WO1999003974A1 - Compositions et procedes servant a identifier des modulateurs de transducisomes, nouvelle categorie de cibles therapeutiques - Google Patents

Compositions et procedes servant a identifier des modulateurs de transducisomes, nouvelle categorie de cibles therapeutiques Download PDF

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Publication number
WO1999003974A1
WO1999003974A1 PCT/US1998/014667 US9814667W WO9903974A1 WO 1999003974 A1 WO1999003974 A1 WO 1999003974A1 US 9814667 W US9814667 W US 9814667W WO 9903974 A1 WO9903974 A1 WO 9903974A1
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protein
signal transduction
transducisome
cell
inad
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PCT/US1998/014667
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English (en)
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Charles S. Zuker
John D. Mendlein
Humei Sun
Susan Tsunoda
Jimena Sierralta
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The Regents Of The University Of California
Aurora Biosciences Corporation
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Priority to US09/462,517 priority Critical patent/US7087388B1/en
Priority to AU84059/98A priority patent/AU8405998A/en
Publication of WO1999003974A1 publication Critical patent/WO1999003974A1/fr
Priority to US11/501,473 priority patent/US20070150970A1/en

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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • 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)
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the invention relates to compositions and methods for identifying modulators of signal transduction in cells, particularly signal transduction related to cell surface receptors and ion channels.
  • GPCRs G-protein coupled receptors
  • GPCRs mediate signal transduction across a cell membrane upon the binding of a ligand to an extracellular portion of a GPCR.
  • the intracellular portion of a GPCR interacts with a G-protein to modulate signal transduction from outside to inside a cell.
  • a GPCR is therefore said to be "coupled" to a G-protein.
  • G-proteins are composed of three polypeptide subunits: an ⁇ subunit, which binds and hydolyzes GTP, and a dimeric ⁇ subunit. In the basal, inactive state, the G-protein exists as a heterotrimer of the ⁇ and ⁇ subunits.
  • GDP guanosine diphosphate
  • GTP guanine triphosphate
  • active G ⁇ subunit disassociates from both the receptor and the dimeric ⁇ subunit.
  • the disassociated, active G ⁇ subunit transduces signals to effectors that are "downstream" in the G-protein signaling pathway within the cell.
  • the G-protein's endogenous GTPase activity returns active G subunit to its inactive state, in which it is associated with GDP and the dimeric ⁇ subunit.
  • Transducisome proteins as described further herein, assembly and organize many types of signal transduction proteins using PDZ domains to permit or enhance signal transduction.
  • Cells respond to a wide variety of external signals mediated by cell surface receptors that transduce extracellular stimuli into an intracellular response. Although receptors that recognize different ligands are known to interact with the same intracellular signaling molecules, the specificity of signaling, often essential to a cell's physiological role, is maintained. How is this specificity maintained, or rather, how is signal cross talk avoided?
  • One solution involves organizing different signaling cascades into physically and functionally distinct signaling units. Such assemblies can permit or enhance signal response time, specificity and selectivity while minimizing cross talk. Until the advent of present invention, know little was known about the architectural organization of the corresponding signaling machinery or how it can be used to discover useful modulators of signal transduction.
  • Transducisome refers to a PDZ domain containing protein that binds at least one signal transduction protein or a PDZ domain containing protein with at least one signal transduction protein bound to it (see FIG. 1 A-C). Other types of transducisomes are described herein.
  • the invention includes methods for identifying modulators of signal transduction comprising contacting a transducisome in a biochemical assay, cell assay or animal assay, with a test chemical and detecting a change in signal transduction.
  • the invention includes animals (e.g., mice and flies), cells (e.g., mammalian and insect), with transducisomes, modified transducisomes and defective transducisomes to use in assays of signal transduction.
  • the invention also includes proteins (as well as polynucleotides encoding the same) corresponding to transducisomes, modified transducisomes or defective transducisomes to use in assays of signal transduction.
  • the invention also includes a screening assay system for detecting protein-protein interactions.
  • This screening assay system comprises a recombinant protein comprising at least one PDZ domain; a PDZ binding protein; and at least one test chemical.
  • the recombinant protein or the PDZ binding protein or both have a label to facilitate the detection of specific binding.
  • the recombinant protein has a donor and the PDZ binding protein has a quencher, wherein the donor and quencher are energy transfer partners, as described herein.
