Two Mutations in Extracellular Loop 2 of the Human GnRH Receptor Convert an Antagonist to an Agonist

Thomas R. Ott, Brigitte E. Troskie, Roger W. Roeske, Nicola Illing, Colleen A. Flanagan and Robert P. Millar

Medical Research Council Human Reproductive Sciences Unit (T.R.O., R.P.M.), Edinburgh EH3 9ET, United Kingdom; Departments of Clinical Laboratory Sciences (T.R.O., B.E.T., C.A.F., R.P.M.) and Medicine (C.A.F.), University of Cape Town Medical School, Observatory 7925, South Africa; Indiana University School of Medicine (R.W.R.), Indianapolis, Indiana 46202; and Department of Molecular and Cellular Biology (N.I.), University of Cape Town, Rondebosch 7700, South Africa

Address all correspondence and requests for reprints to: Robert P. Millar, Medical Research Council Human Reproductive Sciences Unit, 37 Chalmers Street, Edinburgh EH3 9ET, United Kingdom. E-mail: r.millar{at}hrsu.mrc.ac.uk.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH regulates the reproductive system through cognate G protein-coupled receptors in vertebrates. Certain GnRH analogs that are antagonists at mammalian receptors behave as agonists at Xenopus laevis and chicken receptors. This phenomenon provides the opportunity to elucidate interactions and the mechanism underlying receptor activation. A D-Lys(iPr) in position 6 of the mammalian GnRH receptor antagonist is required for this agonist activity (inositol phosphate production) in the chicken and X. laevis GnRH receptors. Chimeric receptors, in which extracellular loop domains of the human GnRH receptor were substituted with the equivalent domains of the X. laevis GnRH receptor, identified extracellular loop 2 as the determinant for agonist activity of one of the mammalian antagonists: antagonist 135-18. Site-directed mutagenesis of nine nonconserved residues in the C-terminal domain of extracellular loop 2 of the human GnRH receptor showed that a minimum of two mutations (Val5.24(197)Ala and Trp5.32(205)His) is needed in this region for agonist activity of antagonist 135-18. Agonist activity of antagonist 135-18 was markedly decreased by low pH (<7.0) compared with GnRH agonists. These findings indicate that D-Lys(iPr)6 forms a charge-supported hydrogen bond with His5.32(205) to stabilize the receptor in the active conformation. This discovery highlights the importance of EL-2 in ligand binding and receptor activation in G protein-coupled receptors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH IS A DECAPEPTIDE [Glu(P)-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2] that plays a central role in reproduction by stimulating pituitary secretion of FSH and LH through activation of its cognate receptor (1). Peptide analogs of GnRH have been used in the treatment of a wide spectrum of hormone-dependent diseases (2). New generation peptide and nonpeptide GnRH antagonists are being actively sought. Delineation of the ligand binding pocket and mechanisms involved in stabilizing the active or inactive conformation of the GnRH receptor may contribute to the development and refinement of these analogs.

GnRH receptors belong to the family of rhodopsin-like G protein-coupled receptors (GPCRs) (3, 4) that are characterized by seven transmembrane domains (TMs) connected by alternating intracellular and extracellular loop (EL) domains. Peptide ligands appear to bind predominantly to extracellular domains and to the TMs (5). Binding of GnRH to its receptor involves Asp2.61(98) (6), Asn2.65(102) (7), Lys3.32(121) (8), and Asp7.32(302) (9), and possibly Trp6.58(279) (10) and Trp2.64(101) (11) (see Ref. 12 for numbering identification). These residues are all in the extracellular domains or close to the extracellular boundaries of the TMs and are conserved in all vertebrate GnRH receptors (1). Antagonists appear to bind to different contact sites than agonists (8, 11, 13, 14) although it is likely that binding sites are at least overlapping (11).

The precise mechanism by which the binding of GnRH (and other peptide ligand GPCRs) to the GnRH receptor is translated into receptor activation is not known. However, a network of interactions involving Asn1.50 in TM-I, Asp2.50 in TM-II, Asn7.49 in TM-VII, and Asn3.49 and Arg3.50 in TM-III are implicated in receptor activation (15). Comparative studies on interactions determining agonism and antagonism provide the potential for further insights into the mechanism of receptor activation.

Mammalian GnRH receptors have more than 85% sequence identity and similar pharmacology (1), whereas nonmammalian GnRH receptors from catfish (16), goldfish (17, 18), frog (19, 20), and chicken (21) exhibit distinctly different pharmacologies. Most significantly certain antagonists at mammalian GnRH receptors behave as agonists at chicken (21), goldfish, and frog (our unpublished results) receptors. D-Lys or D-Lys(iPr) is essential in position 6 of the mammalian GnRH receptor antagonists to act as agonists at the chicken GnRH receptor (21). We now show that one of these analogs, antagonist 135-18, is also an agonist in the Xenopus laevis GnRH receptor and have delineated the ligand-receptor interaction responsible for this phenomenon.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of Domains Involved in Recognizing Antagonist 135-18 as an Agonist or Antagonist
Antagonist 135-18 behaved as an antagonist at the human GnRH receptor as previously described (Ref. 21 and Fig.1AGo). In contrast, it acted as a partial agonist on the X. laevis GnRH receptor in stimulating IP production (Fig. 1AGo), while also partially inhibiting mGnRH-stimulated IP production at high doses of antagonist 135-18 (data not shown).



