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WO1999067292A1 - RECEPTEUR GnRH MODIFIE - Google Patents

RECEPTEUR GnRH MODIFIE Download PDF

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WO1999067292A1
WO1999067292A1 PCT/GB1999/001821 GB9901821W WO9967292A1 WO 1999067292 A1 WO1999067292 A1 WO 1999067292A1 GB 9901821 W GB9901821 W GB 9901821W WO 9967292 A1 WO9967292 A1 WO 9967292A1
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gnrh
leu
ser
phe
modified
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Karin Ann Eidne
Elena Faccenda
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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.
  • GnRH-R gonadotrophin-releasing hormone receptor
  • GnRH decapeptide gonadotrophin-releasing hormone
  • Receptors for GnRH ie GnRH-R
  • 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.
  • 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.
  • GnRH-R 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.
  • a suitable source eg from the pituitary gland
  • 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.
  • 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).
  • transfected cells able to express the nucleotide sequences encoding the particular form of GnRH-R cloned.
  • Tsutsumi et al . used the Xenopus oocyte expression system to express the cloned sequence thus confirming its identity
  • Reinhart et al . also obtained expression in COS-7 cells, although transfection was only transient.
  • Perrin et al . reported expression of both the murine and rat GnRH-Rs using COSM6 cells whilst Kakar et al .
  • the mouse and rat GnRH-Rs have each been successfully cloned in stable expression systems which achieve a useful level of GnRH-R protein.
  • 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.
  • 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.
  • the human form of the receptor is of major interest.
  • GnRH-R preferably human GnRH-R
  • the modified form of GnRH-R, preferably human GnRH-R, of the present invention will have one or more of the following modifications;
  • 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.
  • the modified GnRH-R is produced by genetic engineering, for example is expressed from a recombinant construct.
  • GPCRs G-protein-coupled receptors
  • GnRH-Rs which term refers to GnRH-Rs and gonadoliberin receptors from a non- mammalian source
  • GnRH-Rs from chicken, catfish, goldfish and frog
  • carboxy terminal tails on these primitive receptors.
  • 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 .
  • 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 GH 3 cells relative to the WT rat GnRH-R.
  • the cDNA of the chimeric receptor was expressed only transiently.
  • 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.
  • 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.
  • 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.
  • the modified GnRH-R comprises human GnRH-R plus the C- erminal 51 ammo acids of cf GnRH-R (see SEQ ID No 4) .
  • 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) .
  • 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
  • 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).
  • 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.
  • SEQ ID Nos 4 and 6 the human GnRH-R portion of the chimeric sequence terminates at Ser 326, with the terminal Leu ( ⁇ 327) 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.
  • Figure 2 illustrates a modification of the tail and of its connection to the human sequence.
  • 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) .
  • the additional Phe 339 and Thr 340 may be deleted from the construct.
  • 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.
  • a multi-histidine tag typically comprising 6 to 12 histidine residues
  • WT non-tagged
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides a polynucleotide encoding a GnRH-R modified as described above.
  • 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) .
  • 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.
  • 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.
  • 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
  • cells transformed with such constructs are also encompassed by the present Application.
  • 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.
  • 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 present invention also provides host cells transformed with such constructs and which may express the biologically active modified gene product .
  • 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.
  • 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.
  • 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, GH 3 cells, HEK293 cells and 2.93EBNA cells.
  • 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, GH 3 cells, HEK293 cells and 2.93EBNA cells.
  • 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.
  • 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.
  • 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:
  • 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) .
  • 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.
  • 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) .
  • the expression system may be a transgenic animal .
  • Suitable expression systems include ⁇ K191 hGnRH-R in HEK293 cells (ie the 293-C4 or SCL118 cell line) ; ⁇ K191 hGnRH-R+cf tail in HEK293 cells (ie the 293-C9 or SCL146 cell line) and ⁇ K191 hGnRH-R+10His cf tail in HEK293 cells (ie the 293-C15 or SCL155 cell line) .
  • 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 .
  • the modified GnRH-R may be used as a means of contraception.
  • 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.
  • FIG. 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 125 I-des-Gly 10 , [D-Trp 6 ] GnRH binding levels of the GnRH receptors from the indicated species when expressed in COS-7 cells.
  • FIG. 1 Two-dimensional representation of the human GnRH receptor with an added C-terminal region.
  • 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.
  • FIG. 3A Saturation assay of WT and modified hGnRH-R constructs.
  • Membrane preparations from COS-7 cells were assayed for des-Gly 10 , [D-Trp 6 ] GnRH binding in saturation assays 48 hours post transient transfection of receptor constructs in pcDNA3 vector.
  • FIG. 1 Saturation binding of wild type rat GnRH receptor and +K191rGnRH-R.
  • FIG. 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-Gly 10 , [D-Trp 6 ] GnRH in the presence of increasing amounts of the unlabeled agonist.
  • FIG. 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.
  • FIG. 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.
  • Modification 1 Si te -directed mutagenesis
  • the hGnRH receptor cDNA in pcDNA3 , Invitrogen, DeSchlep, Netherlands
  • 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.
  • 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 .
  • upstream 5'- CGCCATCGATTCGTGCCGACTTGTCC SEQ ID No 19
  • 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 ⁇ K191 hGnRH-R+cf tail .
  • 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 ⁇ K191 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.
  • 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 gCl 2 , pH 7.2). The peptide des-Gly 10 , [D-Trp 6 ] GnRH Ethylamide (D-Trp 6 GnRH) was obtained from Sigma.
  • Displacement assays were performed with radiolabeled 12S I -des-Gly 10 , [D-Trp 6 ] GnRH (100 , OOOcpm) , 30 ⁇ g cell membranes expressing the GnRH-R constructs ⁇ 50 ⁇ l of agonist (doses 0 to 10 *6 M) in a total assay volume of 500 ⁇ l.
  • IP Total inositol phosphate
  • 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, ⁇ K191 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) .
  • ⁇ K191 hGnRH-R expresses approximately twice as many GnRH receptors compared to wild type (WT) ( ⁇ K191 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) .
  • WT wild type
  • ⁇ K191 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.
  • Modifications 2 and 3 Effects of adding cf GnRH receptor tail and lOHis tag to ⁇ K191 hGnRH-R
  • cf GnRH receptor tail and lOHis tag 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 ⁇ K191hGnRH-R increases expression from 1.6 ( ⁇ 0.3) pmol/mg protein to 5.3 ( ⁇ 0.3) pmol/mg protein.
  • the construct ⁇ K191 hGnRH-R+cf tail was expressed both transiently (in COS-7 cells) and stably (in HEK293 cells) and radioligand binding was compared to ⁇ K191 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.
  • COS-7 transient transfections
  • Figure 3B shows levels of specific GnRH agonist binding
  • Binding to a selected HEK293 stable cell line expressing ⁇ K191 hGnRH-R+10His cf tail A stable cell line clone of ⁇ K191hGnRH-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 5 ⁇ g of protein/replicate as shown in Figure 4.
  • 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.
  • 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 .
  • 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..
  • FIG. 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 ⁇ K191hGnRH-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.
  • adding the cf GnRH receptor tail to the WT hGnRH increases expression slightly, although not significantly, above WT alone.
  • WT + lOHis cf tail GnRH-R does express significantly better than either WT alone or WT + cf tail hGnRH-R.
  • 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.
  • BHK baby hamster kidney
  • Trp Tyr Trp Phe Asp Pro Glu Met Leu Asn Arg Leu Ser Asp Pro Val 290 295 300

