EP1137776A2 - Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive - Google Patents

Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive

Info

Publication number
EP1137776A2
EP1137776A2 EP99950301A EP99950301A EP1137776A2 EP 1137776 A2 EP1137776 A2 EP 1137776A2 EP 99950301 A EP99950301 A EP 99950301A EP 99950301 A EP99950301 A EP 99950301A EP 1137776 A2 EP1137776 A2 EP 1137776A2
Authority
EP
European Patent Office
Prior art keywords
endogenous
protein
human
cdna
plasmid
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Withdrawn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
EP99950301A
Other languages
German (de)
English (en)
Inventor
Dominic P. Behan
Karin Lehmann-Bruinsma
Derek T. Chalmers
Ruoping Chen
Huong T. Dang
Martin Gore
Chen W. Liaw
I-Lin Lin
Kevin Lowitz
Carol White
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.)
Arena Pharmaceuticals Inc
Original Assignee
Arena Pharmaceuticals Inc
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
Priority claimed from US09/170,496 external-priority patent/US6555339B1/en
Application filed by Arena Pharmaceuticals Inc filed Critical Arena Pharmaceuticals Inc
Priority to EP05009877A priority Critical patent/EP1676861A3/fr
Priority to EP10007972A priority patent/EP2264068A1/fr
Priority claimed from PCT/US1999/024065 external-priority patent/WO2000022131A2/fr
Publication of EP1137776A2 publication Critical patent/EP1137776A2/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Withdrawn legal-status Critical Current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
    • C07K14/435—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

Definitions

  • receptors and more particularly to human G protein-coupled receptors, and specifically to GPCRs that have been altered to establish or enhance constitutive activity of the receptor.
  • the altered GPCRs are used for the direct identification of candidate compounds
  • receptor agonists as receptor agonists, inverse agonists or partial agonists having potential applicability as receptor agonists, inverse agonists or partial agonists having potential applicability as
  • GPCR G protein-coupled receptor
  • GPCRs GPCRs
  • GPCRs for which the endogenous ligand has been identified are referred to as
  • GPCRs represent an important area for the development
  • each span is identified by number, i. e., transmembrane- 1 (TM-
  • transmebrane-2 (TM-2), etc.).
  • the transmembrane helices are joined by strands of amino acids
  • transmembrane-2 and transmembrane-3 acids between transmembrane-2 and transmembrane-3 , transmembrane-4 and transmembrane-
  • extracellular regions 1 , 2 and 3 (EC- 1 , EC-2 and EC-
  • transmembrane helices are also joined by strands of amino acids
  • activation of the receptor
  • Gq, Gs, Gi, Gz and Go are G proteins
  • signal transduction begins a signaling cascade process (referred to as “signal transduction"). Under normal
  • GPCRs exist in the cell membrane in
  • a receptor in an inactive state is unable to link to the intracellular signaling transduction
  • a receptor may be stabilized in an active state by an endogenous ligand or a compound such as a drug.
  • Figure 1 is a representation of 8XCRE-Luc reporter plasmid (see, Example
  • Figures 2A and 2B are graphic representations of the results of ATP and ADP
  • Figure 3 is a graphic representation of the comparative signaling results of
  • AGONISTS shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor, or enhance GTP binding to
  • PARTIAL AGONISTS shall mean materials (e.g., ligands, candidate compounds)
  • ANTAGONIST shall mean materials (e.g., ligands, candidate compounds) that
  • the intracellular response initiated by the active form of the receptor can thereby inhibit
  • CANDIDATE COMPOUND shall mean a molecule (for example, and not limitation, a chemical compound) that is amenable to a screening technique.
  • a chemical compound for example, and not limitation, a chemical compound that is amenable to a screening technique.
  • COMPOSITION means a material comprising at least one component
  • composition is an example of a composition.
