WO1999067292A1

WO1999067292A1 - MODIFIED GnRH RECEPTOR

Info

Publication number: WO1999067292A1
Application number: PCT/GB1999/001821
Authority: WO
Inventors: Karin Ann Eidne; Elena Faccenda
Original assignee: Medical Research Council
Priority date: 1998-06-20
Filing date: 1999-06-21
Publication date: 1999-12-29
Also published as: AU4378299A; GB9813279D0

Abstract

Modified GnRH receptors (GnRH-Rs), including human GnRH receptors, are described, together with polynucleotides encoding therefor. The modified GnRH-Rs may be expressed in stable cell lines at significantly higher levels than the equivalent wild-type receptor, whilst the binding characteristics of the receptor remain essentially unchanged. Modifications to human GnRH-R which increase expression include deletion of lysine 191 and addition of a catfish C-terminal tail, optionally also including a C-terminal multi-histidine tag.

Description

"Modified GnRH Receptor"

The present invention relates to a modified form of the gonadotrophin-releasing hormone receptor (GnRH-R) , and to genetically engineered host cells able to express the modified GnRH-R.

The decapeptide gonadotrophin-releasing hormone (GnRH) is released from the hypothalamus and acts through receptors to regulate the secretion of gonadotrophins required for reproductive function (see Fink et al . , "Gonadotrophin secretion and its control", The Physiology of Reproduction, E Knobil and I Neill, New York, Raven Press, pages 1349-1377, 1988). Receptors for GnRH (ie GnRH-R) are members of the large G- protein-coupled receptor family and are preferentially coupled to phosphoinositidase C via the G_q/G _l family of G proteins. Typically GnRH-Rs are located in the gonadotroph cells of the anterior pituitary gland (where binding of GnRH leads to release of the gonadotrophins luteinising hormone and follicle- stimulating hormone) , as well as on certain tumours, on the placenta, nervous system and gonads . GnRH receptors may display both up and down regulation and GnRH agonists have been used in management of prostate and breast cancer, as well as to stimulate gonadotrophin secretion in the treatment of infertility.

Expression of mouse and rat GnRH-R was first achieved by injecting poly (A) + mRNA from a suitable source (eg from the pituitary gland) into Xenopus oocytes (see, for example, Eidne et al . , J. Mol. Endocr. Vol 1, pages R9-R12, 1988; Yoshida et al . , Molecular Endocrinology, Vol 3, pages 1953-1960, 1989; and Sealfon et al . , Molecular Endocrinology, Vol 4, pages 119-124, 1990). This system allowed some characterisation of the pharmokinetics of the GnRH-R.

The protein-encoding nucleotide sequence of the murine GnRH-R was first published by Tsutsu i et al . , (Molecular Endocrinology, Vol 6, pages 1163-1169, 1992) together with the deduced amino acid sequence for murine GnRH-R.

Similar work was reported by Reinhart et al . , J. Biol Chem, Vol 267, No 30, pages 21281-21284, 1992 and the sequence was identical to that reported by Tsutsumi et al . except for a single amino acid (M to L at position 250). The protein-encoding nucleotide sequences for rat GnRH-R and human GnRH-R have also been obtained, using portions of the known murine GnRH-R encoding nucleotide sequence or its flanking regions as probes since GnRH-R homology across mammalian species is very high. The protein-encoding nucleotide sequence for rat GnRH-R has been published by Eidne et al . , Molecular and Cellular Endocrinology, Vol 90, pages R5-R9, 1992; by Kaiser et al . , Biochem Biophys Res Comm, Vol 189, No 3, pages 1645-1652, 1992 and Perrin et al . , Biochem Biophys Res Comm, Vol 191, No 3, pages 1139-1144, 1993 and differs from the mouse sequence by 13 amino acids. Perrin et al . also sequenced the murine GnRH-R and obtained identical results to those reported by Reinhart et al . The protein-encoding nucleotide sequence for human GnRH-R has been published by Kakar et al . , Biochem Biophys Res Comm, Vol 189, No 1, pages 289-295, 1992, and differs from the mouse sequence by 38 amino acids.

Reference is also made to O-A-94/00590 of The Mount Sinai School of Medicine which sets out the nucleotide sequences of murine GnRH-R (with M at position 250) and human GnRH-R (see also Kakar et al . , Biochem Biophys Res Comm, Vol 189, pages 289-295, 1993; and Chi et al . , Molecular and Cellular Endocrinology, Vol 91, pages Rl- R3, 1993) . The GnRH receptor has now been cloned from several other mammalian species, including cow (Kakar et al . , Domestic Animal Endocrinology, Vol 19, pages 335-342, 1993) and sheep (Brooks et al . , Molecular and Cellular Endocrinology, Vol 94, pages R1-R6, 1993; and Illing et al . , Biochem Biophys Res Comm, Vol 196, pages 745-751, 1993) . Non-mammalian GnRH-Rs cloned include catfish (Tensen et al . , European Journal of Biochemistry, Vol 243, pages 134-140, 1997) and Drosophila melanogaster (see Hauser et al . , Biochem Biophys Res Comm, Vol 249, pages 822-828, 1998).

Various of the publications referred to above describe transfected cells able to express the nucleotide sequences encoding the particular form of GnRH-R cloned. Thus for example, Tsutsumi et al . (supra) used the Xenopus oocyte expression system to express the cloned sequence thus confirming its identity, whereas Reinhart et al . (supra) also obtained expression in COS-7 cells, although transfection was only transient. Perrin et al . (supra) reported expression of both the murine and rat GnRH-Rs using COSM6 cells whilst Kakar et al . (supra) obtained transient expression of their human GnRH-R clone in COS-7 cells. Eidne et al . , (1988, 1992 supra) reported expression of rat using both Xenopus oocyte and COS-1 cells.

The mouse and rat GnRH-Rs have each been successfully cloned in stable expression systems which achieve a useful level of GnRH-R protein. However, despite the sequence information available and the production of various expression systems for GnRH-R, to date consistent and reliable expression of the human GnRH-R from cloned cDNA in in vi tro cell systems has not been obtained.

We have now produced a modified human GnRH-R cDNA having a modified nucleotide sequence which is expressed at a high level and/or more consistently.

The present invention relates to the production, cloning and expression of a modified form of GnRH-R. The modifications herein described may be applied, where appropriate, to GnRH-R from any (especially mammalian) species. However, the human form of the receptor is of major interest.

The modified form of GnRH-R, preferably human GnRH-R, of the present invention will have one or more of the following modifications;

1) deletion of Lysine 191; and/or 2) inclusion of a carboxy terminal tail of GnRH-R from a non-mammalian species, preferably to modification 1) above; and/or 3) inclusion of a multi-histidine tag at the carboxy terminal, preferably to modification 2) above. Any one of the modifications set out above improves expression of GnRH-R cDNA in in vi tro expression systems, either by increasing the level of expression or by contributing to the stability of the modified construct.

The present invention thus provides modified GnRH-R including at least one of the following characteristics:

a) absence of Lys 191, optionally together with the addition of a non-endogenous C-terminal tail domain derived from non-mammalian GnRH-R or gonadoliberin receptors; and/or

b) a C-terminal portion comprising either:

i) a C-terminal tag itself comprising at least six positively charged amino acids, the tag being attached to a C- terminal tail domain endogenous to the receptor or present as part of modification a) ; or ii) a non-endogenous C-terminal tail domain derived from non-mammalian GnRH-R or gonadoliberin receptors, together with a C-terminal tag attached thereto, said tag comprising at least six positively charged amino acids.

Desirably the modified GnRH-R is produced by genetic engineering, for example is expressed from a recombinant construct.

Surprisingly we have found that deletion of lysine at position 191 (Lys 191) , if present, greatly enhances expression of the modified GnRH-R. This particular amino acid is located on an extracellular loop (EL2) of the receptor (see Fig. 2) and is not believed to be involved in binding GnRH. The term "Lys 191" is to be understood as meaning the lysine residue present on extracellular loop EL2 (see. Figure 2) and normally in the position which corresponds to amino acid 191 having regard to the accepted numbering of the full length human sequence, notwithstanding any deletion, addition or modification of the amino acid sequence N-terminal thereto. Human GnRH-R modified by deletion of lysine 191 has an amino acid sequence as set out in SEQ ID No 2. The present invention thus comprises a polypeptide having the amino acid sequence of SEQ ID No 2.

Mammalian GnRH-Rs are unusual since they are the only example of G-protein-coupled receptors (GPCRs) cloned to date which lack the intracellular cytoplasmic carboxy terminal (or C-terminal) tail. The C-terminal tail has been implicated functionally.

The cloning of non-mammalian GnRH-Rs (which term refers to GnRH-Rs and gonadoliberin receptors from a non- mammalian source) , such as GnRH-Rs from chicken, catfish, goldfish and frog, has shown the existence of carboxy terminal tails on these primitive receptors. In particular, the catfish (cf) GnRH-R expresses highly in in vi tro systems. It is hypothesised that the tail structure may stabilise the receptor protein within the cell membrane resulting in higher levels of cell surface expression. Lin et al . , (see Molecular Endocrinology, Vol 12, pages 161-171, 1998) have shown that a chimeric receptor formed from rat GnRH receptor together with the C-terminal tail of cf GnRH-R expresses at a higher level (approximately 5 fold increase) in GH₃ cells relative to the WT rat GnRH-R. However, the cDNA of the chimeric receptor was expressed only transiently.

Whilst the teaching of Lin et al . (supra) might suggest that inclusion of a cf C-terminal tail to human GnRH-R would likewise promote expression of the chimeric construct in cell lines, our experience indicates that this is not necessarily the case. Fig. 3B illustrates results of experiments in which a chimeric protein was formed from the WT human GnRH-R plus the C-terminal tail of cf GnRH-R. The human GnRH-R/cf C-terminal tail chimera analogous to the rat GnRH-R plus cf C-terminal tail chimera of Lin et al . (supra) was not however stably expressed.

Likewise, a chimeric protein comprising rat GnRH-R together with the C-terminal tail of a different G- protein-coupled receptor (thyrotropin-releasing hormone receptor or TRH-R) has been reported (see Heding et al . , Journal of Biological Chemistry, Vol 273, pages 11472-11477, 1998) . No increased level of expression or stability of the chimeric protein was noted. The improved stability does not appear to be simply related to the presence or absence of the C-terminal tail structure.

A substantial increase in expression and/or stability of human GnRH-R modified by addition of the cf C- terminal tail was however achieved when amino acid 191 (lysine) was deleted from the amino acid sequence. Lysine 191 is known to be located on an extracellular loop of mammalian GnRH-R, whereas the C-terminal tail, when present, is positioned intracellularly . The physical relationship between these two portions of the receptor is thus remote and a functional connection between these two modifications could therefore not have been predicted. However, again, our results show that addition of a lysine at position 191 to rat GnRH-R does not affect expression rates as might be expected.