  • the PDZ binding protein can be selected from the group of a kinase, a phosphatase, a GPCR, a tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion channel, a G-protein, a phospholipase and calcium binding protein. More preferably, the energy transfer partners are a modified GFP FRET partner pair.
  • FIG. 1 A shows an exemplary transducisome in the uncomplexed and complexed state.
  • a transducisome protein "T” has three different PDZ domains that bind different signal transduction proteins such as a phospholipase ("PL”), a phosphokinase ("PK”) and a cell surface receptor (“R”).
  • the interlocking regions on the transducisome protein illustrate PDZ domains that bind signal transduction proteins.
  • the interlocking region on each signal transduction protein illustrates an amino acid region that binds a PDZ domain on the transducisome protein.
  • FIG. IB shows an exemplary transducisome in the uncomplexed and complexed state.
  • a transducisome protein "T” has three different PDZ domains that bind signal transduction proteins such as a phospholipase ("PL"), a phosphokinase ("PK”) and two ion channels of the same type (“IC").
  • the interlocking regions on the transducisome protein illustrate PDZ domains that bind signal transduction proteins.
  • the interlocking region on each signal transduction protein illustrates an amino acid region that binds a PDZ domain on the transducisome protein.
  • FIG. IC shows one embodiment of the invention related monitoring protein-protein interactions in transducisomes in the uncomplexed and complexed state.
  • the hash- marked regions represent a first energy transfer moiety for FRET.
  • the dotted regions represent a second energy transfer moiety for FRET.
  • FRET is substantially occurs in the complexed state between the first and second energy transfer moieties.
  • FIG. 2 A shows a diagram of INAD protein with the locations of and size of each PDZ domain highlighted. Also shown are the relative locations of the three inaD mutations related to photoreceptor activation.
  • FIG. 2B shows an amino acid alignment of PDZ domains from mammalian PSD-95 , nNOS , Drosophila dig and inaD .
  • Black boxes indicate amino acid identities and gray boxes show conservative substitutions.
  • Stars above the sequence indicate residues 27 implicated in substrate binding .
  • the circled residues refer to the site of point mutation in the three Drosophila inaD alleles (see text for details).
  • FIG. 2C shows an immunoblot demonstrating the absence of INAD protein in inaD .
  • FIG. 3A shows that INAD antibodies co-immunoprecipitate TRP, eye-PKC and PLC ⁇ from retinal extracts in an immunoblot.
  • FIG. 3B shows the results obtained in vitro using a full length GST-INAD fusion protein.
  • Full length GST-INAD fusions associate with TRP, PLC ⁇ and PKC in cell
  • FIG. 3B also shows that the third PDZ domain of INAD is specific for TRP , while the fourth domain specifically interacts with eye-PKC and the fifth domain specifically interacts with PLC ⁇ .
  • Overexpression of each of the individual PDZ domains from INAD as GST-PDZ fusions produce highly preferred interactions in biochemical assays.
  • FIG. 4 shows electrophysiology recordings with altered kinetics from wildtype and mutant INAD protein in cells.
  • FIG. 5 A-C show altered kinetics and quantal bumps in photoreceptor cells.
  • Fluorescent donor moiety refers to the radical of a fluorogenic compound, which can absorb energy and is capable of transferring the energy to another fluorogenic molecule or part of a compound.
  • Suitable donor fluorogenic molecules include, but are not limited to, coumarins and related dyes xanthene dyes such as fluoresceins, rhodols, and rhodamines, resorufins, cyanine dyes, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such as luminol and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquinones, and europium and terbium complexes and related compounds.
  • Quencher refers to a chromophoric molecule or part of a compound, which is capable of reducing the emission from a fluorescent donor when attached to the donor. Quenching may occur by any of several mechanisms including fluorescence resonance energy transfer, photoinduced electron transfer, paramagnetic enhancement of intersystem crossing, Dexter exchange coupling, and excitation coupling such as the formation of dark complexes.
  • Acceptor refers to a quencher that operates via fluorescence resonance energy transfer. Many acceptors can re-emit the transferred as energy as fluorescence. Examples include coumarins and related fluorophores, xanthenes such as fluoresceins, rhodols, and rhodamines, resorufms, cyanines, difluoroboradiazaindacenes, and phthalocyanines. Other chemical classes of acceptors generally do not re-emit the transferred energy. Examples include indigos, benzoquinones, anthraquinones, azo compounds, nitro compounds, indoanilines, di- and triphenylmethanes.
