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Molecular Endocrinology, doi:10.1210/me.2006-0150
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Molecular Endocrinology 21 (1): 281-292
Copyright © 2007 by The Endocrine Society

Conserved Amino Acid Residues that Are Important for Ligand Binding in the Type I Gonadotropin-Releasing Hormone (GnRH) Receptor Are Required for High Potency of GnRH II at the Type II GnRH Receptor

Sipho Mamputha, Zhi-liang Lu, Roger W. Roeske, Robert P. Millar, Arieh A. Katz and Colleen A. Flanagan

Medical Research Council/University of Cape Town Research Group for Receptor Biology, Institute for Infectious Diseases and Molecular Medicine and Division of Medical Biochemistry (S.M., R.P.M., A.A.K., C.A.F.), University of Cape Town Faculty of Health Sciences, and Department of Medicine, Groote Schuur Hospital (C.A.F.), Cape Town, South Africa; Medical Research Council Human Reproductive Sciences Unit (Z.L., R.P.M.), The Queen’s Medical Research Institute, Edinburgh EH 16 4TJ, Scotland, United Kingdom; and Indiana University School of Medicine (R.W.R.), Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Dr. Colleen A. Flanagan, Division of Medical Biochemistry, University of Cape Town Faculty of Health Sciences, Private Bag X3, Observatory, 7935, South Africa. E-mail: flanagan{at}curie.uct.ac.za.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the C-O group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE DECAPEPTIDE HORMONE, GnRH I is the central regulator of reproduction. Its high-affinity binding to receptors, designated type I GnRH receptors, leads to synthesis and release of the gonadotropic hormones, which regulate the gonads. A second form of GnRH, GnRH II, is widely conserved among vertebrates, including humans and nonhuman primates (1, 2, 3). GnRH II is found in the central nervous system and a range of peripheral tissues (2, 3, 4, 5, 6, 7). Although the function of GnRH II is not fully understood, studies have suggested roles in neuromodulation, differential release of LH and FSH, reproductive behavior, regulation of energy intake, and regulation of cell proliferation (6, 7, 8, 9, 10, 11, 12). The wide expression and conservation of GnRH II indicate that it has an important function and led to the cloning of specific receptors with high affinity for GnRH II (13, 14).

Consistent with its known function, the type I GnRH receptor has high affinity for GnRH I and lower affinity for GnRH II (15, 16, 17). Genes for a type II GnRH receptor (nomenclature of Ref. 13) have been identified in several mammals (14, 16, 18, 19). Type II GnRH receptors have high affinity for GnRH II and much lower affinity for GnRH I (14, 16). GnRH receptors are members of the rhodopsin-like family of seven-transmembrane domain G protein-coupled receptors. Both GnRH receptor subtypes couple to the Gq/11 family of G proteins, which activate phospholipase C and stimulate production of inositol trisphosphate. The significant activity of the GnRH II peptide at the type I GnRH receptor precludes use of the naturally occurring peptides to distinguish the physiological functions of the different GnRH receptor subtypes. Consequently, identification of high-affinity ligands, particularly antagonists that specifically bind the type II GnRH receptor, would contribute significantly to defining the function of this receptor.

GnRH II differs from GnRH I at three positions, having His5 instead of Tyr5, Trp7 instead of Leu7, and Tyr8 instead of Arg8. One or more of these differences must account for the higher affinity of GnRH II at type II GnRH receptors. Structure-activity studies of type I GnRH receptors have established that the Arg8 residue of GnRH I is critical for high-affinity interactions with the type I GnRH receptor (13, 15). In addition to Arg8, conserved residues at the amino and carboxy termini are necessary for interaction with type I GnRH receptors, and the amino-terminal residues, His2 and Trp3, are particularly important for receptor activation (reviewed in Ref. 20).

Ligand structure-activity relationships have been less thoroughly studied at the type II GnRH receptors. Trp7 and Tyr8 of GnRH II are important for binding to the type II GnRH receptor (21)(Ott, T., Z.-l. Lu, R. Sellar, P. Barran, A.J. Pawson, and R.P. Millar in preparation). As is the case for the type I GnRH receptor, peptide analogs with D-amino acid substitutions at the amino terminus act as antagonists of the type II GnRH receptor (21, 22). This suggests that the conserved amino-terminal residues of GnRH II contribute to activation of type II GnRH receptors.

Ligand binding is the initial step in GnRH receptor activation, but little is known about the ligand binding pocket of the type II GnRH receptor. A study using chimeric receptors (23) has indicated that the extracellular end of transmembrane 7 of the type II GnRH receptor selectively decreases affinity for GnRH I, but it remains unclear which residues of the type II GnRH receptor account for high-affinity recognition of GnRH II.

