86 ± 0.25 nM which was significantly lower (P < 0.005; n = 6) than the IC50 of GnRH I (2.75 ± 0.21 nM).

[His5] GnRH I is the most potent L-amino acid-substituted GnRH analog suppressor of cell growth reported to date. However, it is not the most selective, because it was also very potent in stimulating IP production. Replacing Leu7 by Trp7 led to an analog with the same potency in IP synthesis but higher potency in inhibiting cell number compared with GnRH I. Substitution of Arg8 by Tyr8 produced the most selective antiproliferative/apoptotic agent. Relative to GnRH I, it exhibited a more than 20-fold lower binding affinity and potency in stimulating IP accumulation. However, [Tyr8] GnRH I was 4- to 11-fold more potent in inhibiting cell number. Other GnRH analogs exhibited functional parameters expected from their intermediate structural characteristics between GnRH I and GnRH II.

Because the binding studies and stimulation of inositol stimulation by analogs are acute studies (1–4 h) and the inhibition of cell number is a chronic study (5 d), differential degradation of analogs could contribute to differential activities in the assays. To address this possibility, we replenished the peptides every 12 h in the 5-d study. We also assayed peptide concentrations in the medium during incubation using the receptor binding assay and showed there was no change (data not shown). We further compared the inhibition of cell number by GnRH I in the presence and absence of bacitracin, an inhibitor of GnRH proteolysis (36). The reduction in cell number by GnRH I alone was 46 ± 9% (n =3), which was similar to that observed in the presence of bacitracin 38 ± 9% (n =3). Thus, differential degradation of the GnRH analogs does not contribute to the differential effects on IP production and inhibition of cell number.

What is the explanation for the enhanced selective inhibition of cell number with the substitution of Arg8 by Tyr8 in GnRH I? We propose that the substitution of Arg8 with Tyr8 enables the ligand and receptor to adopt new conformations that better stabilize the receptor in the active state that mediates the antiproliferative/apoptotic effect. In an attempt to understand this, we have compared the structural interactions of GnRH I and GnRH II with the refined molecular model of the GnRH receptor (47). Mammalian GnRH I and GnRH II are perceived as being comprised of three structural domains (1, 35, 48). The N-terminal Glu1-His2-Trp3-Ser4 and C-terminal Pro9-Gly10NH2 sequences have been conserved over 500 million years (1) and are important for receptor binding and the consequent receptor activation. The middle β-II' turn domain is much less conserved among species and among the various GnRH peptide isoforms present in the same species. This region corresponds to Tyr5-Gly6-Leu7-Arg8 in GnRH I and to His5-Gly6-Trp7-Tyr8 in GnRH II. The high-affinity interaction of GnRH I with the type I mammalian GnRHR requires a β-II' turn conformation of the ligand involving these residues (1, 35, 36, 46, 49), which can be stabilized by D-amino acid substitutions. Arg8 has been shown to interact with the conserved Asp302 in the third extracellular loop of the mammalian GnRH receptor, which induces or selects the β-II' turn conformation (50, 51). There is evidence that GnRH II is preconfigured in the β-II' turn conformation, which accounts for its relatively high affinity for all GnRH receptors (43).

When the three-dimensional GnRH I structure derived from NMR studies (52, 53) and PDB code 1YY1 is docked to the cognate receptor binding sites in our recently refined receptor model (54), Arg8 satisfactorily interacts with Asp302 (Fig. 6Go). However, when GnRH II is docked to the receptor, Tyr8 faces away from Asp302 and potentially makes contact with His182 and His199 and possibly other residues in extracellular loop 2. Future studies on the effects of mutating these residues will confirm or negate this proposition. Thus, the findings that substitution of Arg8 with Tyr gives rise to a decrease in IP generation but an increase in the inhibition of cell number suggests that Arg8 stabilizes a receptor conformation with preferential G{alpha}q coupling, whereas its substitution with Tyr favors a receptor conformation associated with signaling to proapoptotic and antiproliferative pathways. Thus, the current study further supports the LiSS concept previously proposed for the GnRH receptor system (21, 29). This is exemplified by the natural GnRH I and GnRH II ligands, which show inverted potency ratios for the stimulation of IP synthesis and inhibition of cell number, and this has been further refined in the [Tyr8] GnRH I analog.