  • Binding pair refers to two moieties (e.g. chemical or biochemical) that have an affinity for one another.
  • binding pairs include antigen/antibodies, lectin/avidin, target polynucleotide/probe oligonucleotide, antibody/anti-antibody, receptor/ligand, enzyme/ligand and the like.
  • One member of a binding pair refers to one moiety of the pair, such as an antigen or ligand.
  • Dye refers to a molecule or part of a compound that absorbs specific frequencies of light, including but not limited to ultraviolet light.
  • the terms “dye” and “chromophore” are synonymous.
  • Fluorophore refers to a chromophore that fluoresces.
  • Membrane-permeant derivative refers a chemical derivative of a compound that has enhanced membrane permeability compared to an underivativized compound. Examples include ester, ether and carbamate derivatives. These derivatives are made better able to cross cell membranes, i.e. membrane permeant, because hydrophilic groups are masked to provide more hydrophobic derivatives. Also, masking groups are designed to be cleaved from a precursor (e.g., fluorogenic substrate precursor) within the cell to generate the derived substrate intracellularly. Because the substrate is more hydrophilic than the membrane permeant derivative it is now trapped within the cells.
  • a precursor e.g., fluorogenic substrate precursor
  • isolated polynucleotide refers a polynucleotide of genomic, cDNA, or synthetic origin or some combination there of, which by virtue of its origin the "isolated polynucleotide” (1) is not associated with the cell in which the "isolated polynucleotide” is found in nature, or (2) is operably linked to a polynucleotide which it is not linked to in nature.
  • isolated protein refers a protein, usually of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated protein” (1) is not associated with proteins that it is normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, e.g. free of human proteins, (4) is expressed by a cell from a different species, or (5) does not occur in nature.
  • isolated naturally occurring protein refers to a protein which by virtue of its origin the “isolated naturally occurring protein” (1) is not associated with proteins that it is normally found with in nature, or (2) is isolated from the cell in which it normally occurs or (3) is isolated free of other proteins from the same cellular source, e.g. free of human proteins.
  • Polypeptide as used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein, fragments, and analogs are species of the polypeptide genus.
  • Preferred transducisome polypeptides include those with the polypeptide sequence represented in the SEQUENCE ID LISTING (as well as human homologs hereof) and any other protein having activity similar to such transducisome proteins as measured by one or more of the assays described herein.
  • SEQ. H) NO.: 1 is a transducisome protein (fly) amino acid sequence
  • SEQ. ID NO. 2 is a transducisome protein (fly) nucleotide sequence.
  • Transducisome polypeptides or proteins can include any protein having sufficient activity for detection in the assays described herein.
  • Naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
  • operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences, such as when the appropriate molecules (e.g., inducers and polymerases) are bound to the control or regulatory sequence(s).
  • Control sequence refers to polynucleotide sequences which are necessary to effect the expression of coding and non-coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Polynucleotide refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred.
  • two protein sequences are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, 1972, Volume 5, National Biomedical Research Foundation, pp. 101-110, and Supplement 2 to this volume, pp. 1-10.
  • the two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 30% identical when optimally aligned using the ALIGN program.
  • Corresponds to refers to a sequence that is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference sequence.
  • reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length protein given in a sequence listing such as a SEQ. ID NO. 1, or may comprise a complete protein sequence. Generally, a reference sequence is at least 400 nucleotides in length, frequently at least 600 nucleotides in length, and often at least 800 nucleotides in length (or the protein equivalent).
  • two proteins may each (1) comprise a sequence (i.e., a portion of the complete protein sequence) that is similar between the two proteins, and (2) may further comprise a sequence that is divergent between the two proteins, sequence comparisons between two (or more) proteins are typically performed by comparing sequences of the two proteins over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window,” as used herein, refers to a conceptual segment of at least 20 contiguous amino acid positions wherein a protein sequence may be compared to a reference sequence of at least 20 contiguous amino acids and wherein the portion of the protein sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
  • sequence identity means that two protein sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acids occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical means that two protein sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, typically share at least 70 percent sequence identity, preferably at least 80 percent sequence identity, more preferably at least 90 percent sequence identity, and most preferably at least 95 percent sequence identity.