View larger version (34K):
[in this window]
[in a new window]
 
Figure 1. Antagonist 135-18-Stimulated IP Production in Chimeric GnRH Receptors

COS-1 cells were transfected with the human/X. laevis GnRH receptor and CHRs 1–7. Transfected cells were incubated without ligand (open bars), or with mGnRH (10-6 M, hatched bars) or antagonist 135-18 (10-8 M, vertically striped bars, 10-5 M, solid bars). IP production is presented as percent of IPmax for mGnRH, from a single experiment that is representative of at least three independent experiments carried out in duplicate. CHRs 1–3 are EL-1, EL-2, and EL-3 of the X. laevis receptor substituted in the human receptor. CHRs 4–7 are consturcts of X. laevis (solid bars) and human (open bars) EL-2 depicted above and described in Fig. 4Go.

 
To investigate whether the extracellular domains of the X. laevis GnRH receptor determined the agonist activity of antagonist 135-18, these domains were individually substituted into the human GnRH receptor using engineered restriction enzyme sites (Fig. 2Go). When EL-1 [chimeric receptor 1 (CHR-1)] and EL-3 (CHR-3) of the human GnRH receptor were substituted with the equivalent X. laevis receptor domains, antagonist 135-18 did not stimulate IPs (Fig. 1AGo). However, when EL-2 was substituted (CHR-2), antagonist 135-18 behaved as a full agonist. The ligand did not inhibit IP production in the presence of mGnRH and, alone, stimulated the production of IPs to levels similar to maximal levels generated by saturating concentrations of mGnRH (Figs. 1AGo and 3AGo). To determine whether the effect of the exchange of EL-2 was specific for antagonists that contain a D-Lys or D-Lys(iPr) in position 6 as has been shown in the chicken GnRH receptor, antagonist 27, which lacks this residue, was tested for its ability to produce IPs. This ligand still behaves as an antagonist on CHR-2 as it is unable to stimulate any IPs, even at high doses, and it inhibits the IP response in the presence of mGnRH (Fig. 3BGo). This correlates with antagonist 27 being a full antagonist in the X. laevis GnRH receptor (data not shown).



View larger version (35K):
[in this window]
[in a new window]
 
Figure 2. Human GnRH Receptor with Engineered Restriction Endonuclease Recognition Sites

Restriction endonuclease recognition sites were engineered near the extracellular ends of TM-III (ScaI), TM-IV (BsrGI), TM-V (StuI), TM-VI (SnaBI), and TM-VII (HpaI) (see Materials and Methods). PflMI near the extracellular end of TM-II is a naturally occurring restriction site.

 


View larger version (18K):
[in this window]
[in a new window]
 
Figure 3. Activity of Antagonist 135-18 and Antagonist 27 in CHR-2

COS-1 cells were transfected with CHR-2 and stimulated with increasing concentrations of antagonist in the presence (10-9 M) ({blacktriangleup}) and absence ({blacksquare}) of mGnRH. IP production is presented as percent of IPmax for mGnRH from a single experiment, which is representative of at least three independent experiments carried out in duplicate.

 
EL-2 of the X. laevis and other nonmammalian GnRH receptors is five amino acids shorter than the human GnRH receptor EL-2. Sequence alignment shows that residues 4.74(188) to 4.78(192) of the human GnRH receptor are not present in the nonmammalian GnRH receptors (Fig. 4Go). To investigate the possible role of this gap and to determine which part of EL-2 is responsible for agonist activity of antagonist 135-18, a further series of chimeric constructs, containing smaller segments of the X. laevis receptor (Fig. 4Go) were tested. All four constructs recognized antagonist 135-18 as a partial agonist (Fig. 1BGo). Antagonist 135-18 alone stimulated IP production, but IPmax levels were lower than those stimulated by mGnRH (Fig. 1BGo). The degree of stimulation was independent of the length of the CHR, indicating that the size of EL-2 is not a crucial determinant for agonist/antagonist activity of antagonist 135-18 (Fig. 1BGo).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 4. Chimeric Receptors Incorporating EL-2 Domains of the X. laevis GnRH Receptor in the Human GnRH Receptor

Sequences of EL-2 of the wild-type X. laevis (top) and human (bottom) GnRH receptors are shown. Bold letters indicate conserved residues. Solid boxes indicate incorporation of X. laevis GnRH receptor residues, whereas open boxes represent the human GnRH receptor residues. The dashed line represents a gap of five amino acids in the X. laevis GnRH receptor, showing that the X. laevis EL-2 and EL-2 of CHR-2, CHR-5, and CHR-7 are shorter than the human GnRH receptor EL-2 by five amino acids.

 
Effects of Point Mutations on the Activity of Antagonist 135-18
As an earlier study identified the necessity of a positively charged residue in position 6 of the mammalian GnRH receptor antagonists for agonist activity at the chicken GnRH receptor (21), a negatively charged counter ion in EL-2 was considered as a target. Comparison of the sequences in EL-2 revealed a Glu residue at the C-terminal end of EL-2 that was conserved among the nonmammalian, but not mammalian, GnRH receptors. To test for a possible interaction with the antagonist, the equivalent residue of the human GnRH receptor (Gln5.35(208)) was mutated to Glu. The resultant receptor showed no agonist response to antagonist 135-18 (Fig. 5AGo). As the C-terminal portion of the X. laevis receptor EL-2 has only seven differences compared with the human GnRH receptor (Fig. 4Go), these residues were investigated for involvement in recognizing antagonist 135-18 as an agonist by individually mutating them in the human GnRH receptor. None showed any IP response in the presence of antagonist 135-18 (Fig. 5AGo). To investigate whether the combined effects of more than one amino acid determine the agonist activity of antagonist 135-18, Ser5.21(194), Val5.24(197), Ser5.30(203), and Trp5.32(205) were mutated in the human GnRH receptor to the residues present in the X. laevis receptor (viz. Thr, Ala, Thr, and His, respectively). This quadruple mutant showed agonist activity in response to antagonist 135-18, which was similar to the activity of CHR-6 (Fig. 5BGo). A series of triple mutations in this region indicated that the minimum requirement for antagonist 135-18 to be recognized as an agonist is an Ala in position 5.24(197) in combination with a His in position 5.32(205) (Fig. 5BGo). This finding was confirmed by creating all possible double mutants in this region and showing that only the Val5.24(197)Ala/Trp5.32(205)His double mutant stimulated IP production in response to antagonist 135-18 (Fig. 5CGo).