  • COMPOUND EFFICACY shall mean a measurement of the ability of a compound
  • CODON shall mean a grouping of three nucleotides (or equivalents to nucleotides)
  • nucleoside adenosine (A), guanosine (G), cytidine (C), uridine
  • CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a receptor subject to
  • a constitutive receptor activation can be endogenous or non-
  • CONSTITUTIVE RECEPTOR ACTIVATION shall mean stabilization of a
  • receptor in the active state by means other than binding of the receptor with its endogenous ligand or a chemical equivalent thereof.
  • CONTACT or CONTACTING shall mean bringing at least two moieties together
  • constitutively activated receptor preferably a constitutively activated o ⁇ han receptor
  • ENDOGENOUS shall mean a material that a mammal naturally produces.
  • NON-ENDOGENOUS in this context shall mean
  • a mammal for example, and not limitation, a human
  • a receptor which is not constitutively active in its
  • non-endogenous, constitutively activated receptor referred to herein as a “non-endogenous, constitutively activated receptor.” Both terms can
  • the endogenous or non-endogenous receptor may be in reference to
  • a mammal has been manipulated to include a non-endogenous constitutively activated
  • PROTEIN in the context of the invention disclosed herein, each mean a non-endogenous
  • o ⁇ han GPCR For example, and not limitation, in an endogenous state, if the G protein
  • Gs ⁇ is the predominate G protein that couples with the GPCR, a GPCR Fusion Protein
  • Gs ⁇ fused to Gs ⁇ ; in some circumstances, as will be set forth below, a non-predominant G protein
  • the G protein can be fused directly to the c-terminus of the
  • GPCR constitutively active GPCR or there may be spacers between the two.
  • HOST CELL shall mean a cell capable of having a Plasmid and/or Vector
  • Plasmid In the case of a prokaryotic Host Cell, a Plasmid is typically replicated
  • Plasmid as a autonomous molecule as the Host Cell replicates (generally, the Plasmid is thereafter
  • Plasmid is integrated into the cellular DNA of the Host Cell such that when the eukaryotic
  • the Host Cell is eukaryotic, more preferably, mammalian, and most
  • INVERSE AGONISTS shall mean materials (e.g., ligand, candidate compound)
  • the baseline intracellular response is inhibited in the presence of the inverse agonist by at least
  • KNOWN RECEPTOR shall mean an endogenous receptor for which the endogenous
  • LIGAND shall mean an endogenous, naturally occurring molecule specific for an
  • a subsequent mutated form of a human receptor is considered to be equivalent to a first mutation of the human receptor if (a) the level of constitutive activation of the
  • the of the receptor is at least about 80%, more preferably at least about 90% and most preferably
  • for achieving constitutive activation includes a single amino acid and/or codon change
  • homology should be at least 98%.
  • NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurring
  • ligand to a receptor activates an intracellular signaling pathway.
  • ORPHAN RECEPTOR shall mean an endogenous receptor for which the
  • PHARMACEUTICAL COMPOSITION shall mean a composition comprising at
  • composition is amenable to investigation for a
  • PLASMID shall mean the combination of a Vector and cDNA.
  • a Plasmid shall mean the combination of a Vector and cDNA.
  • VECTOR in reference to cDNA shall mean a circular DNA capable of inco ⁇ orating
  • At least one cDNA and capable of inco ⁇ oration into a Host Cell at least one cDNA and capable of inco ⁇ oration into a Host Cell.
  • any search for therapeutic compounds should start by screening compounds against
  • GPCRs GPCR (Accession No.) hARE-3 AL033379 1,260 bp 52.3% LPA-R U92642 hARE-4 AC006087 1,119 bp 36% P2Y5 AF000546 hARE-5 AC006255 1,104 bp 32% Oryzias D43633 latipes hGPR27 AA775870 1,128 bp hARE-1 AI090920 999 bp 43% D13626 KIAA0001 hARE-2 AA359504 1,122 bp 53% GPR27 hPPRl H67224 1,053 bp 39% EBI1 L31581 hG2A AA754702 1,113 bp 31% GPR4 L36148 hRTJP3 AL035423 1,005 bp 30% 2133653
  • Receptor homology is useful in terms of gaining an appreciation of a role of the
  • human GPCRs disclosed herein is expressed and/or over-expressed, it is possible to determine
  • human GPCR disclosed herein is based upon the distance from the proline residue at which
  • non-endogenous, constitutively activated GPCR can be identified by the methodologies of this
  • compositions a search for diseases and disorders associated with the GPCR
  • Tissue scans can be used to identify an endogenous ligand to the specific GPCR.