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Abstract

On décrit des récepteurs GnRH modifiés (GnRH-Rs), y compris des récepteurs GnRH humains, ainsi que des polynucléotides codant lesdits récepteurs. Les GnRH-Rs modifiés peuvent être exprimés dans des lignées cellulaires stables à des niveaux considérablement plus élevés que ceux du récepteur équivalent de type sauvage, tandis que les caractéristiques de liaison du récepteur demeurent sensiblement inchangées. Les modifications des GnRH-R humains pouvant augmenter cette expression incluent la délétion de la lysine 191 et l'addition d'une extrémité C-terminale de poisson-chat, y compris, éventuellement, une étiquette multi-histidine C-terminale.
PCT/GB1999/001821 1998-06-20 1999-06-21 RECEPTEUR GnRH MODIFIE Ceased WO1999067292A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU43782/99A AU4378299A (en) 1998-06-20 1999-06-21 Modified gnrh receptor

Applications Claiming Priority (2)

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GBGB9813279.8A GB9813279D0 (en) 1998-06-20 1998-06-20 Modified receptor
GB9813279.8 1998-06-20

Publications (1)

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WO1999067292A1 true WO1999067292A1 (fr) 1999-12-29

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PCT/GB1999/001821 Ceased WO1999067292A1 (fr) 1998-06-20 1999-06-21 RECEPTEUR GnRH MODIFIE

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AU (1) AU4378299A (fr)
GB (1) GB9813279D0 (fr)
WO (1) WO1999067292A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000590A1 (fr) * 1992-06-23 1994-01-06 The Mt. Sinai School Of Medicine Of The City University Of New York CLONAGE ET EXPRESSION D'UN RECEPTEUR D'HORMONE LIBERANT DE LA GONADOTROPINE (R-HLGn)
EP0678577A2 (fr) * 1994-04-19 1995-10-25 Takeda Chemical Industries, Ltd. Méthode pour la production de protéines récepteurs de LH-RH humaines recombinées

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000590A1 (fr) * 1992-06-23 1994-01-06 The Mt. Sinai School Of Medicine Of The City University Of New York CLONAGE ET EXPRESSION D'UN RECEPTEUR D'HORMONE LIBERANT DE LA GONADOTROPINE (R-HLGn)
EP0678577A2 (fr) * 1994-04-19 1995-10-25 Takeda Chemical Industries, Ltd. Méthode pour la production de protéines récepteurs de LH-RH humaines recombinées

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HEDING A. ET AL.: "Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 19, 8 May 1998 (1998-05-08), MD US, pages 11472 - 11477, XP002118585 *
KAKAR S S ET AL: "CLONING, SEQUENCING, AND EXPRESSION OF HUMAN GONADOTROPIN RELEASING HORMONE (GNRH) RECEPTOR", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 189, no. 1, 30 November 1992 (1992-11-30), pages 289 - 295, XP002058778, ISSN: 0006-291X *
LIN X. ET AL.: "Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation", MOLECULAR ENDOCRINOLOGY, vol. 12, no. 2, February 1998 (1998-02-01), pages 161 - 171, XP002118584 *

Also Published As

Publication number Publication date
GB9813279D0 (en) 1998-08-19
AU4378299A (en) 2000-01-10

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