View larger version (34K):
[in this window]
[in a new window]
 
Figure 5. Antagonist 135-18 Stimulated IP Production in Human GnRH Receptor with Mutations in the C-Terminal Region of EL-2

COS-1 cells were transfected with the human wild-type GnRH receptor and receptors with single-point mutations (A), multiple mutations (B), and two mutations (C) and incubated without ligand (open bars), with 10-9 M mGnRH (hatched bars) or 10-6 M antagonist 135-18 (solid bars). IP production is presented as percent of IPmax for mGnRH from a single experiment, which is representative of at least three independent experiments carried out in duplicate.

 
Binding of Antagonist 135-18 to Mutant Receptors
To test the possibility that antagonist 135-18 had gained an additional point of contact with receptors that recognized antagonist 135-18 as an agonist, receptor-binding assays were performed on the human wild-type GnRH receptor and on chimeric and mutant human GnRH receptors. The IC50 for antagonist 135-18 was similar in all receptors (~10 nM, Table 1Go). The total binding of tracer ligand in the absence of cold peptide to mutant GnRH receptors was variable but higher than the binding of tracer ligand to the human wild-type receptor in all mutant receptors (Table 1Go). As IC50 values for [D-Trp6]-GnRH were identical for all mutants and the wild-type GnRH receptor (data not shown), this higher total binding shows that mutants have higher expression levels than the wild-type human GnRH receptor. Binding experiments could not be carried out for the wild-type X. laevis GnRH receptor as no binding of tracer ligands could be detected on COS-1 cells transiently transfected with this receptor, presumably due to low expression (19).


View this table:
[in this window]
[in a new window]
 
Table 1. IC50 of Antagonist 135-18 and Total Counts Bound for Wild-Type and Chimeric and Mutant Receptors, Which Recognize Antagonist 135-18 as an Agonist

 
Effects of pH on the Activity of Antagonist 135-18, mGnRH, and [Trp2]-GnRH at the Mutant Val5.24(197)Ala/Trp5.32(205)His GnRH Receptor
Based on the above results, we propose that the D-Lys(iPr)6 side chain of antagonist 135-18 forms a charge-supported hydrogen bond with His5.32 and that this interaction is responsible for the agonist activity of antagonist 135-18. Decreasing pH results in protonation of His side chains and loss of interaction with hydrogen donor groups. To test the proposed interaction of His5.32 with the D-Lys(iPr)6 side chain, we investigated the effect of pH on the agonist activity of antagonist 135-18, mGnRH, and an agonist, [Trp2]-GnRH, at the mutant Val5.24(197)Ala/Trp5.32(205)His GnRH receptor. IP accumulation in response to antagonist 135-18 was minimal at pH values lower than 7.0, whereas mGnRH and [Trp2]-GnRH substantially stimulated IP accumulation (Fig. 6Go).



View larger version (14K):
[in this window]
[in a new window]
 
Figure 6. Effects of pH on the Ability of Antagonist 135-18, mGnRH, and [Trp2]-GnRH to Stimulate IP Production by the Mutant Val5.24(197)Ala/Trp5.32(205)His GnRH Receptor

COS-1 cells were transfected with the mutant Val5.24(197)Ala/Trp5.32(205)His GnRH receptor and stimulated with 10-5 M antagonist 135-18 ({blacksquare}), mGnRH ({blacktriangleup}), and [Trp2]-GnRH ({blacktriangledown}) at the pH indicated. IP production is presented as percent of IP production at pH 7.5. The graph shown is a typical profile of four independent experiments carried out in duplicate.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We previously demonstrated that mammalian GnRH receptor antagonists that contain a D-Lys or D-Lys(iPr) in position 6 exhibit agonist activity at the chicken GnRH receptor (21). Here we investigated the most active of these, antagonist 135-18, in the X. laevis GnRH receptor. We demonstrated that this mammalian GnRH receptor antagonist is also an agonist at the X. laevis GnRH receptor and have delineated the ligand-receptor interaction responsible for receptor activation.

EL-2 of the X. laevis GnRH Receptor Determines Agonist Activity of Antagonist 135-18
GnRH and its analogs are known to bind primarily to extracellular domains of mammalian receptors (6, 7, 8, 9, 10, 11). To investigate the role of extracellular loop domains in recognizing antagonist 135-18 as either an antagonist or an agonist, EL-1, EL-2, and EL-3 of the human GnRH receptor were substituted individually with the equivalent domains of the X. laevis GnRH receptor. The exchange of EL-2, but not the exchange of EL-1 or EL-3, of the human GnRH receptor with the equivalent domain of the X. laevis GnRH receptor resulted in antagonist 135-18 acting as a full agonist. When only portions of EL-2 were substituted, antagonist 135-18 was a partial agonist. This indicates that the entire EL-2 is required for full agonist activity of antagonist 135-18. The presence of an extra five residues in EL-2 of the human GnRH receptor, compared with nonmammalian receptors, does not prevent antagonist 135-18 from behaving as an agonist, as CHR-4 and CHR-6, which are the same length as the human GnRH receptor, recognize this ligand as an agonist.