  • tissue scans provide a broad range of healthy and diseased tissues. Such tissue scans provide
  • a preferred first step in associating a specific receptor with a disease and/or disorder See, for example, co-pending application (docket number ARE-0050) for exemplary dot-blot and RT-
  • the DNA sequence of the human GPCR is used to make a probe for (a)
  • compared to a normal tissue can be preferably utilized to identify a correlation with a
  • treatment regimen including but not limited to, a disease associated with that disease.
  • Receptors can equally well be localized to regions of organs by this technique. Based on
  • a G protein receptor When a G protein receptor becomes constitutively active, it binds to a G protein (e.g.,
  • Gq Gs, Gi, Gz, Go
  • this assay system is for initial screening of candidate compounds because the system is generically applicable to all G protein-coupled receptors regardless of the particular
  • G protein that interacts with the intracellular domain of the receptor.
  • receptor assay (/. e., an assay to select compounds that are agonists, partial agonists, or inverse
  • a compound identified by the "generic" assay may not bind to the
  • the receptor may instead merely "uncouple" the G protein from the intracellular domain.
  • Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on the other hand,
  • Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus,
  • constitutively activated GPCRs that couple the Gs protein are associated with increased
  • a candidate compound is, e.g., an inverse agonist to the receptor
  • Cyclic AMP drives gene
  • cAMP cAMP -responsive DNA binding protein or transcription factor
  • Reporter systems can be constructed
  • Gs-linked receptor e.g., ⁇ -galactosidase or luciferase.
  • the reporter protein such as ⁇ -galactosidase or luciferase can then be detected using standard
  • Gq and Go are associated with activation of the enzyme phospholipase C, which in
  • DAG diacycloglycerol
  • IP 3 inistol 1,4,5-triphoisphate
  • a candidate compound is, e.g., an inverse agonist to a Gq- or Go-
  • phospholipase C causes activation of genes containing API elements; thus, activated Gq-
  • a preferred approach is the use of a GPCR Fusion Protein.
  • Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed.
  • the GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the non-endogenous GPCR.
  • the GPCR Fusion Protein is preferred for screening with
  • expression vectors and systems offer a variety of approaches that can fit the particular needs
  • endogenous GPCR sequence and the G protein sequence both be in-frame (preferably,
  • sequence for the endogenous GPCR is upstream of the G protein sequence
  • the G protein can also be expressed.
  • the GPCR can be linked directly to the G
  • the G protein is not used to, effectively, upon expression, become a spacer.
  • the G protein is not used to, effectively, upon expression, become a spacer.
  • a construct comprising the sequence of the G protein (i.e., a
  • G protein in an effort to establish a viable cyclase-based assay.
  • a Gz a Gz
  • a GPCR Fusion Protein can be established that utilizes a Gs
  • Candidate compounds selected for further development can be formulated into
  • human GPCRs can also be utilized in research settings. For example, in vitro and in vivo
  • cassettes from one sequence to another e.g. from rat receptor to human receptor or from
  • human receptor A to human receptor B is generally predicated upon sequence alignment
  • GPCRs (Base Pairs) hGPCR27 Mouse AA775870 1,125 bp 17 18 GPCR27 hARE-1 TDAG 1689643 999 bp 19 20 AI090920 hARE-2 GPCR27 68530 1,122 bp 21 22 AA359504 hPPRl Bovine 238667 1 ,053 bp 23 24 PPR1 H67224 hG2A Mouse See Example 2(a), 1,113 bp 25 26 1 179426 below hCHN3 N.A. EST 36581 1.113 bp 27 28 (full length) hCHN4 TDAG 1184934 1 ,077 bp 29 30 AA804531 hCHN6 N.A.