The carboxy terminal tail referred to m modification 2) above is preferably derived from catfish GnRH-R (cf GnRH-R) . The nucleotide sequence encoding for cf GnRH- R has been published by Tensen et al . , European Journal of Biochemistry Vol 243, pages 134-140, 1997 and the tail is comprised of the C-termmal 51 ammo acids. Thus, in a preferred embodiment the modified GnRH-R comprises human GnRH-R plus the C- erminal 51 ammo acids of cf GnRH-R (see SEQ ID No 4) . Alternatively, the carboxy terminal tail may be derived from any other non-mammalian GnRH-R, and suitable examples include goldfish, chicken and frog GnRH-R (see Troskie et al . , Program of the 79th Annual Meeting of The Endocrinology Society, Minneapolis, Minnesota Pl-130, Abstract, 1997; and Pawson et al . , Journal of Endocrinology, Vol 156, pages R9-R12, 1998) .

Preferably the whole of the non-mammalian GnRH-R C- terminal tail is present. At least all of the putative phosphorylation sites (i.e. at threon e and senne residues) present the non-mammalian GnRH-R C- terminal tail should desirably be present. In catfish GnRH-R, the putative phosphorylation sites are located at Ser 331 and Ser 348 ( SEQ ID Nos 4 and 6 the latter residue appears as Ser 345 with a Ser residue corresponding to cf Ser 331 being lost the bridging of the two sequences). Optionally some modifications, including small insertions or single ammo acid deletions or substitutions, may be tolerated without substantially reducing the increased expression/stability achieved by addition of the whole C-termmal tail. Human GnRH-R modified by deletion of Lys 191 and addition of a cf terminal tail has an amino acid sequence as set out in SEQ ID No 4. The present invention thus comprises a polypeptide having the amino acid sequence of SEQ ID No 4. In SEQ ID Nos 4 and 6, the human GnRH-R portion of the chimeric sequence terminates at Ser 326, with the terminal Leu (αα327) being replaced with the bridging amino acids Asp and Arg (Nos 327 and 328 in the chimeric sequence) . The cf c-terminal tail portion commences at Phe 329 onwards and corresponds to the sequence of the WT cf GnRH-R of Phe 332 onwards.

As mentioned above, small variations in the sequence of the C-terminal tail may be tolerated. Figure 2 illustrates a modification of the tail and of its connection to the human sequence. Thus, in the Figure 2 sequence (SEQ ID No 14) , the human portion of the chimeric construct terminates at Leu 328, and the cf C- terminal tail commences with Ser 331 (the putative phosphorylation site) . Desirably, the additional Phe 339 and Thr 340 (not present in the wild type sequence) may be deleted from the construct.

In modification 3), a further increase in receptor stability is obtained. A multi-histidine tag is commonly used to aid purification of a protein, the highly charged histidine tag binding to species such as nickel in affinity chromatography techniques. To our knowledge inclusion of a multi-histidine tag (typically comprising 6 to 12 histidine residues) is normally associated with reduced expression and function. We are not aware of any previous multi-histidine tagged protein experiencing increased expression or stability relative to the WT (non-tagged) protein.

Thus, Fig. 9 shows the results of experiments in which a multi-histidine tag (6 histidine residues) was located either at the N-terminal of WT rat GnRH-R (ATG- His 6) or at the known restriction site (Esp 31) close to the N-terminal (His 6-Esp 31) . Expression of the tagged receptors were reduced by approximately 80% and 60% respectively.

The C-terminal tag desirably comprises at least 6 positively charged amino acid residues, for example histidine residues. The C-terminal tag advantagously comprises only positively charged residues, but this is not absolutely essential and the presence of some other amino acids in the tag sequence may be tolerated. In one embodiment the C-terminal tag is added to the GnRH- R modified by addition of a carboxy terminal tail of a non-mammalian form of GnRH-R. Optionally, Lys 191 (where present) is also deleted and this provides additional benefits in terms of expression. Human GnRH-R modified in this way has an amino acid sequence as set out in SEQ ID No 6. Thus the present invention provides a polypeptide having the sequence of SEQ ID No 6. An alternative sequence where Lys 191 is not deleted is shown in Figure 2 and set out in SEQ ID No 14.

In a further aspect, the present invention provides a polynucleotide encoding a GnRH-R modified as described above. For example, the invention provides a polynucleotide having a sequence substantially as set out in SEQ ID No 1, SEQ ID No 3 or SEQ ID No 5.

SEQ ID No 1 sets out the cDNA sequence of the 293 -C4 cell line. The protein encoding region of the cDNA starts at nucleotide 1 and ends with nucleotide 984 (the stop codon) and the amino acid sequence is set out in SEQ ID No 2. SEQ ID No 3 sets out the cDNA sequence of the 293 -C9 cell line. The cDNA sequence includes flanking regions; the protein encoding region of the cDNA starts at nucleotide 1 and ends with the stop codon at nucleotide 1131 (see SEQ ID No 4 for amino acid sequence) . The cf tail commences at nucleotide 987 onwards .

SEQ ID No 5 sets out the cDNA sequence of the 293-C15 cell line. The protein encoding region of the cDNA starts at nucleotide 1 and ends with the stop codon at nucleotide 1161 (see SEQ ID No 6 for amino acid sequence) .

By "substantially" we include modified sequences retaining GnRH-R function (ie ability to bind GnRH) and having at least 70% homology (preferably 80% homology, especially preferably 85-90% homology) with the nucleotide sequence in question. Functional equivalents of such polynucleotides are also part of this invention. In particular, we include nucleotide substitutions which do not affect the amino acid expressed. Thus, for example, amino acid Glu 8 may be encoded by the codon gag or by the codon gaa and each construct (SEQ ID Nos 1, 3 and 5) may be varied in this way.

The polynucleotides may be in any form (for example DNA or RNA, double or single stranded) but generally double stranded DNA is the most convenient. Likewise the polynucleotides according to the present invention may be part of a recombinant genetic construct, which itself may include a vector (for example an expression vector) or may be incorporated into the genome of a transgenic animal. Any vectors or transgenic animals comprising a polynucleotide as described above form a further aspect of the present invention.

Viewed in a yet further aspect the present invention provides a recombinant expression system able to express the modified GnRH-R described above. DNA constructs (ie a standard vector recombinantly combined with an polynucleotide sequence coding for the modified GnRH-R of interest) and cells transformed with such constructs are also encompassed by the present Application.

The term "expression system" is used herein to refer to a genetic sequence which includes a protein-encoding region and is operably linked to all of the genetic signals necessary to achieve expression of that region. Optionally, the expression system may also include a regulatory element, such as a promoter or enhancer, to increase transcription and/or translation of the protein encoding region or to provide a control over expression. The regulatory element may be located upstream or downstream of the protein encoding region or within the protein encoding region itself.

The term "functional equivalent" used herein refers to any derivative in which nucleotide (s) and/or amino acid(s) have been added, deleted or replaced without a significantly adverse effect on expression of the gene product or on biological function thereof.

There is a substantial body of knowledge concerning the techniques required for the art of genetic engineering and reference is made to Maniatis et al , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 1982 and "Principles of Genetic Engineering", Old and Primrose, fifth addition, 1994. In addition to the recombinant modified GnRH-R construct described above, the present invention also provides host cells transformed with such constructs and which may express the biologically active modified gene product .

In a further aspect, the present invention provides a stable cell-line capable of expressing modified GnRH-R, preferably a modified form of human GnRH-R, as described above. By "stable" we mean that the cell- line retains its ability to express useful quantities of GnRH-R after several (eg 10) generations, with any decrease in the level of GnRH-R expression being sufficiently low not to materially affect the utility of the cell-line.

Desirably the host cell transformed with the construct encoding the modified form of human GnRH-R is of mammalian origin, but other cell types may also be useful. Examples include prokaryotic cells (such as E . coli ) , non-mammalian derived eukaryotic cells (such as insect, yeast or plant cells) . Suitable host cells include COS-1 cells, COS-7 cells, COSM6 cells, CHO cells, BHK cells, GH₃ cells, HEK293 cells and 2.93EBNA cells.

Using a combination of approaches we have generated a range of stable cell lines in both HEK293 and 293EBNA cells expressing WT and modified hGnRH and rGnRH receptors.

The 293EBNA cell line (an isogenic derivative of HEK293 cells expressing the Epstein Barr virus nuclear antigen 1 [EBNA1] ) is permissive to episomal replication and expression of cloned genes in plasmids carrying the Epstein Barr virus origin of replication (EBV oriP) . These cells also express the Ela gene from adenovirus type5 which acts on the CMV immediate early promoter to greatly enhance transcription and subsequent expression of the desired gene. 293EBNA cells have been shown to be suitable for the rapid generation of stable cell lines (Horlick et-al., Protein Expression and Purification, Vol 9, pages 301-308, 1997) resulting in greater levels of protein production than from the parental HEK293 cell line.

Each of the constructs encoding for modified GnRH-R as described above were used to transform COS-7, HEK293 and/or 293EBNA cells. Each of these transformed cells formed a stable cell-line expressing the modified GnRH- R. Examples of stable cell-lines expressing modified GnRH-R according to the invention include 293-C4 (alternatively termed SCL 118) , 293-C9 (alternatively termed SCL146) and 293-C15 (alternatively termed SCL155) (all in HEK293) . These cell-lines continue to express relatively high levels of human GnRH-R and are now several generations old.

The modified GnRH-R can be expressed and used to screen agents of potential therapeutic interest. Thus, the present invention further provides a method of screening an agent for pharmacological activity (i.e. to ascertain its utility in binding to GnRH-R) , said method comprising:

a) providing a modified GnRH-R as described above and exposing said modified GnRH-R to the agent; and b) ascertaining whether said agent interacts with said modified GnRH-R.

An expression system able to produce the modified GnRH- R described above may be used in this method; preferably the expression system will be a transformed cell-line, the host cell usually being of mammalian origin. Preferred cell-lines include the stable HEK293 cell lines 293-C4 (or SCL 118) , 293-C9 (or SCL 146) and 293-C15 (or SCL 155) . Alternatively the expression system may be a transgenic animal.

An expression system able to produce the modified GnRH- R described above may be used to screen agents of potential therapeutic use (such as GnRH agonists or antagonists) . Desirably, therefore the expression system will imitate at least some aspects of GnRH- induced signal transduction. Optionally, the expression system may be a stable cell line, the host cell usually being of mammalian origin. Preferred cell-lines include 293-C4 (or SCL 118) , 293-C9 (or SCL 146) and 293-C15 (or SCL 155) . Alternatively the expression system may be a transgenic animal .