Overexpression of receptors can enhance low intrinsic activity of ligands (26, 27, 28). As some of our mutant receptors that recognize antagonist 135-18 as an agonist exhibited increased expression (e.g. see Table 1Go) this potentially provides an explanation for the observed phenomenon. However, mutant receptor expression did not correlate with the degree of agonism of antagonist 135-18 (Table 1Go and Figs. 1Go and 5Go). The Bmax data were taken as reflecting expression as all of these mutants had similar affinities for antagonist 135-18 (Table 1Go) and the radioligand (data not shown). An example is mutant Glu5.35Gln CHR-2, which was expressed at half the level of the wild-type receptor but still recognized antagonist 135-18 as a full agonist (data not shown). The contrary was also true in that several high expressing mutants did not recognize antagonist 135-18 as an agonist (data not shown).

In recent years a number of studies of GPCRs have demonstrated the conversion of antagonists to agonists. Mutations in TM-III of rhodopsin (29), ß-adrenoceptors (30), V2-vasopressin receptors (31), and AT1A-angiotensin II receptors (32, 33), in TM-IV of opioid receptors (34), in TM-VI of the D1-dopamine receptor (35), and in intracellular loop domain 3 of {alpha}2A-adrenergic receptors (36) all showed the conversion of antagonists to partial or full agonists. In a number of these studies some of the antagonist ligands with very low intrinsic agonist activities at the wild-type receptors had agonist activity at mutant receptors with constitutive activity (31, 32, 33, 35, 36). This characteristic is not apparent for the GnRH receptor mutants described here, as they had no discernible constitutive activity. It is also significant that none of these mutations were in EL domains as for the GnRH receptor. In the absence of increased expression or constitutive activity of the GnRH receptor mutants as explanations for agonist activity of antagonist 135-18, we further explored the possibility of a direct interaction of ligands with residues in EL-2 that might stabilize the active receptor conformation.

Role of EL-2 in Receptor Activation
It has been previously reported that antibodies against EL-2 of the {alpha}1- (37), ß1- (38), and ß2-adrenergic- (39), AT1-angiotensin II- (40), bradykinin B2- (41), M1- (42), and M2-muscarinic-acetylcholine (43) receptors can activate a second messenger response, presumably by stabilizing the receptor in its active conformation. Changes in EL-2 configuration may have the potential to translate into changes in the tertiary structure of the helix bundle. EL-2 is connected to the extracellular end of TM-III by a conserved disulfide bond. As this domain plays an important role in the activation of GPCRs (15, 44, 45), a change in three-dimensional structure of EL-2 through ligand or antibody binding may lead to receptor activation. The crystal structure of rhodopsin revealed recently that EL-2 has a distinct structure intimately associated with, and the potential to perturbate, the TMs (46). Our findings of a crucial role of EL-2 in recognizing antagonist 135-18 as an agonist or antagonist add further support for this developing notion of a generic role of EL-2 in GPCR activation.

TM-III, which is linked to EL-2 by a disulfide bond, is involved in the initial stages of receptor activation, with the highly conserved Asp3.49Arg3.50 motif at the cytoplasmic end of this domain, playing a central role in this process in the GnRH receptors (15, 47) and other GPCRs (44, 45). Ballesteros et al. (15) proposed that there is an ionic interaction between these residues in the inactive state of the GnRH receptor, which is supported by the crystal structure of rhodopsin (46). Protonation of the Asp3.49 residue is proposed to disrupt the interaction with the Arg3.50 side chain, which rotates within the plane of the membrane to establish a new interaction with Asp7.49 in the mammalian GnRH receptors (15). This residue is found in the Asp2.50/Asn7.49 motif in TM-II and TM-VII, which is known to be important for receptor activation (15). These proposed events of receptor activation are centered around rotation of TM-III and provide a rationale for the effects of ligands and antibodies on EL-2 in activating GPCRs because a stabilization of a particular configuration of EL-2 could be translated into the stabilization of the active conformation of TM-III.

Identification of Individual Amino Acids Interacting with Antagonist 135-18 to Confer Agonist Activity
We previously demonstrated that D-Lys(iPr) or D-Lys is needed in position 6 of mammalian GnRH receptor antagonists in order for the ligand to act as an agonist at the chicken GnRH receptor (21). We therefore sought for a single amino acid in EL-2 of the X. laevis receptor which might be a candidate for interaction with D-Lys(iPr)6 of antagonist 135-18. Each of the amino acids that were different in the X. laevis receptor were mutated individually and in combination in the human GnRH receptor. Two mutations, Val5.24(197)Ala and Trp5.32(205)His, are required for antagonist 135-18 to be recognized as an agonist at the human GnRH receptor. His5.32 represented an attractive candidate for interaction with D-Lys(iPr)6 of antagonist 135-18 through a charge-supported hydrogen bond.