  • Mouse EST clone 1179426 was used to obtain a human genomic clone containing all but three amino acid G2A coding sequences. The 5 'of this coding sequence was obtained by
  • the disclosed human G2A was amplified by PCR using the G2A cDNA specific
  • PCR fragment was purified from agarose gel, digested with Hind III and Xba I and cloned into
  • the 5' primer sequence utilized was as follows:
  • the cycle condition was 30 cycles of 94°C for 1 min, 65°C for lmin
  • RUP4 The full length RUP4 was cloned by RT-PCR with human brain cDNA (Clontech) as
  • PCR was performed using TaqPlus PrecisionTM polymerase (Stratagene; manufacturing
  • PCR products were separated on a 1% agarose gel and a 500 bp PCR fragment
  • the 3' RACE product contained a poly(A) tail and a completed open reading frame ending
  • the 5' RACE product contained an incomplete 5' end; i.e., the ATG
  • oligo 3 Based on the new 5' sequence, oligo 3 and the following primer:
  • 5'-GCAATGCAGGTCATAGTGAGC -3' (SEQ.ID.NO.: 52; oligo 5) were used for the second round of 5 ' race PCR and the PCR products were analyzed as above.
  • 5'-GTGATGAGCAGGTCACTGAGCGCCAAG-3' (SEQ.ID.NO.: 54: oligo7).
  • the sequence of the 5' RACE PCR products revealed the presence of the initiation codon ATG, and further round of 5' race PCR did not generate any more 5' sequence.
  • the full length RUP5 was cloned by RT-PCR using a sense primer upstream from
  • step 2 through step 4 repeated 30 times: 94°C for 30 sec; 94° for 15 sec; 69° for 40 sec;
  • Cycles 2 through 4 were repeated 30 times.
  • step 2 to step 4 repeated 30 times: 94 °C for 2 minutes; 94 °C for 15 seconds; 60°C
  • ATI human angiotensin II type 1 receptor
  • each primer 0.25 ⁇ M of each primer, and 0.2 mM of each 4 nucleotides.
  • the cycle condition was 30 cycles of 94°C for 1 min, 55°C for lmin and 72 °C for 1.5 min.
  • the 5' PCR primer contains a Hindlll site with the sequence: 5'-CCCAAGCTTCCCCAGGTGTATTTGAT-3' (SEQ.ID.NO.: 63) and the 3' primer contains a BamHl site with the following sequence:
  • Nucleic acid SEQ.ID.NO.: 65
  • amino acid SEQ.ID.NO.: 66
  • PCR was performed by combining two PCR fragments, using
  • the cycle condition for each PCR reaction was 30 cycles of 94 °C for 1 min, 62 °C for lmin
  • the first fragment was amplified with the 5' PCR primer that contained an end site
  • the second PCR fragment was amplified with a 5' primer having the following sequence: 5'-GTCCGCGTCCTGCTGGTGGTGGTTCTGGCATTTATAATT-3' (SEQ.ID.NO.: 69) and a 3' primer that contained a BamHl site and having the following sequence:
  • the two fragments were used as templates to amplify GPR38, using SEQ.ID.NO.: 67 and
  • SEQ.ID.NO.: 70 as primers (using the above-noted cycle conditions).
  • the resulting 1.44kb PCR fragment was digested with BamHl and cloned into Blunt-BamHI site of pCMV
  • PCR was performed using human genomic cDNA as template and
  • the 5' PCR contained an EcoRI site with the sequence:
  • the 1.0 kb PCR fragment was digest with EcoRI and BamHl and cloned into EcoRI -BamHl
  • PCR was performed using human stomach cDNA as template and
  • the 5' PCR contained a Hindlll site with the sequence:
  • the resulting 1.44 kb PCR fragment was digest with Hindlll and EcoRI and cloned into Hindlll-EcoRI site of pCMV expression vector. Nucleic acid (SEQ.ID.NO.: 77) and amino
  • PCR was performed using genomic DNA as template and rTth
  • each primer and 0.2 mM of each 4 nucleotides.