It is tacitly assumed that functional groups that are conserved in both ligands and receptors will form identical interactions in different ligand-receptor pairs (23, 24). However, the suggestion that different GnRH peptides have distinct intramolecular interactions that affect their conformation and their affinity for the type I GnRH receptor (25) suggests that the ligand binding pockets of different GnRH receptors may differ. This concept is well established in the tachykinin receptor system, where it has been shown that highly homologous peptides have distinct binding contacts in the neurokinin NK1 receptor, and it is believed that different ligands bind to distinct receptor conformations (26, 27). Consequently, functional groups that are conserved between GnRH I and GnRH II may not interact with equivalent residues of the type I and type II GnRH receptors. In the type I GnRH receptor, the Gly10NH2 moiety of GnRH I, which is conserved in all variant forms of GnRH (13, 28), is thought to form a hydrogen bond with the Asn2.65(102) side chain of the receptor (Fig. 1AGo) (29). Asn2.65(102) is conserved in all GnRH receptors, suggesting the potential for a conserved interaction between the conserved receptor and ligand residues in all GnRH receptor-ligand pairs. Like Asn2.65(102), Asp2.61(98) is conserved in all GnRH receptors, and it appears to form a hydrogen bond with the His2 residue of GnRH I (30), which is conserved in GnRH II. However, Asp2.61(98) also forms additional interactions, including an intramolecular interaction with Lys3.32(121) (Fig. 1AGo) (30). Lys3.32(121), which is also conserved, is required for high-affinity interactions with GnRH agonists, but not for GnRH antagonists, and it was suggested that the Lys3.32(121) side chain might interact with one of the conserved amino-terminal residues of agonist ligands (Fig. 1AGo) (31).


Figure 1
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  		Fig. 1. Schematic Receptor-Ligand Complex, Ligand Binding, Expression, and Intracellular Signaling of Wild-Type and Mutant Type II GnRH Receptors

A, Schematic representation of the GnRH receptor-ligand complex shows interactions of conserved receptor residues, Asp2.61, Asn2.65, and Lys3.32 (circles), with conserved amino- and carboxy-terminal ligand residues (circles and bracket). Dotted lines show interactions that are conserved in the GnRH II-type II receptor complex, whereas the dashed line shows the Asp2.61-His2 interaction that is not conserved. Receptor mutations and ligand substitutions used to test this model of the type II receptor-GnRH II complex are indicated in boxes. B, COS-1 cells transiently transfected with wild-type (bullet), Asp2.61(97)Glu ({circ}), Asp2.61(97)Ala ({blacksquare}), Asn2.65(101)Ala ({square}), or Lys3.32(120)Gln ({blacktriangleup}) type II GnRH receptors were incubated with [125I]GnRH II in the presence of increasing concentrations of GnRH II. C, COS-1 cells transiently transfected with FLAG-tagged wild-type and mutant type II GnRH receptors were subjected to PAGE, electroblotted on to polyvinylidene difluoride membranes, and detected using anti-FLAG antibody, M2. D, IP was extracted from COS-1 cells transiently transfected with wild type (bullet), Asp2.61(97)Glu ({circ}), Asp2.61(97)Ala ({blacksquare}), Asn2.65(101)Ala ({square}), or Lys3.32(120)Gln ({blacktriangleup}) type II GnRH receptors incubated with increasing concentrations of GnRH II in the presence of LiCl. dpm, Disingetrations per min; WT, wild type.

 
Improved understanding of GnRH II and its receptor at the molecular level may provide tools to delineate their physiological function, and comparative studies potentially yield insight into the functions of the type I GnRH receptor. We have used site-directed mutagenesis to investigate whether the conserved residues, Asp2.61(97), Asn2.65(101), and Lys3.32(120), are important for ligand interactions of the type II GnRH receptor, and we have used ligand modifications and computational modeling to determine whether intermolecular interactions of these residues are conserved. We show that all three receptor residues are critical for binding of GnRH II. IP accumulation assays indicate that the functions of Asn2.65(101) and Lys3.32(120) are similar to what was found for equivalent residues of the type I GnRH receptor. Although we find that Asp2.61(97) of the type II GnRH receptor contributes to recognition of His2 of GnRH I, it does not distinguish the residues in position 2 of its cognate ligand, GnRH II. Consistent with this, our independently constructed molecular model of the GnRH II-type II GnRH receptor complex shows an interaction of His2 of GnRH II with Lys3.32(120), but not with Asp2.61(97). These results provide evidence that contacts between conserved ligand and receptor residues in one receptor subtype do not necessarily all occur in other receptor subtypes and show that GnRH receptors have subtle differences in ligand recognition.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutation of Conserved Residues Abolishes High-Affinity Binding of GnRH II
To determine whether the conserved residues are important for binding GnRH II, the ability of mutant receptors (Fig. 1AGo) to bind GnRH II was measured using a [125I]GnRH II competition binding assay. The wild-type receptor bound GnRH II with high affinity (IC50, 4.8 nM) (Fig. 1BGo). Mutant receptors with substitutions of Glu or Ala for Asp2.61(97), Ala for Asn2.65(101), or Gln for Lys3.32(120) showed no measurable specific binding of [125I]GnRH II (Fig. 1BGo). This shows that the conserved residues were important for receptor function, but the absence of specific binding of [125I]GnRH II indicates either that the expression of the mutant receptors is compromised or that their affinity for GnRH II is decreased.