  • residue positions, which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.
  • Transducisome protein refers to a protein with the transducisome protein activity of one of the transducisome proteins of the SEQ. ID listing or another PDZ domain containing protein that binds signal transduction proteins.
  • the transducisome protein can functionally bind at least one signal transduction protein.
  • transducisome proteins include INAD, GRIP and other recently identified multi-PDZ domain proteins.
  • signal transduction proteins include GPCRs, tyrosine kinase receptors, tyrosine phosphatase receptors, ion channels, phospholipases, adenylate cyclases, kinases and G-proteins.
  • Transducisome protein activity can be measured with endogenously or heterologously expressed transducisome proteins and signal transduction proteins using the assays described herein.
  • a transducisome protein can functionally bind to at least two different types of signal transduction proteins, more preferably, a transducisome protein can functionally bind to at least three different types of signal transduction proteins.
  • Transducisome proteins permit enhanced signal transduction by signal transduction proteins compared to signal transducton by signal transduction proteins not functionally bound to a transducisome.
  • Transducisome protein activity includes functional binding of a signal transduction protein to a transducisome protein, alteration of signal transduction protein activity when the signal transduction protein is complexed to the transducisome protein (e.g., a increase or decrease in activity or a change in signal transduction protein specificity) and any other activity related to signal transduction induced by a transducisome protein.
  • Mammalian transducisome proteins for use with mammalian signal transduction proteins are preferred, particularly human transducisome proteins.
  • Modulation refers to the capacity to either enhance or inhibit a functional property of biological activity or process (e.g., enzyme activity or receptor binding); such enhancement or inhibition may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or may be manifest only in particular cell types.
  • modulator refers to a chemical (naturally occurring or non-naturally occurring), such as a synthetic molecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Modulators are evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (e.g., agonist, partial antagonist, partial agonist, inverse agonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation-promoting agents, and the like) by inclusion in screening assays described herein. The activity of a modulator may be known, unknown or partially known.
  • Sequence homology refers to the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence from a desired sequence (e.g., SEQ. ID NO. 1) that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
  • test chemical refers to a chemical to be tested by one or more screening method(s) of the invention as a putative modulator. A test chemical is usually not known to bind to the target of interest.
  • control test chemical refers to a chemical known to bind to the target (e.g., a known agonist, antagonist, partial agonist or inverse agonist).
  • test chemical does not include a chemical added as a control condition that alters the function of the target to determine signal specificity in an assay.
  • control chemicals or conditions include chemicals that 1) non- specifically or substantially disrupt protein structure (e.g., denaturing agents (e.g., urea or guandium), charotropic agents, sulfhydryl reagents (e.g., dithiotritol and ⁇ - mercaptoethanol), and proteases), 2) generally inhibit cell metabolism (e.g., mitochondrial uncouplers) and 3) non-specifically disrupt electrostatic or hydrophobic interactions of a protein (e.g., high salt concentrations, or detergents at concentrations sufficient to non-specifically disrupt hydrophobic interactions).
  • test chemical also does not include chemicals known to be unsuitable for a therapeutic use for a particular indication due to toxicity of the subject. Usually, various predetermined concentrations test chemicals are used for screening such as .01 ⁇ M, .1 ⁇ M, 1.0 ⁇ M, and 10.0 ⁇ M.
  • targets refers to a biochemical entity involved a biological process. Targets are typically proteins that play a useful role in the physiology or biology of an organism. A therapeutic chemical binds to target to alter or modulate its function. As used herein targets can include cell surface receptors, G-proteins, kinases, ion channels, phopholipases and other proteins mentioned herein.
  • label refers to incorporation of a detectable marker, e.g., by the incorporation of a radio labeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical, or colorimetric methods).
  • marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical, or colorimetric methods.
  • Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes (e.g., ⁇ , 14 C, 35 S, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, and lanthanide phosphors), enzymatic labels or reporter genes (e.g., horseradish peroxidase, ⁇ -galactosidase, ⁇ -latamase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • Fluorescent label refers to incorporation of a detectable fluorescent marker, e.g., by incorporation of a fluorescent moiety to a chemical entity that binds to a target or attachment to a polypeptide of secondary attachment molecules, such as biotinyl moieties that can be detected by avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity that can be detected by fluorescence detection methods).