Suitable expression systems include ΔK191 hGnRH-R in HEK293 cells (ie the 293-C4 or SCL118 cell line) ; ΔK191 hGnRH-R+cf tail in HEK293 cells (ie the 293-C9 or SCL146 cell line) and ΔK191 hGnRH-R+10His cf tail in HEK293 cells (ie the 293-C15 or SCL155 cell line) .

In another embodiment of the invention, the modified GnRH-R itself could be administered in vivo . The free GnRH-R could competitively bind to GnRH and inhibit its reaction with the native receptor in vivo .

Alternatively, the modified GnRH-R may be used as a means of contraception. For example, a patient may be immunised by injection with the modified GnRH-R. This will induce antibody production to the modified GnRH-R and the antibodies so produced will also interact with native GnRH-R affecting reproductive ability. The invention will now be further described with reference to the non-limiting, examples, and figures in which:

Figure Legends

Figure 1. Alignment of partial amino acid sequences of the GnRH receptor from various species . The amino acid sequence (from residues 183 to 200) indicating position 191 for human, marmoset, sheep, cow, pig, rat and mouse GnRH-R is shown on the left (SEQ ID Nos 7 to 13 respectively) with the bars on the right comparing specific ¹²⁵I-des-Gly¹⁰, [D-Trp⁶] GnRH binding levels of the GnRH receptors from the indicated species when expressed in COS-7 cells. Transient transfections and receptor binding assays were performed as described in the Materials and Methods. The bars are the mean of triplicate observations ± SEM, n=3. n/d = not determined.

Figure 2. Two-dimensional representation of the human GnRH receptor with an added C-terminal region. Two-dimensional representation of the wild type human GnRH receptor locating K191 within extracellular loop 2 (EL2) . The amino acid sequence of the catfish tail plus the carboxy terminal lOHis tag is inserted at the C-terminus. SEQ ID No 14 sets out the amino acid sequence shown.

Figure 3A. Saturation assay of WT and modified hGnRH-R constructs. Membrane preparations from COS-7 cells were assayed for des-Gly¹⁰, [D-Trp⁶] GnRH binding in saturation assays 48 hours post transient transfection of receptor constructs in pcDNA3 vector. The points on the curves represent the mean of triplicate determinations + SEM, n=3.

Figure 3B. Specific binding of WT and modified hGnRH-R constructs. Specific binding determinations of des-Gly¹⁰, [D- Trp⁶]GnRH binding to membranes prepared 48 hours post transient transfection of receptor constructs in pcDNA3 vector. Points represent the means of triplicate determinations + SEM, n=3.

Figure 4. Saturation assay comparing increasing membrane concentrations from a HEK293 stable cell line expressing ΔK191 hGnRH-R +10His cf tail. Saturation assay comparing level of ligand binding in the presence of increasing amounts of membrane protein. A range of one to thirty μg of total membrane protein from the stable cell line was used per triplicate determination measuring self-displacement of ¹²⁵I-des- Gly¹⁰ , [D-Trp⁶] GnRH . n=3 , ±SEM .

Figure 5. Saturation binding of wild type rat GnRH receptor and +K191rGnRH-R. COS-7 membranes expressing wild type rat GnRH receptor or +K191rGnRH-R were assayed for ¹²⁵I-des-GlylO ,.[D- Trp6]GnRH binding. Points are representative of n=2 experiments performed in duplicate, ± SD. Wild type rGnRH receptor is represented by the filled squares and the mutant +K191rGnRH-R by the unfilled squares.

Figure 6. Specific binding to start to stop (s-s) rat and human GnRH receptors. GnRH receptor constructs containing no UTR regions were expressed in COS-1 cells. Membrane preparations from these transiently transfected cells were assayed for binding of ^{1 5}I-des-Gly¹⁰, [D-Trp⁶] GnRH in the presence of increasing amounts of the unlabeled agonist. Specific binding = total binding - non-specific binding. Bars are represented ±SEM, n=3.

Figure 7. Comparison of human GnRH receptor expression comparing two vector systems. Wild type and modified hGnRH receptors were expressed in HEK293 cells using either the mammalian expression vector pcDNA3 or the episomally replicating expression vector pREP4. Results are shown as a percentage of the expression level obtained using WT hGnRH receptor in pcDNA3 after correction for protein concentration. White bars are pcDNA3 and filled bars are pREP4. Errors shown are ±SEM of triplicate determinations, n=2.

Figure 8. Comparison of ligand binding to intact cells and total membrane preparations. Radioligand binding to intact cells (cell surface) or prepared total membrane samples was measured and expressed as a percentage of WT hGnRH receptor binding. White bars represent total membrane binding and filled bars represent cell surface binding to intact cells.

Figure 9. Comparison of expression levels for .WT rat GnRH-R, and two different multi-histidine labelled forms of WT rat GnRH-R. A tag of 6 histidine residues were located either at the N-terminal of WT rat GnRH-R (ATG-His 6) or at a known restriction site (Esp 31) close to the N-terminal (His 6 -Esp 31) . Expression of the tagged receptors were reduced by approximately 80% and 60% respectively. EXAMPLES

Materials and Methods

Modification 1 : Si te -directed mutagenesis The hGnRH receptor cDNA (in pcDNA3 , Invitrogen, DeSchlep, Netherlands) was mutated to remove the codon for lysinel91 using a single-stranded oligonucleotide directed method (Kunkel et al . , PNAS, Vol 82, pages 488-492, 1985, see also Kunkel et al . , Methods in Enzymology, Vol 154, pages 367-382, 1987) using the mutagenic oligonucleotide 5'- CACATTGAGAGAAAACTGTCTGTCCAGAGCTGTC (SEQ ID No 15) . Mutated clones were identified by automated fluorescent DNA sequencing using BigDye Terminator chemistry (ABI , Warrington, UK) . This generated the clone ΔK191 hGnRH- R and the nucleotide sequence is set out in SEQ ID No 1.

The rat GnRH receptor was mutated so as to introduce a lysine at the equivalent position to K191 in the human GnRH receptor sequence (i.e. between alanine 190 and valine 191 of the rat sequence) . This mutagenesis was performed using the QuikChange site-directed mutagenesis kit following the manufacturers instructions (Stratagene, Cambridge, UK) and the oligonucleotide 5 ' -CATTGCGAGAAAACTTTTGCTGGCCCAGAGCCG (SEQ ID No 16) . The underlined nucleotides code for the inserted lysine residue. This generated the mutant +K191 rGnRH-R.

Modification 2 : Tailing the wild- type hGnRH-R and AK191 hGnRH-R Both ΔK191 and WT hGnRH-R constructs were PCR amplified with the upstream oligonucleotide 5'- CCACCATGGCAAACAGTGCCTCTCC (SEQ ID No 17) and the downstream oligonucleotide 5 ' -CTATCAATCGATCAGAAAAATATCC (SEQ ID No 18) which contains a Clal site. The five 5' nucleotides (CCACC) of the upstream primer (SEQ ID No 17) adds a Kozak translation initiation site at the ATG. The CCACC start to the upstream primer (SEQ ID No 17) is optional and these nucleotides can be omitted without affecting primer function. The presence of the Kozak sequence did not have a beneficial effect on expression level. All PCR reactions were performed using the proof-reading DNA polymerase, ULTMA (Perkin Elmer, Warrington, UK) . The resulting PCR bands were A-tailed using Taq DNA polymerase and cloned into the TA vector TOPOII (Invitrogen, DeSchlep, Netherlands) , and incubated with Clal restriction endonuclease . The carboxy terminal tail region of the cf GnRH-R (serine 331-Stop, (Tensen et al . , supra, 1997)) was PCR amplified, again using ULTMA DNA polymerase. Oligonucleotides used were: upstream 5'- CGCCATCGATTCGTGCCGACTTGTCC (SEQ ID No 19) and downstream 5' -CAAGAATCGCAATACAAATCGATCCGGCACCTAC (SEQ ID No 20) . The upstream primer contains a Clal site and the downstream primer anneals across an endogenous Clal site within the 3' untranslated region (UTR; nt 1363) of the cfGnRH receptor sequence. This PCR band encompassing the entire carboxy terminal tail of the cfGnRH receptor was TA cloned as before, released by Clal and gel purified (Gel Purification Kit, Qiagen, Crawley, UK) . The tail fragment was then ligated into Clal digested recipient hGNRH-R DNA and transformed into E. coli for the selection of positive clones. The orientation and integrity of the tail insert were confirmed by DNA sequencing. This modification process generated two constructs: WT hGnRH-R+cf tail and ΔK191 hGnRH-R+cf tail . The nucleotide sequence of construct AK191 hGnRH-R+cf tail (modification 2) is given in SEQ ID No 3. Modification 3 : Addition of l OHistidine (lOHis ) tag to tailed receptor constructs Wild type hGnRH-R+cf tail and ΔK191 hGnRH-R+cf tail DNAs were PCR amplified with the upstream primer 5'- CCACCATGGCAAACAGTGCCTCTCC (SEQ ID No 21) and a downstream primer containing a portion annealing to the 3' end of the cfGnRH receptor tail (deleting the endogenous Stop codon) plus ten histidine codons followed by a Stop codon (5'- TTAATGGTGATGGTGATGATGGTGATGGTGGTGCTGTCCACTGGGTTGGTCACC - SEQ ID No 22) . The five 5' nucleotides (CCACC) of the upstream primer (SEQ ID No 21) adds a Kozak translation initiation site at the ATG. The CCACC start to the upstream primer (SEQ ID No 21) is optional and these nucleotides can be omitted without affecting primer function. The presence of the Kozak sequence did not have a beneficial effect on expression levels. The PCR bands were cloned directly into the T0P03.1 TA cloning vector (Invitrogen, DeSchlep, Netherlands) and incorporation of the tag in a correct in- frame position was confirmed by DNA sequencing. This modification generated two more constructs: WT hGnRH-R+10His cf tail and ΔK191 hGnRH-R+10His cf tail, neither of which contain any UTR sequence. The nucleotide sequence of construct AK191 hGnRH-R+10His of tail is given in SEQ ID No 5. The primary structure of the human GnRH receptor is depicted in Figure 2 which also highlights the location of K191 in the second extracellular loop (EL2) and the amino acid sequence of the cfGnRH receptor tail with the added carboxy terminal lOHis tag.