Quantitative immunoblotting was used to determine expression levels of FLAG epitope-tagged wild-type and mutant receptors and to assess whether the absence of binding of the mutant receptors was due to decreased expression. All epitope-tagged receptor constructs yielded specific bands of approximately 38,000 molecular weight. Expression of the Asp2.61(97)Ala mutant receptor was increased compared with the wild-type receptor, whereas expression of other mutant receptors was not significantly different from wild type (Fig. 1CGo and Table 1Go). This shows that the decreased ligand binding of mutant receptors was not due to decreased expression and must therefore result from decreased affinity for GnRH II. This shows that the mutant receptors have decreased affinity for GnRH II and that the mutated residues are required for high-affinity binding. We have previously found that increasing the concentrations of radiolabeled GnRH ligands in binding assays leads to increased nonspecific binding that makes it difficult to distinguish specific binding from nonspecific (32). Consequently, it is not technically feasible to use increased tracer concentrations or saturation binding assays to determine ligand binding affinities of receptors with decreased affinity for [125I]GnRH II, and we considered whether a functional assay could be used to assess ligand interactions of mutant receptors.


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  		Table 1. GnRH II-Stimulated IP Production and Expression of Wild-Type and Mutant Receptors

 
Mutation of Conserved Receptor Residues Decreases Potency of GnRH II
In our previous studies, which investigated GnRH I interaction with the type I GnRH receptor, receptor mutations also resulted in undetectable ligand binding (29, 30, 31). However, ligand-receptor interactions were sufficient to elicit inositol phosphate (IP) production and allow characterization of mutant receptors. We therefore tested GnRH II stimulation of IP production at mutant type II GnRH receptors. GnRH II stimulated IP production at the wild-type and all mutant receptors, showing that the mutant receptors were functional (Fig. 1DGo and Table 1Go). However, consistent with the apparent decreases in affinity, the potency of GnRH II was decreased at all mutant receptors (EC50 values, 3.5–152 nM) compared with the wild-type receptor (EC50, 0.19 nM). The conservative mutation of the carboxylate Asp2.61(97) side chain to Glu decreased GnRH II potency 17.3-fold compared with the wild-type receptor, whereas mutation to smaller, nonpolar Ala, caused a larger decrease in GnRH II potency (273-fold). GnRH II-stimulated IP production by epitope-tagged and untagged receptors was similar (data not shown). Because expression of the mutant receptors was not significantly decreased, the decreased potency of GnRH II in stimulating IP production, combined with the lack of measurable GnRH II binding, indicates that the mutant receptors are well expressed and able to couple to intracellular signaling, but have decreased affinity for GnRH II. Because the decreased affinity of GnRH II binding precluded use of binding assays, IP assays were used as a proxy for binding assays to further assess interactions of the conserved receptor residues with GnRH II.

Position 2-Substituted GnRH Analogs at the Asp2.61(97)Glu and Asp2.61(97)Ala Type II GnRH Receptors
To assess the contribution of Asp2.61(97) of the type II GnRH receptor to recognition of His2 of GnRH II, we compared IP production mediated by GnRH II and GnRH I, both of which have His2 and the analogs, [Trp2]-GnRH II and [Trp2]-GnRH I, which have Trp substituted for His2. [Trp2]-GnRH I had decreased potency (EC50, 73.8 nM) compared with GnRH I (EC50, 5.9 nM) in the wild-type receptor, showing that His2 of GnRH I was required for maximal potency in the type II GnRH receptor (Fig. 2Go and Table 2Go). [Trp2]-GnRH I showed a smaller decrease in potency at the Asp2.61(97)Glu mutant (10-fold) compared with His2-containing GnRH I (181-fold). This is similar to what was found in the type I GnRH receptor and shows that Asp2.61(97) of the type II GnRH receptor contributes to recognition of His2 of GnRH I. In contrast, [Trp2]-GnRH II and GnRH II potencies were similar in the wild-type receptor (EC50 values, 0.44 nM and 0.19 nM) and in each of the mutant receptors, Asp2.61(97)Glu (EC50 values, 13 nM and 3.5 nM) and Asp2.61(97)Ala (EC50 values, 33 nM and 34 nM). Thus, the Asp2.61(97)Glu mutation resulted in similar decreases in the potency of both GnRH II (17.3-fold) and [Trp2]-GnRH II (26-fold), which were smaller than the potency loss for GnRH I (181-fold). The Asp2.61(97)Ala mutation caused larger decreases in potency for both peptides, suggesting that both the carboxylate functional group and its position relative to the peptide backbone are important for receptor function. These comparable decreases in potency for both peptides at each mutant receptor show that Asp2.61(97) does not discriminate between the presence and absence of His2 in GnRH II. This shows that Asp2.61(97) of the type II GnRH receptor does not contribute to recognition of His2 of GnRH II, although it clearly has some other role in recognizing GnRH II.


Figure 2
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  		Fig. 2. Intracellular Signaling Stimulated by GnRH Analogs with Substitutions for His2 at Type II GnRH Receptors with Mutations of Asp2.61(97)

COS-1 cells transfected with wild type (A), Asp2.61(97)Glu (B), or Asp2.61(97)Ala (C) type II GnRH receptors were incubated with GnRH II (bullet), [Trp2]-GnRH II ({circ}), GnRH I ({blacksquare}), or [Trp2]-GnRH I ({square}) in the presence of LiCl before IP extraction.

