EP1123391A2 - Methodes permettant d'ameliorer la fonction des recepteurs heterologues couples a la proteine g - Google Patents

Methodes permettant d'ameliorer la fonction des recepteurs heterologues couples a la proteine g

Info

Publication number
EP1123391A2
EP1123391A2 EP99944035A EP99944035A EP1123391A2 EP 1123391 A2 EP1123391 A2 EP 1123391A2 EP 99944035 A EP99944035 A EP 99944035A EP 99944035 A EP99944035 A EP 99944035A EP 1123391 A2 EP1123391 A2 EP 1123391A2
Authority
EP
European Patent Office
Prior art keywords
receptor
protein
host cell
nucleic acid
coupled receptor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99944035A
Other languages
German (de)
English (en)
Inventor
Mark Henry Pausch
Jürgen Lab. of Bioorganic Chemistry WESS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Wess Jurgen
Original Assignee
BASF SE
Wess Jurgen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BASF SE, Wess Jurgen filed Critical BASF SE
Publication of EP1123391A2 publication Critical patent/EP1123391A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4722—G-proteins
    • 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
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to mutant G protein-coupled receptors with improved G-protein coupling and receptor response, host cells expressing such receptors, vectors useful for making such cells, and methods of making and using same.
  • G protein-coupled receptors G protein-coupled receptors
  • the regulatory G proteins are comprised of three subunits: a guanylnucleotide binding ⁇ subunit; a ⁇ subunit; and a ⁇ subunit.
  • G proteins cycle between two forms, depending on whether GDP or GTP is bound to the ⁇ subunit.
  • GDP When GDP is bound, the G protein exists as a heterotrimer, the G ⁇ complex.
  • GTP When GTP is bound, the ⁇ subunit dissociates, leaving a G ⁇ complex.
  • a G ⁇ complex operatively associates with an activated G protein coupled receptor in a cell membrane, the rate of exchange of GTP for bound GDP is increased and, hence, the rate of disassociation of the bound G ⁇ subunit from the G ⁇ complex increases.
  • the free G ⁇ subunit and G ⁇ complex are capable of transmitting a signal to downstream elements of a variety of signal transduction pathways.
  • downstream cellular effector proteins include, among others, adenylate cyclases, ion channels, and phospholipases. This fundamental scheme of events forms the basis for a multiplicity of different cell signaling phenomena. KG. Dohlman et al. (1991).
  • HTS high-throughput screening
  • GPCRs transformed with wild-type receptors may perform poorly in growth assays, exhibiting, for example, the inability to interact with the heterotrimeric G protein, inappropriate localization and/or desensitization.
  • Many GPCRs are phosphorylated in response to chronic and persistent agonist stimulation which often leads to desensitization followed by sequestration or internalization of the receptors.
  • Desensitization of GPCRs causes uncoupling from interaction with heterotrimeric G proteins. This process is mediated by a variety of regulatory receptor protein kinases, including G protein-coupled receptor kinases (GRK), protein kinase A (PKA), protein kinase C (PKC), and casein kinases (CK).
  • GRK G protein-coupled receptor kinases
  • PKA protein kinase A
  • PKC protein kinase C
  • CK casein kinases
  • Internalized receptors may be recycled back to the cell surface, or delivered to a lysosomal/vacuolar compartment for degradation.
  • the ubiquitin-mediated degradative pathway is also involved in this process.
  • the ultimate result of receptor phosphorylation and sequestration/internalization is often cell growth arrest, which significantly reduces the utility of the genetically modified microorganism in screening assays.
  • a first aspect of the present invention is directed to nucleotide sequences encoding a G protein-coupled receptor which has been modified to improve the function of the GPCR by causing the receptor to couple more efficiently with the heterotrimeric G protein and/or to fail to interact with the cell desensitization and/or sequestration/internalization machinery, and/or to appropriately localize to the plasma membrane.
  • modifications lead to improved agonist-stimulated growth-promoting ability.
  • One specific modification of the nucleotide sequence encoding a G protein-coupled receptor encompassed by this invention is a mutation in any intracellular domain or membrane region proximal to internal domains.
  • the mutation may be a deletion, including, for example, a point mutation.
  • This invention is also directed to chimeric GPCRs in which intracellular domains of heterologous GPCRs that provide favorable G protein coupling properties or domains not subject to the yeast cell desensitization and/or sequestration/internalization machinery are used to replace comparable domains in GPCRs of interest.
  • This invention also relates to the modified nucleotide sequences encoding the chimeric GPCRs, to expression vectors comprising the modified nucleotide sequences, and to host cells transformed therewith.
  • An additional aspect of this invention is an improved method of assaying compounds to determine effects of ligand binding to the mutant or chimeric GPCRs of this invention by measuring the effect of the test compound on cell growth.
  • the mutant GPCRs prevent or reduce the rate of cell growth arrest due to chronic and persistent agonist stimulation, thereby decreasing the number of false negatives that occur with prior art screening methods and/or increasing the sensitivity of the bioassay.