Start- o-stop constructs The coding regions of rat and human GnRH receptor cDNAs were PCR amplified from the ATG translation start site to the stop codon using primers matching published sequences generating start-to-stop (s-s) receptor constructs which were cloned into pcDNA3 (Invitrogen, DeSchlep, Netherlands) . These constructs therefore lacked any untranslated sequences.

Transient transfection into COS- 7 cells Cells were seeded into T75 tissue culture flasks (3 x 10⁶ cells/flask) on the day prior to transfection. DNA was introduced into monolayer cultures of COS-7 cells using a standard DEAE-dextran protocol (Promega, Madison, USA) using lOμg DNA/flask.

Transfection into HEK293 cells Cells were seeded into 60mm tissue culture plates 24 hours prior to transfection (1.5 x 10⁶ cells/plate). DNA was transfected into the cells using Superfect transfection reagent according to the manufacturers instructions (Qiagen, Crawley, UK) . For stable transfections the respective receptor constructs in pcDNA3 were linearized downstream of the coding region, and 5μg of DNA was used per transfection. After 24 hours the cells were transferred into a T75 tissue culture flask, and then into a T162 tissue culture flask when they reached 60% confluency. On reaching 50-60% confluency in the T162 tissue culture flask G418 (a neomycin analog, Calbiochem, Nottingham, UK) was added to the culture medium at a final concentration of lμg/ml and cells were incubated in antibiotic replenished medium until focal colonies formed. Selected colonies were harvested and expanded to test for receptor expression. Highly expressing clones were maintained for further characterization. Stable cell lines expressing ΔK191hGnRH-R, ΔK191hGnRH-R+cf tail and ΔK191hGnRH-R+10His cf tail were established using this system (stable cell lines (SCL) identified as SCL118, SCL146 and SCL155 respectively) . No stable cell lines expressing WT hGnRH receptor or its modifications were obtained.

Transient and stable transfection into 293EBNA cells All constructs were subcloned into Kpnl/Xhol digested pREP4 vector DNA (Invitrogen, DeSchlep, Netherlands) using standard methods. Transient expression was performed using Superfect transfection reagent at a DNA: reagent ratio of 1:3 following the manufacturers protocol. Cells were assayed for receptor expression 48 hours post transfection. Stable transfection into 293EBNA cells does not require linearization of the plasmid DNA as pREP4 constructs replicate episomally and are passed on to daughter cells. Cell lines generated by this method were not clonally selected but were maintained on lOOμg/ml of hygromycin to select against cells not carrying vector/receptor.

Receptor Binding Assays Membrane assays : Mammalian cell membranes were prepared 48 hours post transfection or from PBS/EDTA harvested stable cell cultures. Membranes were resuspended in assay buffer (AB, 40 mM Tris-HCl, 2 mM gCl₂, pH 7.2). The peptide des-Gly¹⁰, [D-Trp⁶] GnRH Ethylamide (D-Trp⁶GnRH) was obtained from Sigma. Displacement assays were performed with radiolabeled ^12SI -des-Gly¹⁰, [D-Trp⁶] GnRH (100 , OOOcpm) , 30μg cell membranes expressing the GnRH-R constructs ± 50 μl of agonist (doses 0 to 10^*6M) in a total assay volume of 500μl. Saturation binding assays were performed using the radiolabeled GnRH agonist ¹²⁵I-des-Gly¹⁰, [D-Trp⁶] GnRH (0.25 nM to 5.0 nM) + 10^'6M des-Gly¹⁰, [D-Trp⁶] GnRH in a total assay volume of 500μl. Both types of assays were incubated for 2 hours at 4°C prior to filtration onto Whatman GFB filter paper (BDH, Lutterworth, UK) . Receptor dissociation constant (Kd) and receptor number (Bmax) were calculated using a hyperbolic function fit with Sigma Plot (Jandel Scientific) . All assay points were in duplicate/triplicate and experiments were performed at least three times.

Whol e cell (surface receptor) assays : EDTA harvested cells were counted and resuspended in cold HEPES buffered DMEM/0.1% BSA at a concentration of 2xl0⁶ cells/ml, and 500μl aliquoted into 1.5ml centrifuge tubes. Each tube contained 100,000 cpm radioligand to measure total receptor binding. Non-specific binding was determined in the presence of 10^"6M unlabeled GnRH agonist. Cells were pelleted by centrifugation following incubation at 4°C for 2 hours, washed 2x in 1ml cold PBS and the radioactivity bound by the cells was measured.

Total inositol phosphate (IP) measurement Cells were plated out into 24-well plates and labelled with 1.0 μCi/well ³H-myo- inositol in inositol-free DMEM (GIBCO, Paisley, UK), dialysed HIFCS, 0.3 mg/ml glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin. The cells were incubated for a further 48 hours and then treated with GnRH (human, Sigma).. Total IP production was assessed as described elsewhere (Heding et al . , supra, 1998).

Results

Modification 1: Deletion of lysinel91 in the human GnRH receptor Sequence alignments of GnRH receptors from various species revealed an extra lysine residue at position 191 in the hGnRH receptor when compared with other species (Figure 1) . Figure 2 shows the location of this residue in the second extracellular loop (Figure 2) . A deletion mutant of lysinel91 was generated, ΔK191 hGnRH-R, as this extra amino acid appears only in GnRH receptors from the different species with which we have experienced expression level problems. Both rat and mouse GnRH receptors are one amino acid shorter at this position and binding levels are approximately 25 times higher than for either the hGnRH or a primate GnRH receptor (Figure 1) .

When transiently expressed in COS-7 cells ΔK191 hGnRH-R expresses approximately twice as many GnRH receptors compared to wild type (WT) (ΔK191 hGnRH-R 1.6 pmol receptor/mg total protein compared to 0.8 for WT hGnRH- R and 2.5 for WT rat GnRH-R) . ΔK191 hGnRH-R was also used to establish a stable cell line in HEK293 cells (293-C4 and SCL118) which expresses 2.8 pmol receptor/mg total protein. There is no WT stable cell line for comparison, but the previously established rat GnRH-R cell line 293-A2 expresses 6.2 pmol receptor/mg total protein. The affinity of ΔK191 hGnRH-R for the GnRH analogue dTrp6-LHRH is unchanged from that of the WT hGnRH-R (1.1 nM for each) . Table 1 below summarises expression as measured by radioligand binding in saturation assays using membrane preparations.. Measurements of transient transfection were performed 48 hours after transfection with a DEAE-dextran method.

Modifications 2 and 3: Effects of adding cf GnRH receptor tail and lOHis tag to ΔK191 hGnRH-R To test the effect of expressing the hGnRH receptor as a tailed GPCR we engineered the sequence coding for the cfGnRH receptor carboxy terminal tail into the stop codon (Figure 2) . Adding the cf tail to ΔK191hGnRH-R increases expression from 1.6 (±0.3) pmol/mg protein to 5.3 (±0.3) pmol/mg protein. An affinity purification tag consisting of ten histidines (lOHis) was also incorporated into the COOH terminus of the ΔK191 + cf tail hGnRH receptor (Figure 2) . Addition of the lOHis tag further increases expression, resulting in transient expression of ΔK191 + lOHis cf tail hGnRH-R at 11.5 (±0.2) pmol/mg protein. This latter level of expression represents a 14-fold increase compared to WT hGnRH receptor.

The construct ΔK191 hGnRH-R+cf tail was expressed both transiently (in COS-7 cells) and stably (in HEK293 cells) and radioligand binding was compared to ΔK191 hGnRH-R alone and to WT hGnRH-R (see Figures 3A and 3B and Table 1) . Addition of the cf tail increased transient expression by 7 -fold compared to WT human GnRH-R in COS-7 cells, resulting in the production of 5.3 pmol receptor/mg total protein. The stable cell line expressing this chimeric construct (293 -C9) produces 14.4 pmol receptor/mg total protein, a 5-fold increase compared to 293 -C4 cells.

Expression in COS-7 cells of ΔK191 hGnRH-R+10His cf tail gives 11.5 pmol receptor/mg total protein, a 14- fold increase over ΔK191 hGnRH-R alone, and 5-fold increase over the WT rGnRH-R in the same experiments. A stable cell line (termed 293-C15) was established in HEK293 cells which expressed 44.2pmol receptor/mg proteins, a 50-fold increase over WT.

These results are shown graphically in Figure 3A, and are summarized in Table 1. 1 Table 1. Summary of ligand binding parameters of GnRH

2 receptor constructs in transient (COS-7) and stable

3 (HE 293) transfections.

4 Cell membrane preparations were analysed in saturation

5 binding assays using the GnRH agonist dTrp6-LHRH. Bmax

6 values were generated using the programme SigmaPlot and

7 were subsequently used to calculate the number of

8 binding sites as pmol of receptor/mg of membrane

9 protein. COS-7=transient transfections and

10 HEK293=stable cell lines. n/a= not available. The

11 results represent the mean of 3 experiments in each

12 case, + SEM. 13

14 Effects of adding cf GnRH receptor tail and lOHis tag

15 to human WT and rat WT GnRH receptors

16 Figure 3B shows levels of specific GnRH agonist binding

17 to WT and modified human and rGnRH receptors. Adding

18 the cf tail to the rGnRH receptor increases its

19 expression significantly compared to the WT rGnRH

20 receptor. Subsequent addition of the lOHis tag to the

21 tailed rGnRH receptor has no effect on specific binding

22 levels. By comparison, addition of neither the cf GnRH

23 receptor tail nor the lOHis tag has any measurable

24 increasing effect on the expression of the WT hGnRH

25 receptor. However, addition of the cf tail to

26 ΔK191hGnRH-R substantially increases its expression

27 level, giving a construct which expresses as well as the WT rGnRH-R. In contrast to the rat, inclusion of a lOHis tag in the human construct increases its expression level even further, with this construct (ΔK191hGnRH-R+10His cf tail) producing even more membrane bound receptor than the equivalent rat construct. The affinities (Kd values) of the modified receptors for des-Gly¹⁰, [D-Trp⁶] GnRH remain unchanged from WT values. Binding characteristics are summarized in Table 1 (see above) .

Binding to a selected HEK293 stable cell line expressing ΔK191 hGnRH-R+10His cf tail A stable cell line clone of ΔK191hGnRH-R+10His cf tail (SCL155) was expressing in excess of 40 pmol receptor /mg total membrane protein equivalent to 1.8 million receptors/cell. Saturation analysis carried out at different concentrations of membrane preparations from this cell line showed that it is still possible to achieve reasonable results using as little as 5μg of protein/replicate as shown in Figure 4.