 

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  		Table 2. IP Production Stimulated by GnRH I, GnRH II, and Position 2 Substituted Analogs at the Wild-Type, Asp2.61(97)Glu, and Asp2.61(97)Ala Type II GnRH Receptors

 
Carboxy Terminally Modified GnRH Analogs at the Asn2.65(101)Ala Type II GnRH Receptor
Asn2.65(102) of the type I GnRH receptor is thought to interact with the C-terminal glycinamide of GnRH I (20, 29). We compared the IP production at the wild-type and Asn2.65(101)Ala type II GnRH receptors stimulated by GnRH II and GnRH I, which have the C-terminal glycinamide and analogs, [Pro9NHEt]-GnRH II, [Pro9NHEt]-GnRH I, which have an ethylamide C terminus and thus lack the glycinamide. The potencies of [Pro9NHEt]-GnRH II and [Pro9NHEt]-GnRH I (EC50 values, 0.21 and 4.72 nM) were comparable with the potencies of their parental peptides in the wild-type receptor (EC50 values, 0.23 and 8.41 nM) (Fig. 3Go and Table 3Go), but showed much smaller decreases in potency at the Asn2.65(101)Ala mutant (4.33- and 61.8-fold) than GnRH II and GnRH I (133.5- and >2076-fold). This shows that the Asn2.65(101) side chain is required for recognizing the C-terminal glycinamide of GnRH II and GnRH I, as was found for the type I GnRH receptor.


Figure 3
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  		Fig. 3. Intracellular Signaling of the Asn2.65(101)Ala Type II GnRH Receptor Stimulated by GnRH Analogs with Carboxy-Terminal Modifications

COS-1 cells transfected with wild-type (A), or Asn2.65(101)Ala (B) type II GnRH receptors were stimulated with GnRH II (bullet), [Pro9NHEt]-GnRH II ({blacksquare}), GnRH II-OH ({blacktriangleup}), GnRH I ({circ}), [Pro9NHEt]-GnRH I ({square}), or GnRH I-OH ({triangleup}) in the presence of LiCl before IP extraction.

 

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  		Table 3. Peptide-Stimulated IP Production in Cells Expressing Wild-Type and Asn2.65(101)Ala Type II GnRH Receptors

 
To determine whether the carbonyl (C-O) or amine (NH2) group of the glycinamide is important, we tested the potencies of analogs, GnRH II-OH and GnRH I-OH, which have a free carboxyl terminus and thus have a position 10 carbonyl group, but lack the amine group. Like GnRH II and GnRH I, GnRH II-OH showed a large potency decrease (>782-fold) at the Asn2.65(101)Ala mutant, whereas potency of GnRH I-OH was too low to measure. This indicates that the Asn2.65(101) side chain of the type II GnRH receptor contributes to recognition of the carbonyl group at the C terminus of GnRH II.

Agonist and Antagonist Ligands at the Lys3.32(120)Gln Type II GnRH Receptor
Lys3.32(121) of the type I GnRH receptor is required for high-affinity binding of agonist peptides, but not for antagonist activity (31). Three agonists, GnRH II, [His5 D-Tyr6]-GnRH I, and GnRH I stimulated IP production at the wild-type receptor with EC50 values of 0.26, 0.34, and 8.74 nM, respectively (Fig. 4AGo and Table 4Go), but their potencies were decreased between 663- and 1937-fold at the Lys3.32(120)Gln mutant (Fig. 4BGo and Table 4Go) and maximum IP production was lower, suggesting partial uncoupling of the receptor from IP production. This result shows that Lys3.32(120) is required for high agonist potency. Antagonists 03 and 27 inhibited GnRH II-stimulated IP production with IC50 values of 216 and 6169 nM at the wild-type receptor (Fig. 4CGo and Table 4Go). This low potency is not surprising because these synthetic peptides were developed for type I GnRH receptors. The inhibition of GnRH II-stimulated IP production by antagonists 03 and 27 was similar in the Lys3.32(120)Gln mutant (IC50s, 227 nM and 1129 nM) (Fig. 4DGo and Table 4Go), showing that the mutation does not affect interaction of peptide antagonists with the receptor and that the Lys3.32(120) side chain is not required for interaction with antagonist ligands.


Figure 4
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  		Fig. 4. Activities of Agonists and Antagonists at the Lys3.32(120)Gln Type II GnRH Receptor

COS-1 cells transfected with wild-type (A, C, and E) or Lys3.32(120)Gln (B, D, and F) type II GnRH receptors were treated with increasing concentrations of: A, B, GnRH II (bullet), GnRH I ({circ}), or [His5,D-Tyr6]-GnRH I ({blacksquare}); C, antagonist 03 ({triangleup}), antagonist 27 ({circ}), antagonist 03 in the presence of GnRH II (1 nM) ({blacktriangleup}), or antagonist 27 in the presence of GnRH II (1 nM) (bullet); D, antagonist 03 ({triangleup}), antagonist 27 ({circ}), antagonist 03 in the presence of GnRH II (1 µM) ({blacktriangleup}), or antagonist 27 in the presence of GnRH II (1 µM) (bullet); E, antagonist 239-26 ({circ}), antagonist 239-27 ({triangleup}), antagonist 239-26 in the presence of GnRH II (1 nM) (bullet), or antagonist 239-27 in the presence of GnRH II (1 nM) ({blacktriangleup}); F, antagonist 239-26 ({circ}), antagonist 239-27 ({triangleup}), antagonist 239-26 in the presence of GnRH II (1 µM) (bullet), or antagonist 239-27 in the presence of GnRH II (1 µM) ({blacktriangleup}).