  • FIGURE 1A depicts the results of liquid culture assays on yeast cells containing the rat M3 muscarinic acetylcholine receptor (MAR) using MAR agonist carbachol (CCh).
  • MAR muscarinic acetylcholine receptor
  • CCh third intracellular loop
  • FIGURE IB depicts the results of liquid culture fluorescence induction assays on yeast cells containing the rat M3 MAR IC3 ⁇ and the FUS2-GFP reporter plasmid using the MAR agonist carbachol (CCh).
  • a dose-dependent increase in the expression of the green fluorescent protein is observed in response to CCh activation of the M3 MAR IC3 ⁇ expressed in yeast.
  • FIGURE 2 depicts the results of liquid culture assays on yeast cells containing the Drosophila muscarinic acetylcholine receptor using the MAR agonist carbachol (CCh).
  • yeast cells containing a mutated Drosophila MAR containing the M3 MAR IC3 ⁇ produced an agonist-dependent growth response while the wild type Drosophila MAR lacked an agonist-dependent yeast cell growth response.
  • FIGURE 3 depicts the results of an agar-based plate bioassay.
  • FIGURE 3 A shows a robust growth response of yeast cells containing the IC3 ⁇ cholecystokinin CCKB receptor.
  • FIGURE 3B shows only limited growth by yeast cells containing the wild type CCKB receptor, indicating that the deletion of a portion of the third intracellular loop of the CCKB receptor improves its function in yeast.
  • FIGURE 4 depicts yeast cells transformed with rSSTR3 and with rSSTR3 ⁇ IC3.
  • FIGURE 4A demonstrates that yeast cells containing p426GPD- rSSTR3 show a weak response to somatostatin (S-14).
  • FIGURE 4B demonstrates a much stronger response by yeast cells containing p426GPD-rSSTR3 ⁇ IC3 assayed under similar conditions.
  • FIGURE 5 depicts the results of liquid culture assays on yeast cells containing wild type IC3 ⁇ human alpha2A adrenergic receptor using the alpha adrenergic receptor full agonist UK 14304. Yeast cells containing the wild type and IC3 ⁇ human alpha 2 A adrenergic receptor produced a dose-dependent growth response, indicating that the IC3 deletion is functional.
  • FIGURE 6 depicts the results of liquid culture assays on yeast cells containing wildtype and carboxy terminally truncated rat NTl-neurotensin receptors using the neurotensin receptor agonist AcNT8-13. Truncation of the rat NTl- neurotensin receptor produces an agonist-dependent growth response that is more sensitive than that observed with the wildtype receptor.
  • FIGURE 7 depicts the results of liquid culture assays on yeast cells containing the C. elegans serotonin receptor using serotonin (5HT) to stimulate yeast cells growth. Yeast cells containing a mutated C. elegans serotonin receptor containing the M3 MAR IC3 ⁇ produced a 5HT-dependent growth response. The growth response was blocked by addition of the serotonin receptor antagonists lisuride and mianserin.
  • Nucleotide sequences encoding G protein-coupled receptors may be modified to improve the function of the GPCR by causing the receptor to couple more efficiently with the heterotrimeric G protein and/or to fail to interact with the cell desensitization and/or sequestration/internalization machinery. Such modifications lead to improved agonist-stimulated yeast cell growth-promoting ability.
  • the improvement of GPCR-G protein coupling and/or elimination of receptor phosphorylation and/or sequestration/internalization in the host cell provides a means to improve the function of wildtype heterologous GPCRs that fail to stimulate a useful yeast cell growth response.
  • GPCRs that fail to function in their wild type form may be made to work by the methods of this invention.
  • the improvement of GPCR-G protein coupling and or elimination of receptor phosporylation and/or sequestration/internalization in the host cell may be assessed by using routine techniques, such as those described in the Examples set fort below or known to those of skill in the art.
  • improvement of the function of a mutated GPCR over wild type may be quantified as an increase in the signal-to-noise ratio and/or in the sensitivity of the liquid bioassay.
  • the signal-to-noise ratio is determined by comparing the agonist-induced growth rate to the growth rate observed in the absence of agonist.
  • a statistically-significant increase in the signal-to-noise ratio resulting from agonist-stimulation of a mutated GPCR over similar values obtained from cells containing a wild type receptor indicates that the function of the mutated GPCR has been improved.
  • the sensitivity of the liquid bioassay is defined as the agonist concentration necessary to produce a half-maximal growth rate (ED50 or EC50). The sensitivity of the bioassay is increased if a mutated GPCR produces a half-maximal growth rate at an agonist concentration that is less than that required by the wild type GPCR. Similarly, the more qualitative agar based bioassay will reflect increases in signal-to-noise ratio and/or sensitivity due to agonist stimulation of mutated GPCRs.
  • signal-to-noise ratio increases are determined by comparing the extent of growth within the agonist induced growth zone resulting from stimulation of mutated and wild type receptor.
  • the sensitivity of the bioassay is proportional to the radius of the growth zone. Since applied compounds diffuse radially from the site of application to the agar, agonist concentration varies with the square of the radius of the growth zone. Thus, a larger zone of growth in response to agonist activation of mutated GPCRs reflects an increase in sensitivity.
  • G protein-coupled receptor may be employed in practicing this invention.