His side chains are mainly unprotonated under physiological conditions and can therefore act as hydrogen bond acceptors. At low pH, His becomes protonated and loses its ability to act as a hydrogen bond acceptor. If the agonist activity observed for antagonist 135-18 in mutant receptors is caused by the D-Lys(iPr)6 side chain forming a hydrogen bond with His5.32, protonation of His5.32(205) at low pH should prevent this bond from forming and abolish agonist response to antagonist 135-18 in the Val5.24(197)Ala/Trp5.32(205)His mutant receptor. To test this proposal, the Val5.24(197)Ala/Trp5.32(205)His mutant GnRH receptor was stimulated at different pH values with antagonist 135-18, mGnRH, and [Trp2]-GnRH. The latter ligand was used to rule out pH effects on GnRH itself as it contains a His2. Lowering the pH to 6.8 and 6.3 decreased antagonist 135-18 stimulation of IP considerably more than for mGnRH and [Trp2]-GnRH. Antagonist 135-18 lost all of its agonist activity when the pH was lowered to 6.0, which is consistent for the pKa value of 6.2 for His. This finding supports the hypothesis that a charge-supported hydrogen bond between D-Lys(iPr)6 of the antagonist and His5.32(205) is responsible for agonist activity observed in response to antagonist 135-18. Interestingly, there was no significant difference in the effect of pH on the activity of mGnRH and [Trp2]-GnRH. This might indicate that the state of protonation of His2 of GnRH is not significant for the activity of the native ligand.

An increase in binding affinity may have been anticipated if the interaction of His5.32(205) with D-Lys(iPr)6 of antagonist 135-18 is present only in mutants displaying agonism, as it would be an additional interaction. There was no significant increase in binding affinity of the mutants that recognized antagonist 135-18 as an agonist. However, this is not entirely unexpected as a hydrogen bond is anticipated to increase binding affinity about 2- to 3-fold (48), which is a difference not reliably detectable in our binding assays. Most studies of mutations that convert antagonists to agonists also report no change in binding affinity (31, 34, 35). Another explanation for the failure to detect a difference in binding affinities would be that the antagonist 135-18 forms an alternative bond with the mutant receptor while losing another interaction that is formed at the wild-type receptor.

For agonist activity to be realized, a second mutation is needed in position 5.24(197) (Val to Ala). This residue is the equivalent of two turns of an {alpha}-helix away from the His residue. The bulky Val side chain might therefore interfere sterically, either directly with the side chain of His5.32(205) or indirectly through conformational constraints of EL-2, so that the proposed hydrogen bond between His5.32(205) and D-Lys(iPr)6 of antagonist 135-18 cannot be formed.

In summary, EL-2 was identified as a determinant of the recognition of antagonist 135-18 as an agonist or an antagonist. The minimal and essential structural determinants in the C-terminal domain of EL-2 for agonist activity of antagonist 135-18 in the human GnRH receptor is a Trp5.32(205)His mutation combined with a Val5.24(197)Ala substitution. These findings provide direct evidence of the role of EL-2 of a GPCR in receptor activation. Our findings suggest that that by mutating Trp5.32(205) to a His, a charge-supported hydrogen bond can be formed with D-Lys(iPr)6 of antagonist 135-18, which allows the ligand to stabilize the receptor in its active conformation. This study represents the first report of mutations in an extracellular domain that convert an antagonist to an agonist and identifies the ligand and receptor residues involved.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Numbering of Amino Acid Residues
Because GPCRs differ in length, a common numbering system is used so that equivalent residues in different receptors can be compared. In the nomenclature used, the most conserved residue in each TM is assigned the number 50 (12). For example, Arg139 is designated Arg3.50(139).

Silent Mutation of the Human GnRH Receptor
To facilitate the exchange of EL domains, unique restriction enzyme cutting sites were introduced into the human GnRH receptor at the extracellular ends of TMs III–VII (Fig. 2Go) using an adapted method described by Kunkel et al. (22). Mutations were verified by manual DNA sequencing (T7 sequenase kit, version 2.0, Amersham Pharmacia Biotech, Cape Town, South Africa). The resultant receptor showed binding properties and second messenger responses identical with those of the wild-type human GnRH receptor (Table 2Go). This receptor was used for all subsequent mutagenesis.


View this table:
[in this window]
[in a new window]
 
Table 2. Comparison of IC50 and ED50 Values of Various Ligands for the Wild-Type Human GnRH Receptor (GnRH-R) and the Engineered Human GnRH Receptor Containing Silent Restriction Sites

 
Construction of EL Chimeras
Chimeric GnRH receptors were constructed by excising the ELs of the human GnRH receptor and replacing them with those of the X. laevis GnRH receptor using the introduced restriction sites. EL-1, -2 and -3 of the X. laevis GnRH receptor were amplified by PCR using a proofreading DNA polymerase (Deep-Vent, New England Biolabs, Inc., South Africa) and primers flanked by the appropriate restriction endonuclease cutting sites. EL-1, -2, and -3 of the X. laevis GnRH receptor were ligated into the appropriately digested human GnRH receptor in pBluescript SK(-) (Stratagene, supplied through Whitehead Scientific, Cape Town, South Africa) to produce chimeric receptors 1, 2, and 3 (CHR-1, CHR-2, and CHR-3, respectively). The chimeric receptors were subcloned into pcDNAI/Amp for expression in COS-1 cells and pharmacological characterization. All sequences were confirmed by manual sequencing.

The protocol used to obtain CHRs 4–7 was adapted from methods previously described (23). Antisense primers were designed for both the human and X. laevis GnRH receptor containing 18 bases of the other receptor at their 5'-end to produce the constructs shown in Fig. 4Go. These primers were used in combination with a sense primer, which aligned to the N-terminal portion of EL-2 containing the BsrGI recognition sequence at its 5'-end. At the same time the C-terminal section of EL-2 of the other receptor was amplified using a sense primer complementary to the 18 bases on the chimeric primer and an antisense primer, which aligned to the C-terminal section of EL-2 containing the StuI recognition sequence 5'. In a second round of PCR, the two products of the first round of PCR were used in combination with primers containing the BsrGI and StuI recognition sequences (from earlier mutagenesis experiments) to amplify the full-length chimeric EL-2, which was inserted into the human GnRH receptor as described earlier.