  • the cycle condition was 30 cycles of 94°C
  • the 5' PCR primer contained a
  • the resulting 1.1 kb PCR fragment was digested with Hindlll and BamHl and cloned into
  • each primer and 0.2 mM of each 4 nucleotides.
  • the cycle condition was 30 cycles of 94°C
  • the 5' PCR primer contained a Hindlll site
  • the resulting 1.9 kb PCR fragment was digested with Hindlll and BamHl and cloned into
  • Hindlll-BamHI site of pCMV expression vector. H9 contained three potential polymo ⁇ hisms
  • TM6 region of the GPCR, near the TM6/IC3 interface is mutated, most preferably to a
  • Two mutagenesis primers are utilized, most preferably a lysine
  • mutagenesis oligonucleotide that creates the lysine mutation and a selection marker
  • the two mutagenesis primers were used, a lysine mutagenesis oligonucleotide
  • the 5' PCR sense primer used had the following sequence: 5'-CCCAAGCTTCCCCAGGTGTATTTGAT-3' (SEQ.ID.NO.: 95) and the antisense primer had the following sequence: 5'-CCTGCAGGCGAAACTGACTCTGGCTGAAG-3' (SEQ.ID.NO.: 96).
  • the resulting 400 bp PCR fragment was digested with Hindlll site and subcloned into
  • Nl 11 A construct The cycle condition was 25 cycles of 94°C for 1 min, 60°C for lmin
  • 3' untranslated region was generated by using the following sequence: 5'-AGATCTTAAGAAGATAATTATGGCAATTGTGCT-3' (SEQ.ID.NO.: 102) as sense primer and SEQ.ID.NO.: 64 as antisense primer.
  • the PCR condition was 30
  • Fragment A (720 bp) was digested with Hindlll and
  • Fragment B was digested with BamHl and subcloned into pCMV
  • Fragment C was inserted in front of Fragment B through EcoRI and Aflll site.
  • PCR sense primer had the following sequence:
  • the 3 ' PCR sense primer utilized had the following sequence:
  • the PCR condition was the
  • the first PCR fragment (lkb) was amplified by using SEQ.ID.NO.: 75 and an
  • antisense primer comprising a V322K mutation
  • the second PCR fragment (0.44kb) was amplified by using a sense primer comprising the
  • V322K mutation 5'-AGAAGCGCGTGAAGCGCATGCTGCTGGTGATCGTT-3' (SEQ.ID.NO: 114) and SEQ.ID.NO.:
  • Preparation of non-endogenous human GPCRs can also be accomplished by using
  • Endogenous GPCR is preferably used as a template and two mutagenesis
  • primers utilized utilized, as well as, most preferably, a lysine mutagenesis oligonucleotide and a
  • selection marker oligonucleotide (included in kit).
  • codon mutation For convenience, the codon mutation
  • yeast cells upon practicalities, i.e., utilization of, e.g., yeast cells for the expression of a GPCR, while
  • yeast does not) include the receptor-coupling, genetic-mechanism and secretary
  • mammalian cells while of potential use, are not as preferred as that obtained from mammalian
  • COS-7, 293 and 293T cells are particularly preferred, although
  • reaction tubes were prepared (the proportions to follow for each tube are per plate): tube A
  • DNA e.g., pCMV vector; pCMV vector with receptor
  • tube B was prepared by mixing 120 ⁇ l lipofectamine (Gibco BRL) in 1.2ml serum free DMEM. Tubes
  • a and B were admixed by inversions (several times), followed by incubation at room
  • the admixture is referred to as the "transfection mixture”.