  • receptors include, but are not limited to, adenosine receptors, somatostatin receptors, dopamine receptors, cholecystokinin receptors, muscarinic cholinergic receptors, ⁇ -adrenergic receptors, ⁇ -adrenergic receptors, opiate receptors, cannabinoid receptors, growth hormone releasing factor, glucagon, serotonin receptors, vasopressin receptors, melanocortin receptors, and neurotensin receptors.
  • the receptor is a muscarinic acetylcholine receptor and more preferably, the muscarinic acetylcholine receptor is of the M3 subtype.
  • any suitable host cell may be transformed with the nucleotide sequences encoding the modified G protein-coupled receptors of this invention.
  • suitable host cells are yeast cells, mammalian cells, insect cells, and bacterial cells.
  • the host cells are yeast cells.
  • One generalizable method for improving the function of a GPCR expressed in a host cell is by modification or elimination of intracellular domains of the GPCR, such as the third intracellular loop (IC3) sequences of the G protein-coupled receptor.
  • IC3 intracellular loop
  • the desensitization and internalization machinery acts upon the intracellular domains of the GPCR, elimination of the intracellular domains of the GPCRs produces a more stable receptor expression. This has been demonstrated in experiments conducted in mammalian cells. Muscarinic acetylcholine receptors, including the M3 subtype, lacking a domain of their third intracellular loop thought to be involved in receptor internalization, are maintained in the plasma membrane to a greater extent than their wild type counterparts. Moro et al. (1993). Representative embodiments of the invention are described in more detail in the following examples.
  • Example 1 Functional Expression of A Mutated Rat M3 Muscarinic Acetylcholine Receptor (MAR) in Yeast
  • MAR Muscarinic Acetylcholine Receptor
  • the third intracellular loops of GPCRs are thought to interact with and participate in the activation of G proteins upon agonist binding. J. Wess (1997). Mutations in IC3 of the yeast mating pheromone receptors, Ste2 and Ste3 have profound effects on coupling the G proteins. C. Boone et al. (1993) and C. Clark et al. (1994).
  • deletion of portion of the IC3 of mammalian MARs, in particular the rat M3 MAR is correlated with improved functional expression in mammalian cells with retention of full ability to couple to the heterotrimeric G protein, Gq (G ⁇ ).
  • the mutated M3 MAR retains all external loops.
  • Transmembrane domains (TMDs) and internal domains other than the IC3 are unchanged.
  • the IC3 found between 5th and 6th membrane spanning helices, was the only domain modified. The bulk of this domain, 96 amino acids in the center of the
  • IC3 (Ala273-Lys469), were deleted, leaving only 22 amino acids proximal to both the 5th and 6th transmembrane helices.
  • the third intracellular loop of the MAR containing the IC3 deletion (IC3 ⁇ ) is 44 amino acids in length, compared to 240 amino acids in the IC3 of wild type M3 MAR.
  • the improvement in functional expression may due to elimination of domains known to interact with cellular desensitization mechanisms, allowing more functional MAR to be retained at the cell surface.
  • Rat M3 MAR sequences were amplified by PCR using oligonucleotides containing 5' Bgttl (AAAAGATCT AAA ATG TAC CCC TAC GAC GTC CCC) (SEQ ID NO: 1) and 3' Xhol (AAA CTCGAG CTA CAA GGC CTG CTC CGG CAC TCG C) (SEQ ID NO: 2) sites.
  • PCR product was digested with the appropriate restriction endonucleases, purified and ligated into appropriate sites in p426GPD.
  • three M3 MAR fragments were amplified by PCR.
  • An amino-terminal coding fragment was amplified using oligonucleotides containing 5' BgRl (AAAAGATCT AAA ATG TAC CCC TAC GAC GTC CCC) (SEQ ID NO: 1) and 3' Agel (ATAGTCATGATGGTG ACCGGT ATGTAAAAGGCAGCGATC) (SEQ ID NO: 3) sites.
  • a carboxy-terminal coding fragment was amplified using oligonucleotides containing 5' Pmli (GCCTTCATCAT CACGTG GACCCCCTACACC) (SEQ ID NO: 4) and 3' Xhol (AAA CTCGAG CTA CAA GGC CTG CTC CGG CAC TCG C) (SEQ ID NO: 2) sites.
  • An IC3 coding fragment was amplified using oligonucleotides containing 5' Agel (CGATCGCTGCCTTTTACTT ACCGGT CACCATCATGACTAT) (SEQ ID NO:
  • MAR agonist carbachol (CCh).
  • the cells were cultured overnight in 2 ml SC- glucose-ura medium.
  • the cells were diluted 500 fold in SC-glucose-ura-his, pH 6.8 medium containing 5 mM 3-aminotriazole to decrease basal growth rate.
  • Samples of the cell suspension (180 ⁇ l) were dispensed to wells of sterile 96 well microtiter dishes containing 20 ⁇ l of serially-diluted samples (lO ⁇ -lO "8 M) of the muscarinic receptor agonists.
  • the plates were incubated at 30°C for 18 hours with agitation (600 rpm). Growth was monitored by recording increases in OD 620 using a microplate reader.
  • M3 MAR IC3 ⁇ produced an agonist-dependent growth response, demonstrating that the M3 MAR IC3 ⁇ is functional, while the wild type MAR is non-functional, as indicated by the lack of agonist-dependent yeast cell growth.
  • the growth response of the M3 MAR IC3 ⁇ containing cells was dose-dependent giving an EC 50 for carbachol (CCh) equal to 3 ⁇ M. This value matches the K D for CCh obtained in HEK cells
  • IC3 ⁇ may be observed by measuring the agonist-induced increase in fluorescent emission from a green fluorescent protein reporter gene whose expression is stimulated by MAR agonists.
  • Green fluorescent protein (GFP) is a protein from Aequorea that is intrinsically fluorescent when expressed in yeast cells. The fluorescence from GFP is detectable in live yeast calls, making it possible to measure the level of its expression without any deleterious treatment of the yeast cells. This feature is particularly advantageous in the reporter gene assays that do not require additional steps to permit its detection.