 

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  		Table 4. Agonist Stimulation, Antagonist (Ant.) Inhibition, and Potency Losses in IP Assays of the Wild-Type and Lys3.32(120)Gln Receptors

 
In an attempt to generate high-affinity antagonists of the type II GnRH receptor, we designed and synthesized antagonists 239-26 and 239-27, which have the same substitutions as antagonist 03 in positions 1, 2, 3, and 10, but have GnRH II residues in positions 5, 7, and 8, with (antagonist 239-27) or without (antagonist 139-26) D-Lys in position 6. Both peptides acted as antagonists at the type II GnRH receptor, but they showed low potency (212 ± 61.9 nM and 146 ± 44.4 nM), similar to antagonist 03 (Fig. 4EGo and Table 4Go), indicating that they have low affinity for the receptor. They had slightly lower potency at the Lys3.32(120)Gln mutant (860 ± 161 nM and 692 ± 132 nM) (Fig. 4FGo and Table 4Go).

Molecular Modeling
Although it is clear from our experimental results that the mutated residues are required for high-affinity binding of GnRH II, we used an independently constructed computational model of the receptor-ligand complex to assess whether the specific interactions proposed are feasible. GnRH II was docked to a computational model of the marmoset type II GnRH receptor, using contact sites identified for GnRH I binding to the type I GnRH receptor (Gly10NH2 with Asn2.65(101); His2 with both Asp2.61(97) and Lys3.32(120) and pGlu1 with Asn5.39(204)). After energy minimization and molecular dynamics simulation, the GnRH II-type II GnRH receptor complex (Fig. 5Go) showed a hydrogen bond between the carbonyl of the C-terminal glycinamide of GnRH II and the NH2 group of the Asn2.65(101) side chain. There was an intramolecular interaction of Asp2.61(97) with Lys3.32(120), and the NH2 group of the Lys3.32(120) side chain formed a bond with the ionizable {epsilon}-nitrogen of the His2 side chain of GnRH II. However, consistent with our experimental data, no interaction between Asp2.61(97) and His2 of GnRH II was observed.


Figure 5
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  		Fig. 5. Intermolecular Interactions between GnRH II and the Marmoset Type II GnRH Receptor

GnRH II was docked in the ß-II' folded conformation to the receptor model structure built by comparative modeling using the rhodopsin x-ray structure as a template. Intermolecular interactions between His2 and Gly10NH2 (black) of GnRH II and the receptor contact residues Lys3.32(120) and Asn2.65(101) (green) are clearly indicated. A receptor intramolecular interaction between Asp2.61(97) and Lys3.32(120) is also displayed.

 
In summary, we have shown that the conserved residues, Asp2.61(97), Asn2.65(101), and Lys3.32(120) of the type II GnRH receptor are important for binding GnRH II. Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. Asn2.65(101) contributes to recognition of the carbonyl group of the terminal Gly-NH2 of GnRH II. Lys3.32(120) is important for recognition of agonist, but not antagonist, ligands. Consistent with these experimental data, a computational model of the GnRH II-type II GnRH receptor complex showed interactions of the Asn2.65(101) with the Gly10-NH2 of GnRH II and Lys3.32(120) with His2 of GnRH II. Modifying residues in positions 1, 2, 3, and 10 of GnRH II yielded antagonists of the type II GnRH receptor that had low potency, suggesting that one or more of these residues is critical for high-affinity interaction with the receptor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The stringent conservation of GnRH II from bony fish to man suggests it has an important function. Some progress has been made in demonstrating neuromodulatory effects and effects on reproductive behavior and energy status. However, a detailed investigation of its interaction with its receptor has not been undertaken. We have started to define how GnRH II interacts with the recently cloned type II GnRH receptor by assessing whether conserved receptor residues that are thought to interact with conserved residues of GnRH I in the type I GnRH receptor (13, 29, 30, 31) have similar functions in type II GnRH receptor binding of GnRH II.

We have recreated, in the type II GnRH receptor, the mutations that were most informative in defining ligand binding interactions of the type I receptor (i.e. mutations that showed measurable function that was different from wild type) and tested their effects on receptor responses to a range of modified ligands. The severely decreased binding of GnRH II by mutant receptors precluded quantification of binding affinity. However, the demonstration that all mutants are expressed at levels the same as, or higher than wild type, shows that they have decreased affinity for GnRH II. We used ligand-stimulated IP accumulation as a proxy for ligand binding. The 10-fold difference between the IC50 and EC50 values for GnRH II at the wild-type receptor suggests the presence of a receptor reserve. Hence decreases in GnRH II potency at mutant receptors are potentially the product of decreased affinity combined with decreased coupling efficiency/activation of the mutant receptor. Indeed, the decreased Emax values for the Asp2.61(97)Ala and Lys3.32(120)Gln mutants indicate that these two mutants are less efficiently activated than the wild-type receptor. Decreased efficacies are expected when interactions that underlie agonist activity are disrupted (33, 34). In the absence of affinity information, effects of changes in affinity and efficacy cannot be separated, but it is clear that measured changes in ligand potency at mutant receptors qualitatively reflect changes in affinity, although they potentially overestimate the extent. We have used modified ligands lacking the ability to interact with the wild-type receptor residues to support our interpretations of receptor-ligand contacts. The demonstration that mutations have less effect on the potencies of these ligands is consistent with disruption of fewer receptor-ligand interactions.