  • fluorescent labels for polypeptides include, but are not limited to dyes (e.g., FITC and rhodamine), intrinsically fluorescent proteins, and lanthanide phosphors.
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance and in some embodiments to facilitate energy transfer.
  • Reporter gene refers to a nucleotide sequence encoding a protein that is readily detectable either by its presence or activity, including, but not limited to, luciferase, fluorescent protein (e.g., green fluorescent protein), chloramphenicol acetyl transferase, ⁇ -galactosidase, secreted placental alkaline phosphatase, ⁇ -lactamase, human growth hormone, and other secreted enzyme reporters.
  • fluorescent protein e.g., green fluorescent protein
  • chloramphenicol acetyl transferase e.g., chloramphenicol acetyl transferase
  • ⁇ -galactosidase ⁇ -galactosidase
  • secreted placental alkaline phosphatase ⁇ -lactamase
  • human growth hormone and other secreted enzyme reporters.
  • reporter genes encode a polypeptide not otherwise produced by the host cell, which is detectable by analysis of the cell(s), e.g., by the direct fluorometric, radioisotopic or spectrophotometric analysis of the cell(s) and preferably without the need to kill the cells for signal analysis.
  • the gene encodes an enzyme, which produces a change in fluorometric properties of the host cell, which is detectable by qualitative, quantitative or semi- quantitative function of transcriptional activation.
  • Exemplary enzymes include esterases, ⁇ -lactamase, phosphatases, proteases (tissue plasminogen activator or urokinase) and other enzymes whose function can be detected by appropriate chromogenic or fluorogenic substrates known to those skilled in the art or developed in the future.
  • Signal transduction refers to the coupling of an extracellular signal to an intracellular response.
  • Signal transduction detection system refers to a system for detecting signal transduction across a cell membrane, typically a cell plasma membrane. Such systems typically detect at least one activity or physical property directly or indirectly associated with signal transduction.
  • an activity or physical property directly associated with signal transduction is the activity or physical property of either the receptor (e.g., GPCR), or a coupling protein (e.g., a G ⁇ protein).
  • Signal transduction detection systems for monitoring an activity or physical property directly associated with signal transduction include GTPase activity, ion channel activity and conformational changes.
  • An activity or physical property indirectly associated with signal transduction is the activity or physical property produced by a molecule other than by either the receptor (e.g., GPCRs, tyrosine kinase receptors, or tyrosine phoshphatase receptors), ion channel or a coupling protein (e.g., a G ⁇ protein) associated with a receptor (e.g., GPCRs, tyrosine kinase receptors, or tyrosine phoshphatase receptors), or a coupling protein (e.g., a G ⁇ protein).
  • the receptor e.g., GPCRs, tyrosine kinase receptors, or tyrosine phoshphatase receptors
  • a coupling protein e.g., a G ⁇ protein
  • Such indirect activities and properties include changes in intracellular levels of molecules (e.g., ions (e.g., Ca, Na or K), second messenger levels (e.g., cAMP, cGMP and inositol phosphate)), kinase activities, transcriptional activity, enzymatic activity, phospholipase activities, ion channel activities and phosphatase activities.
  • ions e.g., Ca, Na or K
  • second messenger levels e.g., cAMP, cGMP and inositol phosphate
  • kinase activities e.g., transcriptional activity, enzymatic activity, phospholipase activities, ion channel activities and phosphatase activities.
  • Signal transduction assays are further described in commonly owned U.S. applications by Negulescu et al, 60/020,234, filed June 21, 1996 and serial number not yet available June 19, 1997.
  • Transducisome refers to a PDZ domain containing protein that binds at least one signal transduction protein or a PDZ domain containing protein with at least one signal transduction protein bound to it.
  • a transducisome may be an "uncomplexed transducisome” comprising only the PDZ domain containing protein.
  • a transducisome may be a "complexed transducisome” comprising comprising the PDZ domain containing protein and a signal transduction protein.
  • a complexed transducisome may contain at least two different PDZ domains bound to at least two different signal transduction protein partners that separately recognize their respective PDZ domains.
  • a complexed transducisome can enhance signal transduction activity compared 1) signal transduction proteins that are not functionally bound to a transducisome or 2) an uncomplexed transducisome.