  • An inducible reporter gene that is useful in detecting the agonist-activation of heterologous GPCRs expressed in yeast utilizes transcriptional promoters that are activated by the mating signal transduction pathway.
  • FUS2 promoter One such promoter is the FUS2 promoter.
  • FUS2 promoter In the absence of agonist stimulation, little or no expression of the Fus2 protein or any other protein whose expression is directed by the FUS2 promoter is detectable.
  • transcription from the FUS2 promoter After treatment with agonist, transcription from the FUS2 promoter is induced up to 700 fold, leading to substantial increases in Fus2 expression or in the expression of any gene product whose expression is placed under control of the FUS2 promoter.
  • yeast cell fluorescence resulting from GFP expression under the control of the FUS2 promoter from a EC/S2-GFP reporter gene is only observed after agonist activation of a heterologous GPCR.
  • DNA sequences encoding the enhanced GFP (EGFP, Clonetech), FUS2 promoter and FUS2 transcriptional terminator sequences were amplified by PCR.
  • the fragments were assembled into the centromere containing plasmid pRS414 so as to place EGFP expression under control of the pheromone responsive EL7S2 promoter in the centromere containing plasmid pRS414, producing plasmid pMP241.
  • the resulting plasmids were introduced into yeast cells of the kind described in United States Patent 5,691 , 188, that are useful for performing assays of GPCR agonist-stimulated growth of cells containing the M3 MAR IC3 ⁇ . Specifically, the plasmids were introduced into the MPY578fc cells described in Pausch et al. (1998).
  • Yeast cells containing the M3 MAR IC3 ⁇ and the FUS2-EGF? reporter plasmid were assayed in liquid culture using the MAR agonist carbachol (CCh).
  • the cells were cultured overnight in 2 ml SC-glucose-ura medium.
  • the cells were washed and diluted 5 fold in SC-glucose-ura-his, pH 6.8 medium containing 5 mM 3- aminotriazole to decrease basal growth rate.
  • Samples of the cell suspension 180 ⁇ l
  • Example 2 Functional Expression of a Mutated D. melanogaster Muscarinic Acetylcholine Receptor in Yeast
  • Agonist of the G protein-coupled insect muscarinic acetylcholine receptors possess substantial insecticidal and miticidal activity. M.R. Dick et al.
  • the DMAR interacts with the heterotrimeric Gq protein leading to an increase in intracellular calcium in response to muscarinic agonists.
  • One potential explanation for the inactivity of the DMAR in yeast is an inability to efficiently couple to the yeast heterotrimeric G protein.
  • Fragment 1 consisted of the amino terminal coding portion of the Drosophila MAR up to an Agel site within the 5th TMD, amplified by PCR using oligonucleotides (AAA AGATCT AAA ATG TACGGAAACCAGACGAAC) (SEQ ID NO: 7) and (CCA GTA GAG GAA
  • the second fragment of the DMAR was composed of carboxy terminal coding sequences starting with a Pm ⁇ l site in the 6th TMD, amplified by PCR using oligonucleotides (TTCATCATCACGTGGACTCCGTACAACATC) (SEQ ID NO: 9) and (AAA CTCGAG TTATCTAATTGTAGACGCGGC) (SEQ ID NO: 10).
  • the M3 MAR IC3 ⁇ domain was amplified as an Agel-Pmll fragment with coding sequence in frame with fragments 1 and 2, described in Example 1.
  • Yeast cells containing the DMAR and the plasmid containing the wild type DMAR were assayed in liquid culture using the MAR agonist carbachol (CCh). The cells were cultured overnight in 2 ml SC-glucose-ura medium. The cells were diluted
  • Optical density measurements were analyzed using GraphPad Prism and are presented as the mean ⁇ SEM and were plotted vs. agonist concentration.
  • the yeast cells containing the mutated DMAR i.e., the M3 MAR IC3 ⁇
  • produced an agonist-dependent growth response demonstrating that the DMAR-M3 MAR IC3 ⁇ is functional.
  • the wild type DMAR is non-functional, as indicated by the lack of agonist-dependent yeast cell growth.
  • replacing IC3 of the DMAR with the functional deleted IC3 from the rat M3 MAR produces a functional chimeric GPCR when expressed in yeast, indicating that this method of replacing internal domains may be a generalizable method for improving the functional expression of heterologous GPCRs in cell-based assays, such as yeast assays.
  • the DNA sequences encoding the rat wild type and IC3 ⁇ cholecystokinin CCKB receptor were amplified by PCR and cloned into proximity to the glycerol-phosphate dehydrogenase promoter in yeast expression plasmid, p426GPD, by standard methods.
  • the wild type CCKBR was amplified by PCR using oligonucleotides
  • Fragment 1 contained amino terminal coding sequences including 22 amino acids proximal to the 5th TMD, amplified by PCR using oligonucleotides
  • Fragment 2 contained carboxy terminal coding sequences including 22 amino acids proximal to the 6th TMD, amplified by PCR using oligonucleotides (TCCCGTCGACTCAGCCAGGCCCCAGTGTGCTG) (SEQ ID NO: 12) and
  • CGAGACCCAAAGCCGGGCCCGGCCGGGGCCAGTCCCTGGCCCTCG SEQ ID NO: 14
  • the two fragments were fused by amplification by PCR using oligos at 5' and 3' ends of the full length CCKB receptor.
  • the resulting plasmids were introduced into yeast cells useful for performing assays of GPCR agonist-stimulated growth, such as those described in United States Patent 5,691,188, including, specifically, the MPY578fc cells described in Pausch et al. (1998).
  • Yeast strains containing wild type and IC3 ⁇ cholecystokinin CCKB receptor were grown overnight in 2 ml synthetic complete liquid medium containing glucose (2%) and lacking uracil (SCD-ura) medium.