Asn2.65 Has Similar Functions in Type I and Type II GnRH Receptors
Asn2.65(102) of the type I GnRH receptor is thought to form a hydrogen bond interaction with the carboxy-terminal glycinamide of GnRH I (29). This interaction was also predicted in our molecular model of the GnRH II-type II GnRH receptor complex, and our experiments support a similar function of Asn2.65(101) of the type II GnRH receptor. Introduction of Ala, which cannot hydrogen bond, decreased the potencies of GnRH II and GnRH I, which both have the glycinamide, but had less effect on the potencies of [Pro9NHEt]-GnRH II and [Pro9NHEt]-GnRH I, which have an ethyl moiety that cannot form hydrogen bonds. We interpret that the smaller decrease in potency of ethylamide peptides is because there is no disruption of an intermolecular hydrogen bond. These results show that, in the wild-type complex, Asn2.65(101) is required for recognition of peptides containing the glycinamide and support an interaction between Asn2.65(101) and the glycinamide.

The free acid peptide, GnRH II-OH, which retains the C-O functional group of glycinamide, but lacks the amine group, showed a large potency decrease at the Asn2.65(101)Ala mutant. This indicates that the Asn2.65(101) side chain determines recognition of the carbonyl group of the glycinamide. These experimental results are consistent with the hydrogen bond between the carbonyl group of Gly10NH2 in GnRH II and the amine group of the Asn2.65(101) side chain in the molecular model (Fig. 5Go). Disruption of this interaction could account for part of the large decrease in potency of GnRH II at the Asn2.65(101)Ala mutant receptor, whereas the balance of the potency loss may arise from disruption of other receptor ligand interactions or from decreased receptor activation.

Lys3.32(120) of the Type II GnRH Receptor Has a Conserved Role in Discriminating Agonist and Antagonist Peptides
Lys3.32(121) of the type I GnRH receptor is required for binding of agonists but not antagonists (31). Because agonists differ from antagonists at the N terminus, Lys3.32(121) was proposed to interact with an amino-terminal residue of GnRH, and a hydrogen bond with His2 was proposed (31). Although this has been incorporated into models of GnRH I binding to the type I GnRH receptor (35, 36), other interactions have also been proposed (37), and it remains uncertain whether Lys3.32(121) interacts with any amino-terminal functional group of GnRH or stabilizes a high-affinity receptor conformation. In the present study, mutation of Lys3.32(120) led to large decreases in agonist potencies, whereas the IC50 values for antagonists 03 and 27 were unaffected. This shows that Lys3.32(120) of the type II GnRH receptor is required for the high potency of agonists, but has no effect on antagonist activity.

Consistent with a previous report (38), both antagonists had low potency at the type II GnRH receptor. We hypothesized that the low potency was due to the presence of GnRH I residues in positions 5, 7, and 8, and designed antagonists 239-26 and 239-27 to increase affinity for the type II GnRH receptor. The low potencies of these antagonists and of a series of GnRH II antagonists based on the type I receptor antagonist, Cetrorelix (21), indicate that the peptides have low affinity for the receptor. This suggests that one or more of the substitutions in positions 1, 2, 3, and 10 severely disrupts binding of the peptides to the type II GnRH receptor and that a more detailed structure-activity analysis is needed to generate potent, high-affinity antagonists specific for the type II GnRH receptor.

Asp2.61(97) of the Type II GnRH Receptor Recognizes His2 of GnRH I But Not GnRH II
The Asp2.61(98) side chain of the type I GnRH receptor is thought to form a hydrogen bond with His2 of GnRH I (30). If His2 of GnRH II interacts with Asp2.61(97) of the type II GnRH receptor, it is expected that the potency losses of peptides with substitutions at position 2 will be smaller, compared with peptides with His2, when Asp2.61(97) is mutated to Glu or Ala. This was the case when [Trp2]-GnRH I and GnRH I were compared at the Asp2.61(97)Glu mutant, but GnRH II and [Trp2]-GnRH II exhibited similar potency losses at the Asp2.61(97)Glu and Asp2.61(97)Ala mutants. This shows that the determinant for the potency loss was a common feature between GnRH II and [Trp2]-GnRH II and therefore independent of His2. This suggests either that Trp2 can substitute for His2, or that Asp2.61(97) of the type II GnRH receptor does not interact with His2 of GnRH II. Consistent with the latter, GnRH II showed a smaller potency loss than GnRH I (17.9-fold compared with 181-fold) at the Asp2.61(97)Glu mutant, suggesting that fewer intermolecular contacts were disrupted.