  • the receptor couples to a G protein and stimulates the
  • the assay utilizes the ability of G protein coupled receptors to stimulate [ 35 S]GTP ⁇ S
  • the assay can, therefore, be used in
  • the assay is generic and has application
  • the [ 35 S]GTP ⁇ S assay can be incubated in 20 mM HEPES and between 1 and about
  • ⁇ g membrane protein e.g, COS-7 cells expressing the receptor; this amount can be adjusted
  • Flash platesTM and WallacTM scintistrips may be utilized
  • the assay can be utilized for known GPCRs to simultaneously monitor tritiated ligand binding
  • This assay may also be used to detect other types of membrane
  • the assay may be used to determine whether receptor activation events resulting in receptor activation.
  • the assay may be used to determine whether receptor activation events resulting in receptor activation.
  • the assay may be used to determine whether receptor activation events resulting in receptor activation.
  • the scintistrip label comes into proximity with the radiolabeled ligand resulting
  • Flash Plate wells contain a scintillant coating which also contains a specific antibody-
  • the cAMP generated in the wells was quantitated by a direct
  • Transfected cells are harvested approximately three days after transfection.
  • Membranes were prepared by homogenization of suspended cells in buffer containing 20mM
  • HEPES, pH 7.4 and lOmM MgCl 2 Homogenization is performed on ice using a Brinkman
  • Assay Buffer is prepared fresh for screening and contained
  • Assay Buffer can be stored on ice
  • the assay is initiated by addition of 50ul of assay buffer followed by addition
  • a method to detect Gs stimulation depends on the known property of the transcription
  • CREB CREB factor CREB
  • a PathDetectTM CREB trans- Reporting System (Stratagene, Catalogue # 219010) can utilized to assay for Gs coupled
  • Gal4 recognition sequences 40 ng pFA2-CREB (Gal4-CREB fusion protein containing the
  • Gal4 DNA-binding domain 80 ng pCMV -receptor expression plasmid (comprising the
  • CMV-SEAP secreted alkaline phosphatase expression plasmid; alkaline
  • phosphatase activity is measured in the media of transfected cells to control for variations in
  • a method to detect Gq stimulation depends on the known property of Gq-dependent
  • a PathdetectTM AP-1 cis-Reporting System (Stratagene, Catalogue # 219073) can be utilized
  • the components of the calcium phosphate precipitate were 410 ng pAP 1 -Luc. 80 ng pCMV-
  • 293 and 293T cells are plated-out on 96 well plates at a density of 2 x 10 4 cells per well and were transfected using Lipofectamine Reagent (BRL) the following day according
  • pCMV comprising endogenous receptor or non-endogenous receptor or pCMV alone
  • reporter plasmid was prepared as follows: vector SRIF- ⁇ -gal was obtained by cloning the rat
  • somatostatin promoter (-71/+51) at BglV-Hindlll site in the p ⁇ gal-Basic Vector (Clontech).
  • AdpCF126CCRE8 see, 1 Human Gene Therapy 1883 (1996)
  • 8xCRE-Luc reporter plasmid was generated by replacing the beta-galactosidase gene in the
  • DNA/lipid mixture was diluted with 400 ⁇ l of DMEM and lOO ⁇ l of the diluted mixture was
  • a PathdetectTM SRF-Luc-Reporting System (Stratagene) can be utilized to assay
  • Gq coupled activity in, e.g., COS7 cells.
  • Cells are transfected with the plasmid
  • alkaline phosphatase activity is measured in the media of transfected cells to control for
  • cells comprising the receptors can be endogenous and/or non-endogenous.
  • the cells are then incubated for 3-4 hrs at 37°C/5%CO 2 and then the
  • transfection media is removed and replaced with 1 ml/well of regular growth media.
  • the cells are then incubated for 30 min at 37 °C.
  • the cells are then incubated for 30 min at 37 °C.