  • molten (50 °C) SCD-ura-his agar medium 35 ml, adjusted to pH 6.8 by addition of concentrated KOH or NH 4 OH prior to autoclaving) containing 0.5 mM AT (3- aminotriazole) was inoculated with the overnight culture (2 x 10 4 cells/ml) and poured into square (9 x 9 cm) petri plates.
  • Figure 3 A demonstrates the robust growth response of yeast cells containing the IC3 ⁇ cholecystokinin CCKB receptor
  • Figure 3B shows only limited growth by yeast cells containing the wild type CCKB receptor, indicating that the deletion of portion of the third intracellular loop of the CCKB receptor improves its function in yeast.
  • the third intracellular loop participates in many GPCR functions, including G protein coupling, desensitization and interaction with diverse modifying proteins.
  • Somatostatin receptors are encoded in five subtypes, labeled SSTRl-5.
  • SSTR3 subtypes Several amino acids are found in the third intracellular loop of the SSTR3 subtype, but not in the equivalent region of SSTR2 subtype. Since SSTR2 functions efficiently in yeast, deletion of those amino acids from IC3 may impart this functional efficiency upon SSTR3.
  • 8 amino acids, Gln-Trp-Val-Gln-Ala-Pro-Ala-Cys (SEQ ID NO: 15), were deleted from the third intracellular loop of the rSSTR3 cDNA, enabling more efficient receptor signaling in yeast.
  • Rat SSTR3 sequences were amplified by PCR using oligonucleotides containing 5' Bglll and 3' Xhol sites.
  • the resulting PCR product of approximately 1.3 kb was digested with Bglll and Xhol, purified and inserted between the BamHI and Xhol sites in p426GPD to generate the plasmid p426GPD-rSSTR3. Recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing.
  • PCR insert A of approximate size 750 bp was generated using the 5' Bgl oligonucleotide (AAAAAGATCT AAAATGGCCA CTGTTACCTA T) (SEQ ID NO: 16) and the 3' oligonucleotide CTCAGAGCGG CGTCGCCGCT GACACGAGGG CGCCCG (SEQ ID NO: 17).
  • PCR insert B of approximate size 530 bp was generated using the 5' oligonucleotide GCGCCCTCGT GTCAGCGGCG ACGCCGCTCT GAG
  • PCR fragments A and B were gel purified, annealed and amplified by PCR using the flanking 5' Bglll and 3' Xhol oligonucleotides to yield the - 1.3 kb rSSTR3 ⁇ IC3 PCR product.
  • the rSST3 ⁇ IC3 insert was ligated into BamHI-XhoI sites of p426GPD to generate the plasmid p426GPD-rSSTR3 ⁇ IC3. Restriction mapping and DNA sequencing confirmed correct reading frame and sequence.
  • Yeast cells of the type useful for expression of GPCRs described in United States Patent 5,691,188, were transformed with p426GPD-rSSTR3 and p426GPD- rSSTR3 ⁇ IC3, using standard procedures.
  • the cells i.e., the LY296 cells, described in Price et al. (1995)
  • Samples (10 ⁇ l) of Somatostatin (S-14, ImM) were applied to the surface of the selective agar medium containing the yeast cells expressing the SSTR3. The plates were incubated for 3 days at 30°C.
  • GPCRs is correlated with improved functional expression in mammalian and yeast cells with retention of full ability to couple to the heterotrimeric G protein.
  • the mutated MARs, CCKBR, and SSTR3 retain all external loops. Transmembrane domains and internal domains other than the IC3 are unchanged.
  • IC3 of the GPCRs containing the IC3 deletion is 44 amino acids in length.
  • the improvement in functional expression may be due to elimination of domains known to interact with cellular desensitization mechanisms, allowing more functional MAR to be retained at the cell surface.
  • DNA sequences encoding an IC3 ⁇ human alpha2A adrenergic receptor were amplified by PCR and cloned into proximity to the glycerol-phosphate dehydrogenase promoter in yeast expression plasmid, p426GPD, by standard methods.
  • the IC3 ⁇ human alpha2A adrenergic receptor was prepared by fusing two overlapping fragments.
  • Fragment 1 contained amino terminal coding sequences including 39 amino acids proximal to the 5th TMD, amplified by PCR using oligonucleotides (GGCCAGGATCCAAAAATGGGCTCCCTGCAGCCGGACGC) (SEQ ID NO: 20) and (CGGGCCCCGCGGGCGCTCGGGGCCCAGACCGTTGGGC) (SEQ ID NO: 21).
  • Fragment 2 contained carboxy terminal coding sequences including 41 amino acids proximal to the 6th TMD, amplified by PCR using oligonucleotides (CGGGCGACAGCCTGCCGCGGC) (SEQ ID NO: 22) and (AGCGGTCGACTCACACGATCCGCTTCCTGTCCCC) (SEQ ID NO: 23).
  • the two fragments were fused by amplification by PCR using oligos at 5' and 3' ends of the full length alpha2A adrenergic receptor.
  • the resulting plasmids were introduced into yeast cells useful for performing assays of GPCR agonist-stimulated growth, such as those described in United States Patent 5,691,188, including, specifically, the MPY578fc cells described in Pausch et al. (1998).
  • yeast cells containing the wild type IC3 ⁇ human alpha2A adrenergic receptor were assayed in liquid culture using the alpha adrenergic receptor full agonist UK14304 (RBI) and partial agonist clonidine.
  • the cells were cultured overnight in 2 ml SC-glucose-ura medium. The cells were diluted 500 fold in SC-glucose-ura-his, pH 6.8 medium. Samples of the cell suspension (180 ⁇ l) were dispensed to wells of sterile 96 well microtiter dishes containing 20 ⁇ l of serially-diluted samples of the adrenergic receptor agonist, UK14304 (10 "3 - 10 "10 M). The plates were incubated at 30 °C for 18 hours with agitation (600 rpm). Growth was monitored by recording increases in OD620 using a microplate reader. Assays were conducted in duplicate and growth rate measurements obtained during the logarithmic phase of yeast cell growth. Optical density measurements were analyzed using GraphPad Prism and are presented as the mean ⁇ SEM and were plotted vs. agonist concentration.