Agronis -induced total inosi tol phosphate production by wild type and modified human GnRH receptors Total inositol phosphate measurements were carried out on cell lines stably expressing wild type and modified human GnRH receptors following stimulation with GnRH. Cells expressing wild type GnRH-R gave ED50 values of 15nM, while cells expressing ΔK191 hGnRH-R gave ED50 values of 19nM (Table 2) . There is no apparent decrease in the ED50 value arising from GnRH stimulation in cells expressing ΔK191 hGnRH-R while cells expressing ΔK191 hGnRH-R+10His cf tail show a decrease to O.lnM. The maximal stimulation figures (all from basal levels of approximately 800dpm) are indicated in Table 2. There is a large increase in the magnitude of the agonist -induced inositol phosphate response mediated by the ΔK191 hGnRH-R and ΔK191 hGnRH- R+lOHis cf tail when compared with wild-type receptor. These constructs show much higher levels of IP stimulation, giving 48-60 fold increases over basal IP turnover rates compared to the 4-5 fold increase in basal IP rates mediated by the wild-type human GnRH-R.

Table 2. Total Inositol Phosphate Production in stable HEK293 cell lines expressing wild type and modified human GnRH receptors.

GnRH induced total inositol phosphate accumulation in HEK293 cells stably expressing WT and modified human GnRH receptors. Cells in 24-well plates were stimulated for 60min before total IP was measured as described under Materials and Methods. Results generated are the mean of triplicate determinations from a single representative experiment.

Effect of inserting lysine into the rat GnRH receptor (+K191rGnRH-R) Figure 5 shows results of a saturation assay using COS- 7 cell membranes prepared from transfections with wild type rat GnRH receptor and +K191rGnRH-R cDNAs . There was a slight decrease in +K191rGnRH-R Bmax values (7.6 + 0.2 pmol/mg membrane protein) compared with that for the WT GnRH receptor (Bmax = 8.2 + 0.3 pmol/mg membrane protein) which is a significant decrease, but is nowhere near as low as the levels observed for the WT hGnRH receptor. Effect of removing untranslated regions (s-s constructs) A possible explanation for the reduced levels of expression of the human WT GnRH receptor could be the presence of RNA instablity domains contained with the -3' untranslated regions (UTR) of these receptor cDNAs . The hGnRH receptor 3 'UTR has one such domain at nt 1207-1211. In contrast the rGnRH receptor 3 'UTR has three such domains at nt 1774-1778, 1798-1802 and 1840- 1844 and one could therefore imagine this to give a more unstable mRNA than its human counterpart. We therefore wished to establish the effects of removal of the UTRs on both human and rat GnRH receptor binding levels. Receptors were PCR amplified using oligonucleotide primers corresponding to the the start and stop codons generating start to stop (s-s) constructs. Figure 6 shows that removal of the UTRs does not significantly increase the level of binding to the hGnRH receptor. There is a significant increase (by approximately 40%) in specific binding to the rGnRH receptor when the untranslated regions are removed. This holds with the proposition that being more unstable, the rGnRH receptor mRNA benefits more from UTR removal than does the hGnRH receptor mRNA..

Comparison of pcDNA3 and pREP4 expression systems In order to establish whether expression using another mammalian vector system might improve receptor levels, we compared the binding of receptor constructs in pcDNA3 with the same constructs cloned into pREP4. Figure 7 represents specific binding of WT and mutated modified hGnRH receptors. The data are expressed as a percentage of the level of expression achieved using the pcDNA3 system. In almost all cases pREP4 driven expression exceeds pcDNA3 expression levels. The exception is ΔK191hGnRH-R+10His cf tail which is not significantly altered by expression in the pREP4 system. The pREP4 expression system sufficiently raises levels of WT hGnRH receptor expression to permit measurement of the effect of tailing and tagging which was not previously possible with pcDNA3. In fact, having corrected for protein concentration, adding the cf GnRH receptor tail to the WT hGnRH increases expression slightly, although not significantly, above WT alone. However, WT + lOHis cf tail GnRH-R does express significantly better than either WT alone or WT + cf tail hGnRH-R.

Evaluation of the levels of receptor on the cell surface compared to total membrane receptor content Specific binding to either intact cells or membrane fractions prepared from the same numbers of cells was measured. Compared to WT hGnRH receptors, mutated and modified hGnRH receptors show the same trend in improved expression in whole cell binding as measured previously with membrane preparations. The percentage of binding compared to WT remained constant for each construct, suggesting an over all increase in receptor expression.

Comparison of receptor binding to various GnRH compounds We compared the binding characteristics of WT and modified hGnRH receptors to GnRH agonists (des-Gly¹⁰, D- Trp⁶, GnRH, D-Lys6 and a GnRH antagonist ([Ac-3,4- dhydro-Pro¹ D-p-F-Phe², D-Trp³'⁶] -GnRH) . Receptor EC50 values remained unchanged from WT values for each succesive modification: ΔK191 mutation, addition of the cf tail or lOHis tag incorporation. Functional binding parameters are summarized in Table 3. 1 Table 3. Agonist and antagonist binding to wild type

2 and modified human GnRH receptors.

3 ^{1 S}I-des-Gly¹⁰, [D-Trp⁶] GnRH membrane binding was displaced 4 by increasing concentrations of the following unlabeled 5 compounds: D-Trp6 (desGly¹⁰, [D-Trp⁶] -GnRH-ethylamide) ; 6 GnRH; D-Lys6 ( [D-Lys⁶] -GnRH) ; and Antag ([Ac-3,4- 7 dehydro-Pro^D-p-F-Phe², D-Trp³'⁶] -GnRH) . Data are 8 represented ± SEM in each case, n=3. 9 10 Discussion 11 Mammalian cell lines stably expressing the rat GnRH 12 receptor produce levels of receptor of approximately 3 13 pmol/mg of membrane protein (Heding et al . , supra, 14 1998) . Achieving similar levels of the human GnRH 15 receptor in mammalian cell culture systems has not 16 previously been possible. By identifying a crucial 17 amino acid within the hGnRH receptor protein we have 18 been able to express this receptor in a high, 19 reproducible and stable fashion. Further modifications 20 (adding a carboxy terminal tail and a lOHis tag) to the 21 mutated receptor further increased expression, 22 augmenting the effect of mutation. Moreover, none of 23 the modifications have any significant effect on the 24 binding affinity of the receptors for several GnRH 25 agonists and a GnRH antagonist. However the ability to 26 couple to the second messenger pathway was enhanced in 27 cells expressing hGnRH receptor (with a deletion at 28 position 191 and extended carboxy terminal tail and a 29 lOHis tag) . To date only relatively low level 30 expression of the hGnRH receptor has been reported in oocytes (Tsutsumi et al . , supra, 1992), various mammalian cell lines (Kakar et al . , supra, 1992; Chi et al . , Molecular and Cellular Endocrinology, Vol 91, pages R1-R3, 1993; Beckers et al . , Journal of Biochemistry, Vol 231, pages 535-543, 1995) and most recently in insect cells (Marheineke et al . , Cellular and Molecular Neurobiology, Vol 18, pages 509-523, 1998) . Marheineke et al . also expressed the hGnRH receptor in baby hamster kidney (BHK) cells where an expression level of 0.42 pmol receptor/mg membrane protein was measured, a level only half of our wild type expression level using a pcDNA3/HEK293 system. Myburgh et al . (European Journal of Endocrinology, Vol 139, pages 438-447, 1998) recently reported a single amino acid substitution in transmembrane domain VI of the hGnRH receptor which increased their expression approximately 3-fold (Bmax values not given) , in comparison with our deletion mutant ΔK191hGnRH-R which doubles expression compared to WT hGnRH receptor. This is all in contrast to the ΔK191hGnRH-R+10His cf tail described above, which produces around 40 pmol/mg membrane protein, 100 fold greater than any previous report.

Comparing the level of ligand binding of whole cells in suspension to that measured in total membrane preparations we can see that the ratios remain almost the same indicating an overall increase in receptor numbers associated with the receptor modifications. This suggests that the modified receptors are neither retained within the Golgi nor processed preferentially to the membrane. An explanation for this could be that for some reason the mRNAs for the modified receptors are more stable or more translatable, or that the protein molecules themselves are more stable and therefore survive within the cells for longer than normal. However, putative stabilization of the GnRH receptor mRNAs by removal of mRNA instability domains in the 3 'UTR did not significantly increase expression of the hGnRH receptor, but had a more significant effect with the rGnRH receptor, increasing expression by approximately 40%.

Expression of the WT hGnRH-R was increased greatly using the episomal vector system. However, stable cell lines expressing ΔK191hGnRH-R+10His cf tail generated using this system did not produce any more receptor than the best pcDNA3/HEK293 cell line. It may be that the cells are physically incapable of producing any greater amount of extra protein without losing integrity.

The fact that inserting a lysine at the same position in the rat GnRH-R does not diminish expression to levels comparable to the wild type human GnRH receptor is intriguing. This observation suggests that K191 may not be acting alone in its putative regulatory role and that the amino acids surrounding this location in a confor ationally folded and constrained receptor may also be important.

In conclusion, we have achieved the generation of stable cell lines expressing high levels of functionally active hGnRH receptors. Such cell lines will be of value to drug discovery, and with the inclusion of a histidine purification tag, these cell lines may be used to isolate the hGnRH receptor for X- ray crystallization studies. SEQUENCE LISTING