Because the LiSS concept invokes the existence of different active conformations of GnRH receptor that are preferentially stabilized by specific GnRH analogs and preferably recruit different signaling pathways, it should be possible to produce GnRH receptor mutants that change ligand selectivity. We have mutated a series of transmembrane domain residues that are predicted to interact in our pursuit of creating different receptor conformations. Some of these mutations did indeed change ligand selectivity because they increased the binding affinity of GnRH II about 10-fold without significantly affecting GnRH I affinity (47, 55). Furthermore, the studies showed that Tyr8 was the residue responsible for the increased affinity of the mutant receptors. These findings parallel the SAR for the inhibition of cell number in the current study and suggest that the conformation of the receptor stabilized by these mutations is the same as that which mediates the inhibition of cell number.

It is interesting that all of the analogs exhibited preferential inhibition of cell number over IP production when compared with GnRH I. This is anticipated because they all incorporated substitutions of amino acids in GnRH II, which is about 10 times more potent than GnRH I in inhibiting cell number but less potent in stimulating IP production. However, in a new wider series of analogs we have begun to characterize, there are some that exhibit preferential IP stimulation over inhibition of cell number.

A crucial question is whether or not the in vitro effects of the GnRH analogs translate to effects in vivo. We therefore first tested the ability of GnRH II to inhibit tumors derived from the HEK293 cells stably expressing the rat GnRH receptor but found no effect. Because native GnRHs are known to be rapidly degraded, we then tested D-Arg and D-Lys6 GnRH II analogs that are protected from degradation (35, 36). D-Arg6 GnRH II halted tumor growth, and D-Lys6 GnRH II impressively caused tumor regression. These effects are evidently directly on the tumors and not through steroid hormone inhibition. First, the HEK293 cells lack sex steroid hormone receptors. Second, the antagonist, antide, which has no direct effects in vitro but inhibits steroid hormones, was ineffective on the tumors. It is therefore evident that the SARs we have determined for GnRH analog effects in vitro must now be accompanied by D-amino acid substitutions in position six to create analogs with in vivo efficacy. We are currently pursuing this approach and aim to extend the study to human reproductive-tract tumors that express GnRH receptors.

In conclusion, we have systematically explored the structural features of GnRH that convey preferential inhibition of cell growth compared with the stimulation of IP production. His5 substitution for Tyr5 results in high potency for both outputs, with a degree of preferential antiproliferative effect. Tyr8 substitution for Arg8 results in reduced potency for IP generation but increased antiproliferative potency at both the rat and human receptor, thus producing the most selective analog for inhibition of cell growth. These analogs thus provide useful molecules for studying recruitment of different signaling pathways in research and are also a point of departure for developing selective analogs for clinical application.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
GnRH I and GnRH II were purchased from Sigma-Aldrich Co. Ltd. (Poole, Dorset, UK). GnRH analogs were synthesized using solid-phase synthesis and were purified using reverse-phase HPLC to more than 98% purity as previously described (45, 51). Anti-cleaved PARP antibody (Asp214/Gly215; human specific) was from Cell Signaling Technology, Inc. (Beverly, MA). Anti-ERK2 and secondary antibodies were from Sigma.

Cell Culture
The HEK293 cell lines stably expressing the rat and human GnRH receptors (named HEK293/rGnRHR and HEK293/hGnRHR, respectively) were produced and used as previously described (56, 57). Cells were maintained in DMEM (Sigma) supplemented with 10% fetal bovine serum, 2% glutamine, and 1% penicillin (10,000 U/ml)/streptomycin (10,000 µg/ml) at 37 C in a humidified 5% CO2 atmosphere. Cell treatments were performed at 37 C in serum-containing medium with varying GnRH and GnRH analog concentrations and time periods, as indicated in the figure legends.