  • the yeast cells containing the wild type IC3 ⁇ human alpha 2 A adrenergic receptor produced a dose-dependent growth response, indicating that this IC3 deletion is functional ( Figure 5).
  • modification of the third intracellular loop leads to improvement in functional expression of a variety of heterologous GPCRs expressed in yeast.
  • Agonist induced desensitization of the GPCRs is also mediated in part by
  • GPCR internal domains other than the third intracellular loop such as the intracellular carboxy-terminal tail.
  • the rat NTR1 was modified by deleting the 52 amino acids that constitute the carboxy terminal tail, leaving a shortened receptor 372 amino acids in length.
  • the resulting plasmids were introduced into yeast cells of the kind described in United States Patent 5,691,188 that are useful for performing assays of GPCR agonist-stimulated growth, including, specifically, the MPY578fc cells described in Pausch et al. (1998).
  • Yeast cells containing the NTRls were assayed in liquid culture using the NT receptor agonist acetyl neurotensin 8-13 (AcNt8-13). The cells were cultured overnight in 2 ml SC-glucose-ura medium. The cells were diluted 500 fold in SC- glucose-ura-his, pH 6.8 medium containing 2 mM 3-aminotriazole to decrease basal growth rate. Samples of the cell suspension (180 ⁇ l) were dispensed to wells of sterile 96 well microtiter dishes containing 20 ⁇ l of serially-diluted samples (10 *3 -10 "10 M) of AcNT8-13. The plates were incubated at 30 °C for 18 hours with agitation (600 rpm). Growth was monitored by recording increases in OD 620 using a microplate reader. Growth rate measurements were obtained during the logarithmic phase of yeast cell growth. Optical density measurements were analyzed using GraphPad
  • the yeast cells containing the NTRls produced an agonist-dependent growth response demonstrating that both the wild type and carboxy terminally truncated NTRls were functional.
  • the growth response of the rNTRl C-term ⁇ containing cells was dose-dependent giving an EC50 for AcNT8-13 equal to 520 nM. This value is five fold lower than observed for cells expressing the wild type NTR1 (2.1 ⁇ M).
  • the carboxy terminal deletion has produced a rNTRl that responds to a lower concentration of NTR agonist improving the sensitivity of the yeast bioassay.
  • deletion of a portion of the carboxy terminal intracellular domain of the rat NTR1 has produced a functional GPCR with increased agonist sensitivity when expressed in yeast, suggesting that modification of this internal domain is a generalizable method for improving the function of heterologous GPCRs expressed in yeast.
  • Example 7 Functional Expression of a Mutated C. elegans Serotonin Receptor in Yeast
  • Ce 5HTR G protein-coupled C. elegans serotonin receptor
  • Fragment 1 contains the amino terminal- coding portion of the Ce 5HTR to the intracellular interface of the 5th TMD, amplified by PCR using oligonucleotides (AAAAGATCTAAAATGATCGACGAGACGCTTC) (SEQ ID NO: 27) and
  • the second fragment of the Ce 5HTR contains carboxy-terminal coding sequences starting proximal to the intracellular interface of the 6th TMD, amplified by PCR using oligonucleotides (AAAACTCGAGTCAATAATCGTGAATAAGGCA) (SEQ ID NO: 29) and (GGCCTACAGGCCTCTGGGACAGAAACCAGAAGTCAGATCACCAAGCGGA
  • oligonucleotides (AAAACTCGAGTCAATAATCGTGAATAAGGCA) (SEQ ID NO: 29) oligonucleotides.
  • the resulting fragment was digested with appropriate restriction endonucleases and assembled in p426GPD to place the mutated Ce 5HTR under control of the GPD promoter.
  • the resulting plasmids were introduced into yeast cells useful for performing assays of GPCR agonist-stimulated growth, such as those described in United States Patent 5,691,188, including, specifically, the MPY578fc cells described in Pausch et al. 1998.
  • yeast cells containing the Ce 5HTR were assayed in liquid culture.
  • the cells were cultured overnight in 2 ml SC-glucose-ura medium.
  • the cells were diluted 500 fold in SC-glucose-ura-his, pH 6.8 medium containing 2 mM 3-aminotriazole to decrease basal growth rate.
  • Samples of the cell suspension 200 ⁇ l were dispensed to wells of sterile 96 well microtiter dishes containing 2.0 ⁇ l of serially-diluted samples (10" 2 -10" 9 M) of serotonin (5HT).
  • the serotonergic antagonists lisuride and mianserin were added to each well at 10 ⁇ M.
  • the plates were incubated at 30°C for 18 hours with agitation (600 rpm).
  • replacing IC3 of the Ce 5HTR with the functional deleted IC3 from the rat M3 MAR produces a functional chimeric GPCR when expressed in yeast, indicating that this method of replacing internal domains may be a generalizable method for improving the functional expression of heterologous GPCRs in cell-based assays, such as yeast assays.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Endocrinology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne des récepteurs mutants couplés à la protéine G, lesquels présentent un meilleur couplage à la protéine G et une meilleure réponse de réception. L'invention concerne également des cellules de levure exprimant ces récepteurs, des vecteurs convenant à la fabrication de ces cellules et des méthodes de fabrication et d'utilisation associées.
EP99944035A 1998-09-01 1999-09-01 Methodes permettant d'ameliorer la fonction des recepteurs heterologues couples a la proteine g Withdrawn EP1123391A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US9870498P 1998-09-01 1998-09-01
US98704P 1998-09-01
PCT/US1999/020013 WO2000012705A2 (fr) 1998-09-01 1999-09-01 Methodes permettant d'ameliorer la fonction des recepteurs heterologues couples a la proteine g