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Met Ala Asn Ser Ala Ser Pro Glu Gin Asn Gin Asn His Cys Ser Ala 1 5 10 15 ate aac aac age ate cca ctg atg cag ggc aac etc ccc act ctg ace 96 He Asn Asn Ser He Pro Leu Met Gin Gly Asn Leu Pro Thr Leu Thr 20 25 30 ttg tct gga aag ate cga gtg acg gtt act ttc ttc ctt ttt ctg etc 144 Leu Ser Gly Lys He Arg Val Thr Val Thr Phe Phe Leu Phe Leu Leu 35 40 45 tct gcg ace ttt aat get tct ttc ttg ttg aaa ctt cag aag tgg aca 192 Ser Ala Thr Phe Asn Ala Ser Phe Leu Leu Lys Leu Gin Lys Trp Thr 50 55 60 cag aag aaa gag aaa ggg aaa aag etc tea aga atg aag ctg etc tta 240 Gin Lys Lys Glu Lys Gly Lys Lys Leu Ser Arg Met Lys Leu Leu Leu 65 70 75 80 aaa cat ctg ace tta gcc aac ctg ttg gag act ctg att gtc atg cca 288 Lys His Leu Thr Leu Ala Asn Leu Leu Glu Thr Leu He Val Met Pro 85 90 95 ctg gat ggg atg tgg aac att aca gtc caa tgg tat get gga gag tta 336 Leu Asp Gly Met Trp Asn He Thr Val Gin Trp Tyr Ala Gly Glu Leu 100 105 110 3b

etc tgc aaa gtt etc agt tat eta aag ctt ttc tec atg tat gcc cca 384 Leu Cys Lys Val Leu Ser Tyr Leu Lys Leu Phe Ser Met Tyr Ala Pro 115 120 125 gcc ttc atg atg gtg gtg ate age ctg gac cgc tec ctg get ate acg 432 Ala Phe Met Met Val Val He Ser Leu Asp Arg Ser Leu Ala He Thr 130 135 140 agg ccc eta get ttg aaa age aac age aaa gtc gga cag tec atg gtt 480 Arg Pro Leu Ala Leu Lys Ser Asn Ser Lys Val Gly Gin Ser Met Val 145 150 155 160 ggc ctg gcc tgg ate etc agt agt gtc ttt gca gga cca cag tta tac 528 Gly Leu Ala Trp He Leu Ser Ser Val Phe Ala Gly Pro Gin Leu Tyr 165 170 175 ate ttc agg atg att cat eta gca gac age tct gga cag aca gtt ttc 576 He Phe Arg Met He His Leu Ala Asp Ser Ser Gly Gin Thr Val Phe 180 185 190 tct caa tgt gta aca cac tgc agt ttt tea caa tgg tgg cat caa gca 624 Ser Gin Cys Val Thr His Cys Ser Phe Ser Gin Trp Trp His Gin Ala 195 200 205 ttt tat aac ttt ttc ace ttc age tgc etc ttc ate ate cct ctt ttc 672 Phe Tyr Asn Phe Phe Thr Phe Ser Cys Leu Phe He He Pro Leu Phe 210 215 220 ate atg ctg ate tgc aat gca aaa ate ate ttc ace ctg aca egg gtc 720 He Met Leu He Cys Asn Ala Lys He He Phe Thr Leu Thr Arg Val 225 230 235 240 ctt cat cag gac ccc cac gaa eta caa ctg aat cag tec aag aac aat 768 Leu His Gin Asp Pro His Glu Leu Gin Leu Asn Gin Ser Lys Asn Asn 245 250 255 ata cca aga gca egg ctg aag act eta aaa atg acg gtt gca ttt gcc 816 He Pro Arg Ala Arg Leu Lys Thr Leu Lys Met Thr Val Ala Phe Ala 260 265 270 act tea ttt act gtc tgc tgg act ccc tac tat gtc eta gga att tgg 864 Thr Ser Phe Thr Val Cys Trp Thr Pro Tyr Tyr Val Leu Gly He Trp 275 280 285 tat tgg ttt gat cct gaa atg tta aac agg ttg tea gac cca gta aat 912 Tyr Trp Phe Asp Pro Glu Met Leu Asn Arg Leu Ser Asp Pro Val Asn 290 295 300 cac ttc ttc ttt etc ttt gcc ttt tta aac cca tgc ttt gat cca ctt 960 His Phe Phe Phe Leu Phe Ala Phe Leu Asn Pro Cys Phe Asp Pro Leu 305 310 315 320 ate tat gga tat ttt tct ctg tga ttg 987 He Tyr Gly Tyr Phe Ser Leu 325

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Met Ala Asn Ser Ala Ser Pro Glu Gin Asn Gin Asn His Cys Ser Ala 1 5 10 15

He Asn Asn Ser He Pro Leu Met Gin Gly Asn Leu Pro Thr Leu Thr 20 25 30

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Ser Ala Thr Phe Asn Ala Ser Phe Leu Leu Lys Leu Gin Lys Trp Thr 50 , 55 60

Gin Lys Lys Glu Lys Gly Lys Lys Leu Ser Arg Met Lys Leu Leu Leu 65 70 75 80

Lys His Leu Thr Leu Ala Asn Leu Leu Glu Thr Leu He Val Met Pro 85 90 95

Leu Asp Gly Met Trp Asn He Thr Val Gin Trp Tyr Ala Gly Glu Leu 100 105 110

Leu Cys Lys Val Leu Ser Tyr Leu Lys Leu Phe Ser Met Tyr Ala Pro 115 120 125

Ala Phe Met Met Val Val He Ser Leu Asp Arg Ser Leu Ala He Thr 130 135 140

Arg Pro Leu Ala Leu Lys Ser Asn Ser Lys Val Gly Gin Ser Met Val 145 150 155 160

Gly Leu Ala Trp He Leu Ser Ser Val Phe Ala Gly Pro Gin Leu Tyr 165 170 175

He Phe Arg Met He His Leu Ala Asp Ser Ser Gly Gin Thr Val Phe 180 185 190

Ser Gin Cys Val Thr His Cys Ser Phe Ser Gin Trp Trp His Gin Ala 195 200 205

Phe Tyr Asn Phe Phe Thr Phe Ser Cys Leu Phe He He Pro Leu Phe 210 215 220

He Met Leu He Cys Asn Ala Lys He He Phe Thr Leu Thr Arg Val 225 230 235 240

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He Pro Arg Ala Arg Leu Lys Thr Leu Lys Met Thr Val Ala Phe Ala 260 265 270

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Tyr Trp Phe Asp Pro Glu Met Leu Asn Arg Leu Ser Asp Pro Val Asn 290 295 300

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<400> 3 atg gca aac agt gcc tct cct gaa cag aat caa aat cac tgt tea gcc 48

Met Ala Asn Ser Ala Ser Pro Glu Gin Asn Gin Asn His Cys Ser Ala 1 5 10 15

^"ate aac aac age ate cca ctg atg cag ggc aac etc ccc act ctg ace 96 He Asn Asn Ser He Pro Leu Met Gin Gly Asn Leu Pro Thr Leu Thr 20 25 30 ttg tct gga aag ate cga gtg acg gtt act ttc ttc ctt ttt ctg etc 144 Leu Ser Gly Lys He Arg Val Thr Val Thr Phe Phe Leu Phe Leu Leu 35 40 45 tct gcg ace ttt aat get tct ttc ttg ttg aaa ctt cag aag tgg aca 192 Ser Ala Thr Phe Asn Ala Ser Phe Leu Leu Lys Leu Gin Lys Trp Thr 50 55 ^" 60 cag aag aaa gag aaa ggg aaa aag etc tea aga atg aag ctg etc tta 240 Gin Lys Lys Glu Lys Gly Lys Lys Leu Ser Arg Met Lys Leu Leu Leu 65 70 75 80 aaa cat ctg ace tta gcc aac ctg ttg gag act ctg att gtc atg cca 288 Lys His Leu Thr Leu- Ala Asn Leu Leu Glu Thr Leu He Val Met Pro 85 90 95 ctg gat ggg atg tgg aac att aca gtc caa tgg tat get gga gag tta 336 Leu Asp Gly Met Trp Asn He Thr Val Gin Trp Tyr Ala Gly Glu Leu 100 105 110 etc tgc aaa gtt etc agt tat eta aag ctt ttc tec atg tat gcc cca 384 Leu Cys Lys Val Leu Ser Tyr Leu Lys Leu Phe Ser Met Tyr Ala Pro 115 120 125 gcc ttc atg atg gtg gtg ate age ctg gac cgc tec ctg get ate acg 432 Ala Phe Met Met Val Val He Ser Leu Asp Arg Ser Leu Ala He Thr 130 135 140 agg ccc eta get ttg aaa age aac age aaa gtc gga cag tec atg gtt 480 Arg Pro Leu Ala Leu Lys Ser Asn Ser Lys Val Gly Gin Ser Met Val 145 150 155 160 ggc ctg gcc tgg ate etc agt agt gtc ttt gca gga cca cag tta tac 528 Gly Leu Ala Trp He Leu Ser Ser Val Phe Ala Gly Pro Gin Leu Tyr 165 170 175 ate ttc agg atg att cat eta gca gac age tct gga cag aca gtt ttc 576 He Phe Arg Met He His Leu Ala Asp Ser Ser Gly Gin Thr Val Phe 180 185 190 tct caa tgt gta aca cac tgc agt ttt tea caa tgg tgg cat caa gca 624 Ser Gin Cys Val Thr His Cys Ser Phe Ser Gin Trp Trp His Gin Ala 195 200 205 ttt tat aac ttt ttc ace ttc age tgc etc ttc ate ate cct ctt ttc 672 Phe Tyr Asn Phe Phe Thr Phe Ser Cys Leu Phe He He Pro Leu Phe 210 215 220 ate atg ctg ate tgc aat gca aaa ate ate ttc ace ctg aca egg gtc 720 He Met Leu He Cys Asn Ala Lys He He Phe Thr Leu Thr Arg Val 225 230 235 240 ctt cat cag gac ccc cac gaa eta caa ctg aat cag tec aag aac aat 768 Leu His Gin Asp Pro His Glu Leu Gin Leu Asn Gin Ser Lys Asn Asn 245 250 . 255 ata cca aga gca egg ctg aag act eta aaa atg acg gtt gca ttt gcc 816 He Pro Arg Ala Arg Leu Lys Thr Leu Lys Met Thr Val Ala Phe Ala 260 265 270 act tea ttt act gtc tgc tgg act ccc tac tat gtc eta gga att tgg 864 Thr Ser Phe Thr Val Cys Trp Thr Pro Tyr Tyr Val Leu Gly He Trp 275 280 285 tat tgg ttt gat cct gaa atg tta aac agg ttg tea gac cca gta aat 912 Tyr Trp Phe Asp Pro Glu Met Leu Asn Arg Leu Ser Asp Pro Val Asn 290 295 300 cac ttc ttc ttt etc ttt gcc ttt tta aac cca tgc ttt gat cca ctt 960 His Phe Phe Phe Leu Phe Ala Phe Leu Asn Pro Cys Phe Asp Pro Leu 305 310 315 320 ate tat gga tat ttt tct gat cga ttc cgt gcc gac ttg tec aga tgt 1008

He Tyr Gly Tyr Phe Ser Asp Arg Phe Arg Ala Asp Leu Ser Arg Cys 325 330 335 ttc tgt tgg agg aac caa aat get tea gcc aaa tct ctg cca cac ttc 1056

Phe Cys Trp Arg Asn Gin Asn Ala Ser Ala Lys Ser Leu Pro His Phe 340 345 350 tct ggt cat agg agg gag gtt agt ggg gaa gca gag tea gac ttg ggc 1104

Ser Gly His Arg Arg Glu Val Ser Gly Glu Ala Glu Ser Asp Leu Gly 355 360 365 agt ggt gac caa ccc agt gga cag taagctcaaa tgatttgaac ttgataatgt 1158