These findings show that mutation of Asp2.61 has distinct effects on ligand-stimulated signaling in type I and type II GnRH receptors. Using ligand potency as a proxy for ligand affinity, this leads to the conclusion that the interactions between the conserved type II GnRH receptor residues and GnRH II differ somewhat from those between the equivalent residues in the type I GnRH receptor and GnRH I. Furthermore, the interaction of GnRH II with the type II GnRH receptor differs from the interaction of GnRH I with the same receptor. A recent report that mutations of the extracellular loop 3 -transmembrane helix 7 region of the type II GnRH receptor increased affinity for GnRH I, but had no effect on GnRH II (23), supports our conclusion that GnRH I and GnRH II have partially distinct, although overlapping, binding surfaces on the type II GnRH receptor. There is also evidence to suggest that ligand binding surfaces for GnRH I and GnRH II differ in the type I GnRH receptor, because mutations remote from the ligand binding site enhanced affinity for GnRH II, but not for GnRH I (36), and mutation of extracellular loop 3 decreased affinity for GnRH I, but not GnRH II (39). Although the complexity of peptide ligands makes it difficult to fully define ligand contact surfaces of peptide receptors, there is precedent for distinct ligand binding interactions of other closely related peptide receptors. In the NK1, NK2, and NK3 tachykinin receptors, distinct residues are important for recognition of the three related peptides, substance P, neurokinin A and neurokinin B (26, 40, 41, 42, 43, 44), whereas distinct regions of the µ-, {kappa}-, and {delta}-opioid receptors determine subtype selectivity of these receptors (45), and it has been concluded that every ligand of the {delta}-opioid receptor has a "distinct binding fingerprint" (46).

To summarize, we have shown that amino acid residues that are important for ligand binding in the type I GnRH receptor, and conserved in the type II GnRH receptor, are important for ligand binding in the type II GnRH receptor. As in the type I receptor, Asn2.65(101) is important for recognizing the carboxy-terminal C-O functional group and Lys3.32(120) is important for responses to agonists, but not antagonists. Although we show that Asp2.61(97) contributes to GnRH II binding, it does not contribute to recognition of the His2 side chain. Combined with the molecular model, ligand binding and ligand-stimulated signaling results lead to the conclusion that the ligand binding interactions of the two GnRH receptors are broadly similar, but that conservation of amino acids between receptors and ligands does not necessarily lead to conservation of contacts between them. Defining the similarities and differences between the type I and type II GnRH receptors is useful in understanding how both receptors interact with and respond to their ligands.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Peptides
Peptides, GnRH I (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2); GnRH II [His5,Trp7,Tyr8]-GnRH); [Trp2]-GnRH II; [Trp2]-GnRH I; [Pro9NHEt]-GnRH II; the free acid peptides, GnRH II-OH and GnRH I-OH; [His5,D-Tyr6]-GnRH I; antagonist 03 ([Ac-D-Phe1,D-p-ClPhe2,D-Trp3,D-Trp6,D-Ala10NH2]-GnRH) and antagonist 27 ([Ac-D-Nal1,D-Me-4-Cl-Phe2,D-Trp3,Ipr-Lys5,D-Tyr6,D-Ala10NH2]-GnRH) were prepared by conventional solid phase synthesis, as were the novel GnRH II-based analogs, antagonist 239–26 ([Ac-D-Phe1,D-p-Cl-Phe2,D-Trp3,His5,Trp7,Tyr8,D-Ala10NH2]-GnRH) and antagonist 239–27 ([Ac-D-Phe1,D-p-Cl-Phe2,D-Trp3,His5,D-Lys6,Trp7,Tyr8,D-Ala10NH2]-GnRH). [Pro9NHEt]-GnRH I was purchased from Bachem (Bubendorf, Switzerland).

Receptor Amino Acid Residue Numbering
A consensus amino acid residue numbering scheme (47) has been used to identify equivalent amino acid residues in different G protein-coupled receptors. The amino acid name is followed by an identifier consisting of the transmembrane segment number and the position of that residue relative to the most conserved residue in that segment, which is assigned the number 50, and sequence number in parentheses. For example, Asp97 of the type II GnRH receptor is designated Asp2.61(97) because it is 11 residues C-terminal of the most conserved residue of the TMS 2, Asp2.50(86). The equivalent residue of the type I GnRH receptor is Asp2.61(98). Mutant receptors are identified by the wild-type residue followed by the replacement residue, e.g. Asp2.61(97)Glu has a Glu substitution for Asp2.61(97).

Receptor Constructs
Mutations were based on the most informative mutations previously done in the type I GnRH receptor (29, 30, 31). The mutations, Asp2.61(97)Glu, Asp2.61(97)Ala, Asn2.65(101)Ala, and Lys3.32(120)Gln were introduced into the cDNA of the wild-type marmoset type II GnRH receptor, using PCR-based site-directed mutagenesis. PCR primers contained the required mutant codons and silent restriction enzyme sites to allow identification of mutant receptors by restriction analysis. Mutant receptors were subcloned into the pcDNA3.1(+) expression vector (Invitrogen, Carlsbad, CA). The wild-type and mutant receptor constructs were also subcloned into the pFLAG-CMV-2 (Sigma-Aldrich, St. Louis, MO) epitope-tagged expression vector to assess receptor expression by immunoblotting. DNA was prepared using the Nucleobond DNA purification kit (Macherey-Nagel, Duren, Germany). Sequences of all mutant receptor constructs were confirmed by automated sequencing.