Publications (1)

Publication Number Publication Date
EP1123391A2 true EP1123391A2 (fr) 2001-08-16

Family

ID=22270543

Family Applications (2)

Application Number Title Priority Date Filing Date
EP99944033A Withdrawn EP1112359A2 (fr) 1998-09-01 1999-09-01 Expression fonctionnelle amelioree des recepteurs couples a la proteine g
EP99944035A Withdrawn EP1123391A2 (fr) 1998-09-01 1999-09-01 Methodes permettant d'ameliorer la fonction des recepteurs heterologues couples a la proteine g

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP99944033A Withdrawn EP1112359A2 (fr) 1998-09-01 1999-09-01 Expression fonctionnelle amelioree des recepteurs couples a la proteine g

Country Status (5)

Country Link
EP (2) EP1112359A2 (fr)
JP (2) JP2002523090A (fr)
AU (2) AU756244B2 (fr)
CA (2) CA2344591A1 (fr)
WO (2) WO2000012705A2 (fr)

Families Citing this family (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6528271B1 (en) 1997-06-05 2003-03-04 Duke University Inhibition of βarrestin mediated effects prolongs and potentiates opioid receptor-mediated analgesia
JP2004500843A (ja) 2000-04-24 2004-01-15 ワイエス トランスジェニック動物
EP1337852A1 (fr) * 2000-10-26 2003-08-27 The University of British Columbia Essai fonctionnel a base de cellules d'insectes concernant les recepteurs coupl s aux proteines g
ATE357457T1 (de) * 2000-11-01 2007-04-15 Astellas Pharma Inc Verfahren zum screenen eines plättchenhemmers
US7018812B2 (en) 2000-11-03 2006-03-28 Duke University Modified G-protein coupled receptors
US7163800B2 (en) 2000-11-03 2007-01-16 Molecular Devices Corporation Methods of screening compositions for G protein-coupled receptor desensitization inhibitory activity
DE10056899A1 (de) 2000-11-16 2002-05-29 Aventis Pharma Gmbh Promotor zur funktionellen Charakterisierung von G-Protein gekoppelten Rezeptoren in der Hefe Saccharomyces cerevisiae
AU2002228844A1 (en) * 2000-12-06 2002-06-18 Deltagen, Inc. Transgenic mice containing targeted gene disruptions
GB0030038D0 (en) * 2000-12-08 2001-01-24 Univ Warwick Yeast-based assay
US7279324B2 (en) 2001-01-23 2007-10-09 Duke University Nucleic acid encoding G-protein coupled receptor with modified DRY motif
EP1336655A1 (fr) * 2002-01-12 2003-08-20 Aventis Pharma Deutschland GmbH Méthode pour l'identification des mutants ayant une actività constitutive
DE10233516A1 (de) * 2002-07-23 2004-02-12 Aventis Pharma Deutschland Gmbh Verfahren zur Identifizierung von Substanzen
WO2006104254A1 (fr) * 2005-03-29 2006-10-05 Bio-Energy Corporation Procede pour le criblage d'une substance de liaison des recepteurs
JP5774264B2 (ja) 2005-04-27 2015-09-09 アリーナ ファーマシューティカルズ， インコーポレイテッド 肥満および糖尿病、ならびにそれらに関連する病態の処置のため、ならびに血中ｇｌｐ−１レベルを増大させることによって緩和される病態の処置のための、併用療法
NZ564759A (en) 2005-06-30 2011-08-26 Prosidion Ltd GPCR agonists
DE602007010420D1 (de) 2006-04-11 2010-12-23 Arena Pharm Inc Verfahren zur verwendung des gpr119-rezeptors zur identifizierung von verbindungen zur erhöhung der knochenmasse bei einer person
AR064736A1 (es) 2007-01-04 2009-04-22 Prosidion Ltd Agonistas de gpcr
CL2008000018A1 (es) 2007-01-04 2008-08-01 Prosidion Ltd Compuestos derivados de heterociclos de nitrogeno y oxigeno, agonistas de gpcr; composicion farmaceutica que comprende a dicho compuesto; y uso del compuesto para el tratamiento de la obesidad, diabetes, sindrome metabolico, hiperlipidemia, toleranci