Ser Gly Asp Gin Pro Ser Gly Gin 370 375 tctttgtttc aagggaaatg tgatgattaa ctgtagtatt aatcatgtac tgccttatct 1218 ttttcacgtσ gtctggtatt ctaaatgaga aaaattaaaa atggaateta agaattgtta 1278 aatgeetgtt agggatggga ateaceaggg aeeaectatt aeaatattat cacgatacct 1338 actgtaggtg ccggatcgat 1358

<210> 4

<211> 376

<212> PRT

<213> Artificial Sequence <223> Description of Artificial Sequence: Sequence coding for a modified form of the human gonadotrophin-releasing hormone receptor (GnRH-R)

<400> 4

Met Ala Asn Ser Ala Ser Pro Glu Gin Asn Gin Asn His Cys Ser Ala 1 5 10 15

He Asn Asn Ser He Pro Leu Met Gin Gly Asn Leu Pro Thr Leu Thr 20 25 30

Leu Ser Gly Lys He Arg Val Thr Val Thr Phe Phe Leu Phe Leu Leu 35 40 45

Ser Ala Thr Phe Asn Ala Ser Phe Leu Leu Lys Leu Gin Lys Trp Thr 50 55 60

Gin Lys Lys Glu Lys Gly Lys Lys Leu Ser Arg Met Lys Leu Leu Leu - 65 70 75 80

Lys His Leu Thr Leu Ala Asn Leu Leu Glu Thr Leu He Val Met Pro 85 90 95

Leu Asp Gly Met Trp Asn He Thr Val Gin Trp Tyr Ala Gly Glu Leu 100 105 110

Leu Cys Lys Val Leu Ser Tyr Leu Lys Leu Phe Ser Met Tyr Ala Pro 115 120 125

Ala Phe Met Met Val Val He Ser Leu Asp Arg Ser Leu Ala He Thr 130 135 140

Arg Pro Leu Ala Leu Lys Ser Asn Ser Lys Val Gly Gin Ser Met Val 145 150 155 160

Gly Leu Ala Trp He Leu Ser Ser Val Phe Ala Gly Pro Gin Leu Tyr 165 170 175

He Phe Arg Met He His Leu Ala Asp Ser Ser Gly Gin Thr Val Phe 180 185 190

Ser Gin Cys Val Thr His Cys Ser Phe Ser Gin Trp Trp His Gin Ala 195 200 205

Phe Tyr Asn Phe Phe Thr Phe Ser Cys Leu Phe He He Pro Leu Phe 210 215 220

He Met Leu He Cys Asn Ala Lys He He Phe Thr Leu Thr Arg Val 225 230 235 240

Leu His Gin Asp Pro His Glu Leu Gin Leu Asn Gin Ser Lys Asn Asn 245 250 255

He Pro Arg Ala Arg Leu Lys Thr Leu Lys Met Thr Val Ala Phe Ala 260 265 270

Thr Ser Phe Thr Val Cys Trp Thr Pro Tyr Tyr Val Leu Gly He Trp 275 280 285

Tyr Trp Phe Asp Pro Glu Met Leu Asn Arg Leu Ser Asp Pro Val Asn 290 295 300

His Phe Phe Phe Leu Phe Ala Phe Leu Asn Pro Cys Phe Asp Pro Leu 305 310 315 320 He Tyr Gly Tyr Phe Ser Asp Arg Phe Arg Ala Asp Leu Ser Arg Cys 325 330 335

Phe Cys Trp Arg Asn Gin Asn Ala Ser Ala Lys Ser Leu Pro His Phe 340 345 350

Ser Gly His Arg Arg Glu Val Ser Gly Glu Ala Glu Ser Asp Leu Gly 355 360 365

Ser Gly Asp Gin Pro Ser Gly Gin 370 375

<210> 5

<211> 1161

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Sequence of a modified form of the human gonadotrophin-releasing hormone receptor (GnRH-R)

<220>

<221> CDS

<222> (1) .. (1158)

<400> 5 atg gca aac agt gcc tct cct gaa cag aat caa aat cac tgt tea gcc 48

Met Ala Asn Ser Ala Ser Pro Glu Gin Asn Gin Asn His Cys Ser Ala

1 5 10 15 ate aac aac age ate cca ctg atg cag ggc aac etc ccc act ctg ace 96 He Asn Asn Ser He Pro Leu Met Gin Gly Asn Leu Pro Thr Leu Thr 20 25 30 ttg tct gga aag ate cga gtg acg gtt act ttc ttc ctt ttt ctg etc 144 Leu Ser Gly Lys He Arg Val Thr Val Thr Phe Phe Leu Phe Leu Leu 35 40 45 tct gcg ace ttt aat get tct ttc ttg ttg aaa ctt cag aag tgg aca 192 Ser Ala Thr Phe Asn Ala Ser Phe Leu Leu Lys Leu Gin Lys Tro Thr 50 55 60 cag aag aaa gag aaa ggg aaa aag etc tea aga atg aag ctg etc tta 240 Gin Lys Lys Glu Lys Gly Lys Lys Leu Ser Arg Met Lys Leu Leu Leu 65 70 75 80 aaa cat ctg ace tta gcc aac ctg ttg gag act ctg att gtc atg cca 288 Lys His Leu Thr Leu Ala Asn Leu Leu Glu Thr Leu He Val Met Pro 85 90 95 ctg gat ggg atg tgg aac att aca gtc caa tgg tat get gga gag tta 336 Leu Asp Gly Met Trp Asn He Thr Val Gin Trp Tyr Ala Gly Glu Leu 100 105 110 etc tgc aaa gtt etc agt tat eta aag ctt ttc tec atg tat gcc cca 384 Leu Cys Lys Val Leu Ser Tyr Leu Lys Leu Phe Ser Met Tyr Ala Pro 115 120 125 gcc ttc atg atg gtg gtg ate age ctg gac cgc tec ctg get ate acg 432 Ala Phe Met Met Val Val He Ser Leu Asp Arg Ser Leu Ala He Thr 130 135 140 agg ccc eta get ttg aaa age aac age aaa gtc gga cag tec atg gtt 480 Arg Pro Leu Ala Leu Lys Ser Asn Ser Lys Val Gly Gin Ser Met Val 145 150 155 160 ggc ctg gcc tgg ate etc agt agt gtc ttt gca gga cca cag tta tac 528 Gly Leu Ala Trp He Leu Ser Ser Val Phe Ala Gly Pro Gin Leu Tyr 165 170 175 ate ttc agg atg att cat eta gca gac age tct gga cag aca gtt ttc 576 He Phe Arg Met He His Leu Ala Asp Ser Ser Gly Gin Thr Val Phe 180 185 190 tct caa tgt gta aca cac tgc agt ttt tea caa tgg tgg cat caa gca 624 Ser Gin Cys Val Thr His Cys Ser Phe Ser Gin Trp Trp His Gin Ala 195 200 205 ttt tat aac ttt ttc ace ttc age tgc etc ttc ate ate cct ctt ttc 672 Phe Tyr Asn Phe Phe Thr Phe Ser Cys Leu Phe He He Pro Leu Phe 210 215 220 ate atg ctg ate tgc aat gca aaa ate ate ttc ace ctg aca egg gtc 720 He Met Leu He Cys Asn Ala Lys He He Phe Thr Leu Thr Arg Val 225 230 235 240 ctt cat cag gac ccc cac gaa eta caa ctg aat cag tec aag aac aat 768 Leu His Gin Asp Pro His Glu Leu Gin Leu Asn Gin Ser Lys Asn Asn 245 250 255 ata cca aga gca egg ctg aag act eta aaa atg acg gtt gca ttt gcc 816 He Pro Arg Ala Arg Leu Lys Thr Leu Lys Met Thr Val Ala Phe Ala 260 265 270 act tea ttt act gtc tgc tgg act ccc tac tat gtc eta gga att tgg 864 Thr Ser Phe Thr Val Cys Trp Thr Pro Tyr Tyr Val Leu Gly He Trp 275 280 285 tat tgg ttt gat cct gaa atg tta aac agg ttg tea gac cca gta aat 912 Tyr Trp Phe Asp Pro Glu Met Leu Asn Arg Leu Ser Asp Pro Val Asn 290 295 300 cac ttc ttc ttt etc ttt gcc ttt tta aac cca tgc ttt gat cca ctt 960 His Phe Phe Phe Leu Phe Ala Phe Leu Asn Pro Cys Phe Asp Pro Leu 305 310 315 320 ate tat gga tat ttt tct gat cga ttc cgt gcc gac ttg tec aga tgt 1008

Phe Cys Trp Arg Asn Gin Asn Ala Ser Ala Lys Ser Leu Pro H s Phe 340 345 350 tct ggt cat agg agg gag gtt agt ggg gaa gca gag tea gac ttg ggc 1104

Ser Gly His Arg Arg Glu Val Ser Gly Glu Ala Glu Ser Asp Leu Gly

355 360 365 agt ggt gac caa ccc agt gga cag cac cac cat cac cat cat cac cat 1152

Ser Gly Asp Gin Pro Ser Gly Gin His His His His His His H s His 370 375 380 cac cat taa

1161

His His 385

<210> 6

<211> 386

<212> PRT

<213> Artificial Sequence

<400> 6

Met Ala Asn Ser Ala Ser Pro Glu Gin Asn Gin Asn His Cys Ser Ala 1 5 10 15

He Asn Asn Ser He Pro Leu Met Gin Gly Asn Leu Pro Thr Leu Thr 20 25 30

Leu Ser Gly Lys He Arg Val Thr Val Thr Phe Phe Leu Phe Leu Leu 35 40 45

Ser Ala Thr Phe Asn Ala Ser Phe Leu Leu Lys Leu Gin Lys Trp Thr 50 55 60

Gin Lys Lys Glu Lys Gly Lys Lys Leu Ser Arg Met Lys Leu Leu Leu 65 70 75 80

Lys His Leu Thr Leu Ala Asn Leu Leu Glu Thr Leu He Val Met Pro 85 90 95

Leu Asp Gly Met Trp Asn He Thr Val Gin Trp Tyr Ala Gly Glu Leu 100 105 110

Leu Cys Lys Val Leu Ser Tyr Leu Lys Leu Phe Ser Met Tyr Ala Pro 115 120 125

Ala Phe Met Met Val Val He Ser Leu Asp Arg Ser Leu Ala He Thr 130 135 140

Arg Pro Leu Ala Leu Lys Ser Asn Ser Lys Val Gly Gin Ser Met Val 145 150 155 160

Gly Leu Ala Trp He Leu Ser Ser Val Phe Ala Gly Pro Gin Leu Tyr 165 170 175

He Phe Arg Met He His Leu Ala Asp Ser Ser Gly Gin Thr Val Phe 180 185 190

Ser Gin Cys Val Thr His Cys Ser Phe Ser Gin Trp Trp His Gin Ala 195 200 205

Phe Tyr Asn Phe Phe Thr Phe Ser Cys Leu Phe He He Pro Leu Phe 210 215 220

He Met Leu He Cys Asn Ala Lys He He Phe Thr Leu Thr Arg Val 225 230 235 240

Leu His Gin Asp Pro His Glu Leu Gin Leu Asn Gin Ser Lys Asn Asn 245 250 255

He Pro Arg Ala Arg Leu Lys Thr Leu Lys Met Thr Val Ala Phe Ala 260 265 270

Thr Ser Phe Thr Val Cys Trp Thr Pro Tyr Tyr Val Leu Gly He Trp 275 280 285 Tyr Trp Phe Asp Pro Glu Met Leu Asn Arg Leu Ser Asp Pro Val Asn 290 295 300