Point Mutations in the Human GnRH Receptor
Point mutations in the human GnRH receptor were produced as previously described (23). The mutant receptors were then cloned into pcDNAI/Amp and sequences were confirmed by DNA sequencing.

Transfection, IP, and Binding Assays
Plasmid DNA for transfection was prepared using QIAGEN (West Sussex, UK) Maxipreps. COS-1 cells were maintained in DMEM (Sigma, UK) containing 10% FCS. Cells (200,000–300,000 per well) were transfected as previously described (24). Transfected cells were grown for 48 h in DMEM containing 10% FCS and antibiotics before IP and binding experiments.

IP assays were carried out as previously described (24). Briefly, transfected COS-1 cells were labeled for 16–18 h in 0.5 ml DMEM without inositol (Life Technologies, Inc., Paisley, UK), containing 1% FCS and 2 µCi/ml myo[2-3H]inositol (Amersham Pharmacia Biotech, Buckinghamshire, UK). Cells were incubated in the presence and/or absence of agonists and antagonists in 0.5 ml buffer I (140 mM NaCl, 4 mM KCl, 20 mM HEPES, 8.3 mM D-glucose, 1 mM MgCl2, 1 mM CaCl2, 0.1% BSA, pH 7.4, unless stated otherwise). Total IPs were extracted at 4 C with 10 mM formic acid for a minimum of 30 min (25) and separated using a Dowex ion exchange resin. Basal IP production was measured in the absence of ligand. No significant difference was observed between basal IP and mock-transfected cells stimulated with 1 µM mGnRH.

[D-Trp6]-GnRH was radioiodinated using Iodogen (Pierce Chemical Co. and Warriner, Chester, UK) and purified on a Sephadex G25 column using 0.01 M acetic acid/0.1% BSA for elution. Transfected cells were incubated with 200,000 cpm 125I-labeled [D-Trp6]-GnRH in 500 µl buffer I in the presence/absence of unlabeled ligand for 2 h on ice. Cells were washed twice with ice-cold PBS before being dissolved in 1.0 ml 0.1 M NaOH and radioactivity counted. Nonspecific binding was determined by performing the assay on vector-transfected cells.

GnRH Analogs
The following GnRH analogs: Glu(P)-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2 (mGnRH), [Ac-D-Nal(2)1, D-4-Cl-Phe2, D-Pal(3)3, Ile5, D-Lys(iPr)6, Lys(iPr)8, D-Ala10NH2]-GnRH (antagonist 135-18); [Ac-D-Nal(2)1, D-{alpha}-Me-4-Cl-Phe2, D-Trp3, Lys(iPr)5, D-Tyr6, D-Ala10NH2]-GnRH (Antagonist 27); [Gln8]-GnRH (chicken GnRH I), [His5, Trp7, Tyr8]-GnRH (GnRH II) and [D-Ala6, N-Me-Leu7, Pro9NHEt]-GnRH (GnRH A), [D-Trp6]-GnRH were prepared by solid-phase synthesis and purified by C-18 reverse-phase chromatography.

Data Analysis
Experiments were carried out in duplicate. Graphs shown are representative of at least three independent experiments. Data were plotted and IC50 and EC50 values were calculated using the One site competition formula of Prism (version 3.02, GraphPad Software, Inc., San Diego, CA) for the IC50 values and Sigmoidal dose response formula of Prism (version 3.02, GraphPad Software, Inc.) for the EC50 values.


    ACKNOWLEDGMENTS
 
We thank Dr. Dan Donelly for insightful discussions, Dr. Adam Pawson for supplying the Cys5.27(200)Ser mutant, as well as Robin Sellar and Nicola Miller for excellent technical assistance.


    FOOTNOTES
 
This work was supported by the Medical Research Council and the National Research Foundation of South Africa, the University of Cape Town and the Medical Research Council of the United Kingdom.

Abbreviations: CHR, Chimeric receptor; GPCR: G protein-coupled receptor; EL, extracellular loop domain; IP, inositol phosphate; TM, transmembrane domain.