ES2373181T3 (es) 2007-01-04 2012-02-01 Prosidion Ltd Agonistas de gpcr de piperidina.
EP3926045A1 (fr) * 2007-03-22 2021-12-22 Heptares Therapeutics Limited Protéines mutantes et leurs procédés de sélection
JP2010539152A (ja) 2007-09-10 2010-12-16 プロシディオン・リミテッド 代謝障害の治療のための化合物
GB0720389D0 (en) 2007-10-18 2008-11-12 Prosidion Ltd G-Protein Coupled Receptor Agonists
GB0720390D0 (en) 2007-10-18 2007-11-28 Prosidion Ltd G-Protein coupled receptor agonists
EP2108960A1 (fr) 2008-04-07 2009-10-14 Arena Pharmaceuticals, Inc. Procédés d'utilisation d'un récepteur couplé à protéine G pour identifier les secrétagogues de peptide YY (PYY) et composés utiles dans le traitement des conditions modulées de secrétagogues BY (PYY) et composés utiles dans le traitement des conditions par PYY
GB0904285D0 (en) 2009-03-12 2009-04-22 Prosidion Ltd Compounds for the treatment of metabolic disorders
GB0904284D0 (en) 2009-03-12 2009-04-22 Prosidion Ltd Compounds for the treatment of metabolic disorders
GB0904287D0 (en) 2009-03-12 2009-04-22 Prosidion Ltd Compounds for the treatment of metabolic disorders
JP7422498B2 (ja) 2019-07-25 2024-01-26 芳武 村山 ペプチド、細胞遊走抑制剤、細胞浸潤抑制剤、がん細胞転移抑制剤、及び医薬

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5691188A (en) * 1994-02-14 1997-11-25 American Cyanamid Company Transformed yeast cells expressing heterologous G-protein coupled receptor
CA2193809A1 (fr) * 1994-06-29 1996-01-11 Catherine D. Strader Recepteurs modifies lies a des proteines g
WO1998038217A1 (fr) * 1997-02-27 1998-09-03 Milt Teitler Recepteurs de serotonine actives par constitution

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0012705A3 *

Also Published As

Publication number Publication date
CA2344591A1 (fr) 2000-03-09
JP2002523091A (ja) 2002-07-30
WO2000012705A3 (fr) 2000-10-05
EP1112359A2 (fr) 2001-07-04
WO2000012705A9 (fr) 2000-07-13
AU5700999A (en) 2000-03-21
WO2000012705A2 (fr) 2000-03-09
WO2000012704A9 (fr) 2000-06-08
CA2343046A1 (fr) 2000-03-09
WO2000012704A3 (fr) 2000-11-30
AU756244B2 (en) 2003-01-09
WO2000012704A2 (fr) 2000-03-09
JP2002523090A (ja) 2002-07-30
AU5701199A (en) 2000-03-21

Similar Documents

Publication Publication Date Title
AU756244B2 (en) Methods for improving the function of heterologous G protein-coupled receptors
US20080153124A1 (en) Methods for improving the function of heterlogous G protein-coupled receptors
US6406871B1 (en) Method for detecting ligand binding to G protein coupled receptors
Price et al. Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway
US5739029A (en) Vectors for expression of G protein coupled receptors in yeast
JP5108062B2 (ja) Ｇタンパク質キメラ体
Sarramegna et al. Optimizing functional versus total expression of the human μ-opioid receptor in Pichia pastoris
Ladds et al. Modified yeast cells to investigate the coupling of G protein‐coupled receptors to specific G proteins
US6291177B1 (en) Assay for agents which alter G-protein coupled receptor activity
Reiländer et al. Large-Scale Expression of Receptors in Yeasts
WO2002083934A1 (fr) Recepteur p2yac implique dans l'agregation plaquettaire

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17P Request for examination filed

Effective date: 20010426

17Q First examination report despatched

Effective date: 20030924

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040406