Assay for Cell Number
HEK293/rGnRHR and HEK293/hGnRHR cells were seeded into 96-well plates at 5000 cells (in 100 µl) per well and cultured with continuous agonist exposure (peptide replenishment every 12 h) for 5 d as previously described (21). After 5 d, 10 µl WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate} reagent (Roche Diagnostics Ltd., Lewes, East Sussex, UK) was added directly to each well, and after 3 h at 37 C, absorbance was read at 450 nm (with a reference at 690 nm) against a background control as blank using a microplate ELISA reader. We compared this index with manual counting of cell number and showed they were directly correlated.

Thymidine Incorporation Assay
HEK293/rGnRHR cells were seeded into 24-well poly-L-lysine-coated plates and cultured in the presence of 100 nM GnRH I or GnRH II for 24, 48, or 96 h (fresh peptide was applied every day). At the indicated time intervals, medium was removed and 0.5 µCi [3H]thymidine (GE Healthcare, Munich, Germany) in complete fresh medium was added to each well. After an overnight incubation, the medium was removed, the cells washed three times with PBS, fixed in 5% trichloroacetic acid for 15 min at room temperature, and centrifuged. The supernatant was removed, precipitate was dissolved in 0.5 ml 0.1 N NaOH, transferred to a scintillation vial with 2 ml Optiphase HiSafe 3 cocktail (PerkinElmer, Wellesley, MA), and 3H counts were measured in a liquid scintillation 1450 Wallac MicroBeta TriLux counter (GMI Inc., Minneapolis, MN).

Immunoblotting
After stimulation of HEK293/rGnRHR cells with GnRH I or GnRH II (100 nM), cell monolayers were placed on ice, washed twice in ice-cold Dulbecco’s PBS, and lysed in an Nonidet P-40-based solubilization buffer described previously (21, 58). Solubilized lysates were clarified by centrifugation at 15,000 rpm for 15 min. Lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane (NEN Life Sciences, Boston, MA) for protein immunoblotting. Polyvinylidene difluoride membranes were blocked in a 4% BSA, 50 mM Tris-HCl (pH 7.0), 0.05% Tween 20, and 0.05% Nonidet P-40 blocking solution. Caspase 3 activation was determined by incubating the membrane with rabbit anti-cleaved PARP antibody (Cell Signaling) at a 1:1000 dilution at 4 C with gentle shaking overnight. ERK2 was detected with a 1:1000 dilution of the antibody (New England Biolabs Ltd., Hitchin, Hertfordshire, UK). An alkaline phosphatase-conjugated IgG (Sigma) was employed as a secondary antibody for both anti-PARP and anti-ERK2. Visualization of alkaline phosphatase-labeled proteins was performed using enzyme-linked chemifluorescence (GE Healthcare) and quantified using a Typhoon 9400 Phosphorimager (GE Healthcare).

Binding Assay
Specific binding of 80 pM 125I-labeled [His5,D-Tyr6] GnRH I to HEK293 cells expressing GnRH receptor was calculated as the difference between the amount of labeled GnRH I bound in the absence and presence of various doses of unlabeled ligands (59). Monolayers of HEK293 cells stably expressing human or rat GnRH receptors on 12-well poly L-lysine-coated plates were incubated in binding buffer (10 mM HEPES, 1% BSA in DMEM) containing 125I-labeled [His5,D-Tyr5] GnRH I (100,000 cpm) and 10–6 to 10–2 M unlabeled ligand. After incubation for 4 h at 4 C, cells were washed and then lysed in 0.1 M NaOH, and the radioactivity in the extract was measured as described above.