Received for publication May 2, 2001. Accepted for publication December 21, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. 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]
  2. Millar RP, Assefa D, Ott T, Pawson A, Troskic B, Wakefield I, Katz A 1998 GnRH and GnRH analogues: structure, actions and clinical applications. Horm Frontier Gynaecol 5:77–83
  3. 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]
  4. Kaiser UB, Zhao D, Cardona GR, Chin WW 1992 Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor. Biochem Biophys Res Commun 189:1645–1652[Medline]
  5. Ji TH, Grossmann M, Ji I 1998 G protein-coupled receptors. I. Diversity of receptor-ligand interactions. J Biol Chem 273:17299–17302[Free Full Text]
  6. Flanagan CA, Rodic V, Konvicka K, Yuen T, Chi L, Rivier JE, Millar RP, Weinstein H, Sealfon SC 2000 Multiple interactions of the Asp2.61(98) side chain of the gonadotropin-releasing hormone receptor contribute differentially to ligand interaction. Biochemistry 39:8133–8141[CrossRef][Medline]
  7. Davidson JS, McArdle CA, Davies P, Elario R, Flanagan CA, Millar RP 1996 Asn102 of the gonadotropin-releasing hormone receptor is a critical determinant of potency for agonists containing C-terminal glycinamide. J Biol Chem 271:15510–15514[Abstract/Free Full Text]
  8. Zhou W, Rodic V, Kitanovic S, Flanagan CA, Chi L, Weinstein H, Maayani S, Millar RP, Sealfon SC 1995 A locus of the gonadotropin-releasing hormone receptor that differentiates agonist and antagonist binding sites. J Biol Chem 270:18853–18857[Abstract/Free Full Text]
  9. Flanagan CA, Becker II, Davidson JS, Wakefield IK, Zhou W, Sealfon SC, Millar RP 1994 Glutamate 301 of the mouse gonadotropin-releasing hormone receptor confers specificity for arginine 8 of mammalian gonadotropin-releasing hormone. J Biol Chem 269:22636–22641[Abstract/Free Full Text]
  10. Chauvin S, Berault A, Lerrant Y, Hibert M, Counis R 2000 Functional importance of transmembrane helix 6 Trp(279) and exoloop 3 Val(299) of rat gonadotropin-releasing hormone receptor. Mol Pharmacol 57:625–633[Abstract/Free Full Text]
  11. Hoffmann SH, ter Laak T, Kuhne R, Reilander H, Beckers T 2000 Residues within transmembrane helices 2 and 5 of the human gonadotropin-releasing hormone receptor contribute to agonist and antagonist binding. Mol Endocrinol 14:1099–1115[Abstract/Free Full Text]
  12. Ballesteros J, Weinstein H 1995 Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein coupled receptors. Methods Neurosci 25:366–428
  13. Assefa D, Pawson AJ, McArdle CA, Millar RP, Flanagan CA, Roeske R, Davidson JS 1999 A new photoreactive antagonist cross-links to the N-terminal domain of the gonadotropin-releasing hormone receptor. Mol Cell Endocrinol 156:179–188[CrossRef][Medline]
  14. Cui J, Smith RG, Mount GR, Lo JL, Yu J, Walsh TF, Singh SB, DeVita RJ, Goulet MT, Schaeffer JM, Chang K 2000 Identification of Phe313 of the gonadotropin-releasing hormone (GnRH) receptor as a site critical for the binding of nonpeptide GnRH antagonists. Mol Endocrinol 14:671–681[Abstract/Free Full Text]
  15. Ballesteros J, Kitanovic S, Guarnieri F, Davies P, Fromme BJ, Konvicka K, Chi L, Millar RP, Davidson JS, Weinstein H, Sealfon SC 1998 Functional microdomains in G-protein-coupled receptors. The conserved arginine-cage motif in the gonadotropin-releasing hormone receptor. J Biol Chem 273:10445–10453[Abstract/Free Full Text]
  16. Tensen C, Okuzawa K, Blomenrohr M, Rebers F, Leurs R, Bogerd J, Schulz R, Goos H 1997 Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor. Eur J Biochem 243:134–140[Abstract]
  17. Illing N, Troskie BE, Nahorniak CS, Hapgood JP, Peter RE, Millar RP 1999 Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus). Proc Natl Acad Sci USA 96:2526–2531[Abstract/Free Full Text]
  18. Yu KL, He ML, Chik CC, Lin XW, Chang JP, Peter RE 1998 mRNA expression of gonadotropin-releasing hormones (GnRHs) and GnRH receptor in goldfish. Gen Comp Endocrinol 112:303–311[CrossRef][Medline]
  19. Troskie BE, Hapgood JP, Millar RP, Illing N 2000 Complementary deoxyribonucleic acid cloning, gene expression, and ligand selectivity of a novel gonadotropin-releasing hormone receptor expressed in the pituitary and midbrain of Xenopus laevis. Endocrinology 141:1764–1771[Abstract/Free Full Text]
  20. Wang L, Bogerd J, Choi HS, Seong JY, Soh JM, Chun SY, Blomenrohr M, Troskie BE, Millar RP, Yu WH, McCann SM, Kwon HB 2001 Three distinct types of gonadotropin-releasing hormone receptor characterized in a single diploid species. Proc Natl Acad Sci USA 98:361–366[Abstract/Free Full Text]
  21. Sun YM, Flanagan CA, Illing N, Ott TR, Sellar R, Fromme BJ, Hapgood J, Sharp P, Sealfon SC, Millar RP 2001 A chicken gonadotropin-releasing hormone receptor which confers agonist activity to mammalian antagonists: identification of D-Lys6 in the ligand and extracellular loop two of the receptor as determinants. J Biol Chem 276:7754–7761[Abstract/Free Full Text]
  22. Kunkel TA, Bebenek K, McClary J 1991 Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol 204:125–139[Medline]
  23. Mullis KB, Faloona FA 1987 Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–350[Medline]
  24. Millar RP, Davidson JS, Flanagan CA, Wakefield I 1995 Ligand binding and second-messenger assays for cloned Gq/G11-coupled neuropeptide receptors: the GnRH receptor. Methods Neurosci 25:145–163