  • the lysate is then transferred into 1.5 ml eppendorf tubes and 1 ml of
  • the transfected cells were grown in media containing serum for an assay
  • the serum-free media was comprised solely
  • DME Dulbecco's Modified Eagle's
  • the media with serum contained the following: 10%
  • a 96-well Adenylyl Cyclase Activation FlashplateTM was used (NEN: #SMP004A).
  • CMV or TDAG8 were harvested 24 (assay detection in serum media) or 48 hours post-

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  • Zoology (AREA)
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Abstract

La présente invention se rapporte à des récepteurs transmembranaires, notamment au récepteur de la protéine G humaine pour lequel le ligand endogène est connu ('récepteurs GPCR orphelins') et, plus particulièrement, à des versions mutées (non endogènes) des GPCR humains pouvant révéler une activité constitutive.
EP99950301A 1998-10-13 1999-10-13 Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive Withdrawn EP1137776A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP05009877A EP1676861A3 (fr) 1998-10-13 1999-10-13 Récepteurs non-endogènes de la protéine G humaine ayant une activité constitutive
EP10007972A EP2264068A1 (fr) 1998-10-13 1999-10-13 Récepteurs non-endogènes de la protéine G humaine ayant une activité constitutive

Applications Claiming Priority (65)

Application Number Priority Date Filing Date Title
1998-01-20
US170496 1998-10-13
US09/170,496 US6555339B1 (en) 1997-04-14 1998-10-13 Non-endogenous, constitutively activated human protein-coupled receptors
US10802998P 1998-11-12 1998-11-12
US108029P 1998-11-12
US10921398P 1998-11-20 1998-11-20
US109213P 1998-11-20
US11006098P 1998-11-27 1998-11-27
US110060P 1998-11-27
US12041699P 1999-02-16 1999-02-16
US120416P 1999-02-16
US12185299P 1999-02-26 1999-02-26
US121852P 1999-02-26
US12394499P 1999-03-12 1999-03-12
US12394599P 1999-03-12 1999-03-12
US12395199P 1999-03-12 1999-03-12
US12394999P 1999-03-12 1999-03-12
US12394899P 1999-03-12 1999-03-12
US12394699P 1999-03-12 1999-03-12
US123946P 1999-03-12
US123951P 1999-03-12
US123949P 1999-03-12
US123944P 1999-03-12
US123948P 1999-03-12
US123945P 1999-03-12
US13713199P 1999-05-28 1999-05-28
US13643999P 1999-05-28 1999-05-28
US13643699P 1999-05-28 1999-05-28
US13712799P 1999-05-28 1999-05-28
US13756799P 1999-05-28 1999-05-28
US13643799P 1999-05-28 1999-05-28
US137127P 1999-05-28
US136439P 1999-05-28
US137131P 1999-05-28
US137567P 1999-05-28
US136436P 1999-05-28
US136437P 1999-05-28
US14144899P 1999-06-29 1999-06-29
US141448P 1999-06-29
US15111499P 1999-08-27 1999-08-27
US151114P 1999-08-27
US15252499P 1999-09-03 1999-09-03
US152524P 1999-09-03
US15665399P 1999-09-29 1999-09-29
US15663499P 1999-09-29 1999-09-29
US15663399P 1999-09-29 1999-09-29
US15655599P 1999-09-29 1999-09-29
US156633P 1999-09-29
US156555P 1999-09-29
US156634P 1999-09-29
US15729399P 1999-10-01 1999-10-01
US15729499P 1999-10-01 1999-10-01
US15728299P 1999-10-01 1999-10-01
US15728199P 1999-10-01 1999-10-01
US15728099P 1999-10-01 1999-10-01
US157280P 1999-10-01
US157293P 1999-10-01
US157282P 1999-10-01
US157294P 1999-10-01
US157281P 1999-10-01
US41676099A 1999-10-12 1999-10-12
US41704499A 1999-10-12 1999-10-12
US417044 1999-10-12
US416760 1999-10-12
PCT/US1999/024065 WO2000022131A2 (fr) 1998-10-13 1999-10-13 Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive

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