His Phe Phe Phe Leu Phe Ala Phe Leu Asn Pro Cys Phe Asp Pro Leu 305 310 315 320

He Tyr Gly Tyr Phe Ser Asp Arg Phe Arg Ala Asp Leu Ser Arg Cys 325 330 335

Phe Cys Trp Arg Asn Gin Asn Ala Ser Ala Lys Ser Leu Pro His Phe 340 345 350

Ser Gly His Arg Arg Glu Val Ser Gly Glu Ala Glu Ser Asp Leu Gly 355 360 365

Ser Gly Asp Gin Pro Ser Gly Gin His His His His His His His His 370 375 380

His His 385

<210> 7 <211> 18 <212> PRT <213> Human

<400> 7

Leu Ala Asp Ser Ser Gly Gin Thr Lys Val Phe Ser Gin Cys Val Thr 1 5 10 15

His Cys

<210> 8 <211> 18 <212> PRT <213> Marmoset

<400> 8

Leu Ala Asp Ser Ser Gly Gin Thr Lys Val Phe Ser Gin Cys Val Thr 1 5 10 15

His Cys

<210> 9 <211> 18 <212> PRT <213> Sheep

<400> 9

Leu Ala Asp Asp Ser Gly Gin Thr Glu Gly Phe Ser Gin Cys Val Thr 1 5 10 15

His Cys

<210> 10 <211> 18 <212> PRT <213> Cow

<400> 10

Leu Ala Asp Asp Ser Gly Gin Thr Glu Gly Phe Ser Gin Cys Val Thr 1 5 10 15

His Cys

<210> 11 <211> 18 <212> PRT <213> Pig

<400> 11

Leu Ala Asp Ser Ser Gly Gin Thr Glu Gly Phe Ser Gin Cys Val Thr 1 5 10 15

His Gly

<210> 12 <211> 17 <212> PRT <213> Rat

<400> 12

Leu Val Asp Gly Ser Gly Pro Ala Val Phe Ser Gin Cys Val Thr His 1 5 10 15

Cys

<210> 13 <211> 17 <212> PRT <213> Mouse

<400> 13

Leu Ala Asp Gly Ser Gly Pro Thr Val Phe Ser Gin Cys Val Thr His 1 5 10 15

Cys

<210> 14

<211> 389

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: hGnRH-R having the catfish tail and plus ten histidine residues inserted at the C-terminus

<400> 14 Met Ala Asn Ser Ala Ser Pro Glu Gin Asn Gin Asn His Cys Ser Ala 1 5 10 15

He Asn Asn Ser He Pro Leu Met Gin Gly Asn Leu Pro Thr Leu Thr 20 25 30

Leu Ser Gly Lys He Arg Val Thr Val Phe Phe Leu Phe Leu Leu Ser 35 40 45

Ala Thr Phe Asn Ala Ser Phe Leu Leu Lys Leu Gin Lys Trp Thr Gin 50 55 60

Lys Lys Glu Lys Gly Lys Leu Ser Arg Met Lys Leu Leu Leu Lys His 65 70 75 80

Leu Thr Leu Ala Asn Leu Leu Glu Thr Leu He Val Met Pro Leu Asp 85 90 95

Gly Met Trp Asn He Thr Val Gin Trp Tyr Ala Gly Glu Leu Leu Cys 100 105 110

Lys Val Leu Ser Tyr Leu Lys Leu Phe Ser Met Tyr Ala Pro Ala Phe 115 120 125

Met Met Val Val He Ser Leu Asp Arg Ser Leu Ala He Thr Arg Pro 130 135 140

Leu Ala Leu Lys Ser Asn Ser Lys Val Gly Gin Ser Met Val Gly Leu 145 150 155 160

Ala Trp He Leu Ser Ser Val Phe Ala Gly Ala Gly Pro Gin Leu Tyr 165 170 175

He Phe Arg Met He His Leu Ala Asp Ser Ser Gly Gin Thr Lys Val 180 185 190

Phe Ser Gin Cys Val Thr His Cys Ser Phe Ser Gin Trp Trp His Gin 195 200 205

Ala Phe Tyr Asn Phe Phe Thr Phe Ser Cys Leu Phe He He Pro Leu 210 215 220

Phe He Met Leu He Cys Asn Ala Lys He He Phe Thr Leu Thr Arg 225 230 235 240

Val Leu His Gin Asp Pro His Glu Leu Gin Leu Asn Gin Ser Lys Asn 245 250 255

Asn He Pro Arg Ala Arg Leu Lys Thr Leu Lys Met Thr Val Ala Phe 260 265 270

Ala Thr Ser Phe Thr Val Cys Trp Thr Pro Tyr Tyr Val Leu Gly He 275 280 285

Trp Tyr Trp Phe Asp Pro Glu Met Leu Asn Arg Leu Ser Asp Pro Val 290 295 300

Asn His Phe Phe Phe Leu Phe Ala Phe Leu Asn Pro Cys Phe Asp Pro 305 310 315 320

Leu He Tyr Gly Tyr Phe Ser Leu Ser Phe Arg Ala Asp Leu Ser Arg 325 330 335

Cys Phe Phe Thr Cys Trp Arg Asn Gin Asn Ala Ser Ala Lys Ser Leu 340 345 350 Pro His Phe Ser Gly His Arg Arg Glu Val Ser Gly Glu Ala Glu Ser 355 360 365

Asp Leu Gly Ser Gly Asp Gin Pro Ser Gly Gin His His His His His 370 375 380

His His His His His 385

<210> 15

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Mutagenic oligonucleotide used to generate DeltaK 191 modified hGnRH-R.

<400> 15 cacattgaga gaaaactgtc tgtccagagc tgtc

<210> 16

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Mutagenic oligonucleotide used to introduce a lysine in the rat GnRH-R cDNA at a position equivalent to K191 in human GnRH-R.

<400> 16 cattgcgaga aaacttttgc tggcccagag ccg 33

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Upstream oligonucleotide primer used to PCR amplify hGnRH-R construct

<400> 17 ccaccatggc aaacagtgcc tctcc 25

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Primers

<400> 18 ctatcaatcg atcagaaaaa tatcc 25 <210> 19

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Upstream oligonucleotide primer used to PCR amplify

C-terminal tail region of catfish GnRH-R

<400> 19 cgccatcgat tcgtgccgac ttgtcc 26

<210> 20

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Downstream oligonucleotide primer used to PCR amplify

C-terminal tail region of catfish GnRH-R

<400> 20 caagaatcgc aatacaaatc gatccggcac ctac 34

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Upstream oligonucleotide primer used to PCR amplify hGnRH-R/ catfish tail constructs.

<400> 21 ccaccatggc aaacagtgcc tctcc 25

<210> 22

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Downstream oligonucleotide primer to PCR amplify and add 10

His + stop codon to hGnRH-R/ catfish tail constructs .

<400> 22 ttaatggtga tggtgatgat ggtgatggtg gtgetgtcca etgggttggt cace 54

Claims

1. Modified GnRH-R including at least one of the following characteristics:

b) a C-terminal portion comprising either:

2. Modified GnRH-R as claimed in Claim 1 wherein said C-terminal tag comprises at least six histidines.

3. Modified GnRH-R as claimed in Claim 2 wherein said C-terminal tag comprises at least ten histidines.

4. Modified GnRH-R as claimed in any one of Claims 1 to 3 wherein said tag comprises at least six consecutive positively charged amino acids.

5. Modified GnRH-R as claimed in Claim 4 wherein said tag comprises at least ten consecutive histidines.

6. Modified GnRH-R as claimed in any one of Claims 1 to 5 wherein the non-endogenous C-terminal tail is derived from catfish GnRH-R.

7. Modified GnRH-R as claimed in Claim 6 wherein said non-endogenous C-terminal tail includes amino acid Nos 331 onwards of catfish GnRH-R.

8. Modified mammalian GnRH-R as claimed in any one of Claims 1 to 7.

9. Modified human GnRH-R as claimed in any one of Claims 1 to 8.

10. Modified human GnRH-R having an amino acid sequence as set out in any one of SEQ ID Nos 2, 4, 6 or 14.

11. A recombinant polynucleotide encoding a modified GnRH-R as claimed in any one of Claims 1 to 10.

12. A recombinant polynucleotide having a nucleotide sequence as set out in any one of SEQ ID Nos 1, 3 or 5.

13. An expression vector comprising a recombinant polynucleotide as claimed in either one of Claims 11 and 12.

14. A pcDNA3 expression vector as claimed in Claim 13.

15. A pREP4 expression vector as claimed in Claim 13.

16. A transformed host cell able to express modified GnRH-R as claimed in any one of Claims 1 to 10.

17. A transformed mammalian host cell as claimed in Claim 16.

-18. A host cell as claimed in Claim 17 selected from the group consisting of COS-1, COS-7, COSM6 , CHO, BHK, GH₃, HEK293 and 292EBNA cells.

19. Transformed HEK293 cells as claimed in Claim 18.

20. Transformed 293EBNA cells as claimed in Claim 18.

21. A stable cell line able to express modified GnRH-R as claimed in any one of Claims 1 to 10.

22. A transgenic animal having a recombinant polynucleotide as claimed in either one of Claims 11 and 12 stably integrated into its genome.

2 . A method of screening agents for pharmacological activity, said method comprising

a) providing a modified GnRH-R as described above and exposing said modified GnRH-R to the agent ; and b) ascertaining whether said agent interacts with said modified GnRH-R.

24. A method as claimed in Claim 23 wherein said modified GnRH-R is expressed by a transformed host cell as claimed in any one of Claims 16 to 20.

25. A method as claimed in either one of Claims 23 and 24 wherein an aspect of GnRH-induced signal transduction is measured in step b) .