Accumulation of Total Inositol Phosphates (IPs)
HEK293/rGnRHR and HEK293/hGnRHR cells growing on 12-well poly-L-lysine-coated plates were prelabeled with 1 µCi/ml myo-[H3]inositol (GE Healthcare) in inositol-free DMEM (Sigma) for 48 h. Cells were then washed with assay buffer (140 mM NaCl, 20 mM HEPES, 8 mM glucose, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mg/ml BSA), preincubated with 10 mM LiCl (in assay buffer) for 30 min, and stimulated with different concentrations of the peptides (in the same buffer) for 1 h at 37 C. Incubations were terminated by removing the culture medium and lysing the cells in ice-cold 10 mM formic acid for 30 min. IPs were separated by chromatography with an anion exchange resin (AG 1-X8; Bio-Rad, Hemel Hempstead, UK) by elution with 1 M ammonium formate and 0.1 M formic acid (60). Scintillant was added to the eluate, and [3H]IPs were measured in the {gamma}-counter (1450 Wallac MicroBeta counter).

Inhibition of Tumor Growth in Nude Mice
Cultured cells were grown in vitro in the presence of 500 µg/ml G418 before implantation into nude mice. Cells (5–10 million) were implanted sc into the flanks of groups of adult female nude mice. Pharmacological treatments (daily doses of GnRH analog, 10 µg/d in 20% propylene glycol, or vehicle) were initiated when tumors were 50–100 mm3 in size. At least five animals bearing bilateral tumors were studied per treatment group. Tumor dimensions were measured in two diameters using calipers and volumes calculated, where V = {pi} x D x d2/6, where D and d represent the larger and smaller diameters, respectively. Tumor volumes were expressed as a ratio relative to the volume on d 0 of treatment. Changes in mean tumor volumes were plotted using Prism software (GraphPad, San Diego, CA).

Data Transformation and Analysis
Bar graphs and curves were generated using Prism 3.0 (GraphPad). IC50 and EC50 values were determined by nonlinear regression analysis. Curves were best-fitted to a one-site model. The figures shown represent one of at least three independent experiments for which each point represents the mean of three to six values with SEM displayed as error bars, unless otherwise stated. Values were normalized as specified in the figure legends. Statistical significances were assessed by unpaired t test analysis using GraphPad.

GnRH Docking to the Human GnRH Receptor
The human GnRH receptor model was built by homology modeling through MODELER within DS Modeling (version 1.6; Accelrys, San Diego, CA) as described previously (54, 55) using the crystal structure of a photoactivated deprotonated intermediate state of bovine rhodopsin (PDB code 2137) (61) as a template. A βII'-type turn conformation of GnRH I (derived from an NMR structure, PDB code 1YY1) and of GnRH II was docked into the model (1, 54, 62) according to the experimentally identified or putative contact points between GnRH and receptor, i.e. pGlu1 with Asn212(5.39) (63), His2 with Asp98(2.61)/Lys121(3.32) (64), and Gly10NH2 with Asn102(2.65) (65). The GnRH-receptor complex was then optimized by energy-minimization and MD simulations of 150 psec by means of the CHARMM program (66) using a similar set-up as described for the oxytocin receptor (67) with harmonic restraints on the receptor backbone atoms, except for extracellular loop 2 and its covalently linked N-terminal domain (55).


   FOOTNOTES
 
Present address for R.L.d.M.: Centre for Applied Medical Research (CIMA), University of Navarra, Division of Neuroscience, Pamplona-Iruñea 31008, Spain.

Present address for S.M.: National Institute on Aging, Johns Hopkins Medical Center, Gerontology Research Center, 5600 Nathan Shock Drive, Baltimore, Maryland 21224.

Disclosure Summary: The authors have nothing to declare.

First Published Online May 8, 2008

Abbreviations: IP, Inositol phosphate; LiSS, ligand-induced selective signaling; NMR, nuclear magnetic resonance; PARP, poly[ADP-ribose]polymerase; SAR, structure-activity relationship; WST-1, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate.

Received for publication December 15, 2006. Accepted for publication May 1, 2008.


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