  25. Berg KA, Clarke WP, Chen Y, Ebersole BJ, McKay RD, Maayani S 1994 5-Hydroxytryptamine type 2A receptors regulate cyclic AMP accumulation in a neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms. Mol Pharmacol 45:826–836[Abstract]
  26. Pohjanoksa K, Jansson CC, Luomala K, Marjamaki A, Savola JM, Scheinin M 1997 {alpha}2-Adrenoceptor regulation of adenylyl cyclase in CHO cells: dependence on receptor density, receptor subtype and current activity of adenylyl cyclase. Eur J Pharmacol 335:53–63[CrossRef][Medline]
  27. Hermans E, Challiss RA, Nahorski SR 1999 Effects of varying the expression level of recombinant human mGlu1{alpha} receptors on the pharmacological properties of agonists and antagonists. Br J Pharmacol 126:873–882[Abstract/Free Full Text]
  28. Gazi L, Bobirnac I, Danzeisen M, Schupbach E, Langenegger D, Sommer B, Hoyer D, Tricklebank M, Schoeffter P 1999 Receptor density as a factor governing the efficacy of the dopamine D4 receptor ligands, L-745,870 and U-101958 at human recombinant D4.4 receptors expressed in CHO cells. Br J Pharmacol 128:613–620[Abstract/Free Full Text]
  29. Han M, Lou J, Nakanishi K, Sakmar TP, Smith SO 1997 Partial agonist activity of 11-cis-retinal in rhodopsin mutants. J Biol Chem 272:23081–23085[Abstract/Free Full Text]
  30. Strader CD, Candelore MR, Hill WS, Dixon RA, Sigal IS 1989 A single amino acid substitution in the ß-adrenergic receptor promotes partial agonist activity from antagonists. J Biol Chem 264:16470–16477[Abstract/Free Full Text]
  31. Morin D, Cotte N, Balestre MN, Mouillac B, Manning M, Breton C, Barberis C 1998 The D136A mutation of the V2-vasopressin receptor induces a constitutive activity which permits discrimination between antagonists with partial agonist and inverse agonist activities. FEBS Lett 441:470–475[CrossRef][Medline]
  32. Noda K, Feng YH, Liu XP, Saad Y, Husain A, Karnik SS 1996 The active state of the AT1 angiotensin receptor is generated by angiotensin II induction. Biochemistry 35:16435–16442[CrossRef][Medline]
  33. Groblewski T, Maigret B, Larguier R, Lombard C, Bonnafous JC, Marie J 1997 Mutation of Asn111 in the third transmembrane domain of the AT1A angiotensin II receptor induces its constitutive activation. J Biol Chem 272:1822–1826[Abstract/Free Full Text]
  34. Claude PA, Wotta DR, Zhang XH, Prather PL, McGinn TM, Erickson LJ, Loh HH, Law PY 1996 Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists. Proc Natl Acad Sci USA 93:5715–5719[Abstract/Free Full Text]
  35. Cho W, Taylor LP, Akil H 1996 Mutagenesis of residues adjacent to transmembrane prolines alters D1-dopamine receptor binding and signal transduction. Mol Pharmacol 50:1338–1345[Abstract]
  36. Wurch T, Colpaert FC, Pauwels PJ 1999 G-protein activation by putative antagonists at mutant Thr373Lys {alpha}2A adrenergic receptors. Br J Pharmacol 126:939–948[Abstract/Free Full Text]
  37. Fu ML, Herlitz H, Wallukat G, Hilme E, Hedner T, Hoebeke J, Hjalmarson A 1994 Functional autoimmune epitope on {alpha}1-adrenergic receptors in patients with malignant hypertension. Lancet 344:1660–1663[Medline]
  38. Magnusson Y, Wallukat G, Waagstein F, Hjalmarson A, Hoebeke J 1994 Autoimmunity in idiopathic dilated cardiomyopathy. Characterization of antibodies against the ß1-adrenoceptor with positive chronotropic effect. Circulation 89:2760–2767[Abstract]
  39. Lebesgue D, Wallukat G, Mijares A, Granier C, Argibay J, Hoebeke J 1998 An agonist-like monoclonal antibody against the human ß2-adrenoceptor. Eur J Pharmacol 348:123–133[CrossRef][Medline]
  40. Fu ML, Schulze W, Wallukat G, Elies R, Eftekhari P, Hjalmarson A, Hoebeke J 1998 Immunohistochemical localization of angiotensin II receptors (AT1) in the heart with anti-peptide antibodies showing a positive chronotropic effect. Receptors Channels 6:99–111[Medline]
  41. abu Alla S, Quitterer U, Grigoriev S, Maidhof A, Haasemann M, Jarnagin K, Muller-Esterl W 1996 Extracellular domains of the bradykinin B2 receptor involved in ligand binding and agonist sensing defined by anti-peptide antibodies. J Biol Chem 271:1748–1755[Abstract/Free Full Text]
  42. Borda E, Leiros CP, Bacman S, Berra A, Sterin-Borda L 1999 Sjogren autoantibodies modify neonatal cardiac function via M1-muscarinic acetylcholine receptor activation. Int J Cardiol 70:23–32[CrossRef][Medline]
  43. Masuda MO, Levin M, De Oliveira SF, Dos Santos Costa PC, Bergami PL, Dos Santos Almeida NA, Perdrosa RC, Ferrari I, Hoebeke J, Campos de Carvalho AC 1998 Functionally active cardiac antibodies in chronic Chagas’ disease are specifically blocked by Trypanosoma cruzi antigens. FASEB J 12:1551–1558[Abstract/Free Full Text]
  44. Gether U, Kobilka BK 1998 G protein-coupled receptors. II. Mechanism of agonist activation. J Biol Chem 273:17979–17982[Free Full Text]
  45. Hulme EC, Lu ZL, Ward SD, Allman K, Curtis CA 1999 The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm. Eur J Pharmacol 375:247–260[CrossRef][Medline]
  46. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshirm H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M 2000 Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745[Abstract/Free Full Text]
  47. Zhu SZ, Wang SZ, Hu J, el-Fakahany EE 1994 An arginine residue conserved in most G protein-coupled receptors is essential for the function of the M1-muscarinic receptor. Mol Pharmacol 45:517–523[Abstract]
  48. Fersht AR 1987 Dissection of the structure and activity of the tyrosyl-tRNA synthetase by site-directed mutagenesis. Biochemistry 26:8031–8037[Medline]