Cell Culture and Transfection
COS-1 cells were maintained in DMEM (Invitrogen) containing 10% fetal calf serum (Delta Bioproducts, Kempton Park, South Africa) in a 10% carbon dioxide atmosphere and seeded onto 12-well plates (2 x 105 cells per well). Cells were transfected using a modification of the diethylaminoethyl-dextran method as previously described (48, 49).

IP Assays
Ligand-stimulated production of IP was assessed as previously described (48). Cells were labeled 2 d after transfection for 20 h in 0.5 ml medium 199 (Invitrogen Ltd., Paisley, Scotland, UK) containing 2% FCS and 2 µCi/ml myo-[2-3H]inositol (Amersham Biosciences). Cells were treated with various concentrations of peptides for 1 h at 37 C in buffer (140 mM NaCl; 20 mM HEPES; 4 mM KCl; 8 mM glucose; 1 mM MgCl2; 1 mM CaCl2; 1 mg/ml BSA, pH 7.4) containing 10 mM LiCl. IP was extracted with 10 mM formic acid for at least 30 min at 4 C, separated on Dowex ion exchange columns, and counted by scintillation counting. Every experiment included the wild-type receptor stimulated with GnRH II as a reference curve to control for any variation in transfection efficiency. Insofar as Emax in IP assays reflects receptor expression, the Emax of mutant receptors relative to wild type was consistent and reproducible (Table 1Go). It can be seen from the raw data presented in Figs. 2Go and 4Go that mutant receptor Emax (% wild type) was constant throughout the study, although counts per min values varied among experiments. From this we believe that relative expression of wild-type and mutant receptors was constant in all experiments.

Ligand Binding Assays
GnRH II was radioiodinated by a variation of the chloramine-T method as previously described for 125I-[His5,D-Tyr6]-GnRH (32). Transfected COS-1 cells were incubated with 125I-GnRH II (100 000 cpm/well) and varying concentrations of unlabeled GnRH II in a volume of 0.5 ml HEPES-buffered DMEM for 16 h at 4 C. Cells were washed twice with ice-cold PBS, and bound radioactivity was collected in 1 M NaOH and counted by {gamma} counting.

Quantitative Immunoblotting
Membranes were prepared from transfected COS-1 cells, solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, electrophoresed, and blotted as previously described (50). Blots were blocked with 5% milk powder in Tris-buffered saline and incubated overnight with the anti-FLAG M2 antibody (Sigma-Aldrich Corp.). Horseradish peroxidase-linked antimouse IgG was used as secondary antibody and detected using the ECL Plus Western Blotting kit (Amersham, Arlington Heights, IL). The AlphaEaseFC analysis software (Alpha Innotech Corp. San Leandro, CA) was used for quantification.

Molecular Modeling
A model of the marmoset type II GnRH receptor was built by comparative modeling through MODELLER as described previously for the human GnRH receptor (36) and the M1 muscarinic acetylcholine receptor (51), by using the 2.8 Å x-ray crystal structure of bovine rhodopsin as a template (52) (root mean square deviation, 1.25 Å for 296 C{alpha}-atoms). The model omitted 18 amino acids in the middle of the intracellular loop 3 (Arg236-Asp253) and the last 44 amino acids in the C-terminal tail. A ß-II' conformation of GnRH II was first docked manually into the putative conserved binding sites of the marmoset type II GnRH receptor derived from the human GnRH receptor binding sites of GnRH I (i.e. pGlu1 with Asn5.39(204), His2 with Asp2.61(97)/Lys3.32(120), and Gly10NH2 with Asn2.65(101)) (13), by using the Swiss-PdbViewer (Version 3.7 sp5, http://www.expasy.org/spdbv/). The GnRH II manually docked to the marmoset type II GnRH receptor model was then subjected to in vacuo energy minimization and molecular dynamics simulations by means of the CHARMM program (53) using a setup similar to that described by Fanelli and colleagues (54, 55).

Data Analysis
Competition binding assays, immunoblotting, and IP assays were performed in duplicate in at least three independent experiments. IC50 and EC50 values were calculated by nonlinear regression using PRISM graphing software (GraphPad, San Diego, CA). Statistical analysis of immunoblot results was performed using a two-tailed Student’s t test.


   FOOTNOTES
 
This work was supported by grants from the South African Medical Research Council (to S.M., C.A.F., and A.A.K.), the South African National Research Foundation (to C.A.F.), the University of Cape Town (to C.A.F. and A.A.K.), and the U.K. Medical Research Council (to Z.-L.L and R.P.M.).

Disclosure Summary: The authors have nothing to declare.

First Published Online September 14, 2006

Abbreviation: IP, Inositol phosphate.

Received for publication April 3, 2006. Accepted for publication September 5, 2006.


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