  • Transducisome protein only refers to the uncomplexed transducisome.
  • Cells respond to a wide variety of external signals mediated by cell surface receptors that transduce extracellular stimuli into an intracellular response.
  • Receptors vary in type based on their properties, including structural similarities, signaling properties and intracellular pathways (e.g., G protein coupled receptors, tyrosine kinase receptors, and tyrosine phophatase receptors).
  • G protein coupled receptors e.g., G protein coupled receptors, tyrosine kinase receptors, and tyrosine phophatase receptors.
  • receptors that recognize different ligands are known to interact with the same intracellular signaling molecules, the specificity of signaling, often essential to a cell's physiological role is maintained. How is this specificity maintained, or rather, how is signal cross talk avoided?
  • One solution involves organizing different signaling cascades into physically and functionally distinct signaling units. Such assemblies can permit or enhance signal response time, specificity and selectivity while minimizing cross talk.
  • Transducisome refers to a PDZ domain containing protein that binds at least one signal transduction protein or a PDZ domain containing protein with at least one signal transducton protein bound to it.
  • the invention includes methods for identifying modulators of signal transduction comprising contacting a transducisome in a biochemical assay, cell assay or animal assay, with a test chemical and detecting a change in signal transduction.
  • the invention includes animals (e.g., mice and flies), cells (e.g., mammalian and insect), with transducisomes, modified transducisomes and defective transducisomes to use in assays of signal transduction.
  • the invention also includes proteins (as well as polynucleotides encoding the same) corresponding to transducisomes, modified transducisomes or defective transducisomes to use in assays of signal transduction.
  • the Drosophila phototransduction is described herein as an example of signal transduction, as it was used by the inventors to demonstrate for the first time the function and structure of a transducisome.
  • the phototransduction cascade is a G protein-coupled
  • Drosophila photoreceptor neurons also show a high degree of architectural organization, with most of the molecules involved in phototransduction localized to the rhabdomeres, a specialized subcellular compartment consisting of approximately 60,000 tightly packed
  • PLC ⁇ catalyzes the breakdown of phosphatidylinositol bisphosphate (PIP2) into the two intracellular messengers inositol triphosphate (IP3) and diacylglycerol (DAG), which leads to the
  • InaD is one of the many loci that have been identified in genetic screens designed to dissect this
  • a single mutant allele, InaD was isolated and shown to have a dominant negative phenotype on photoreceptor deactivation ' .
  • the INAD protein was shown to contain at least two PDZ domain , and to interact with the TRP ion
  • the present invention shows that the INAD protein is composed of five distinct PDZ domains, and functions as the organizing scaffold for photoreceptor signaling complexes in vivo.
  • the genetic, physiological and cell biological studies of the inventors demonstrate the presence of a multivalent adapter protein that links multiple signaling components within the same cascade, and provides a fundamental role for PDZ domains in the assembly of transduction complexes in vivo.
  • a cell can optimize, and tune, its responses to different pathways by controlling the recruitment of different signaling molecules into the different transduction complexes, thus enhancing specificity and speed, while minimizing cross talk.
  • An example is the yeast mating response, in which the product of the sterile 5 gene functions as a scaffold protein, coordinating the recruitment of several kinases within the same signaling pathway.
  • the inventors used phototransduction in Drosophila as a model to study the organization of G protein-coupled transduction complexes in vivo and in vitro in the Examples. In this signaling pathway, photoreceptor neurons report activity with extraordinar sensitivity and specificity.
  • Photoreceptors achieve superb temporal resolution by ensuring that the transduction machinery is reset quickly after generating a response.
  • Phototransduction in Drosophila is the fastest known G protein-coupled cascade, taking just a few tens of milliseconds to go from light activation of rhodopsin to the generation of a receptor potential, and less than 100 ms to shut-off following termination of the
  • the transducisome protein, INAD coordinates the recruitment of components involved both in activation (PLC ⁇ and TRP), as well as in deactivation (eye-
  • a photoreceptor neuron attains high sensitivity by having an exceptionally large number
  • each G protein would need only to sample a small number of receptors in the membrane (i.e. an ultra-microdomain of signaling) and report their activity to the downstream transduction complexes.
  • transducisomes while PKC, TRP ion channel and PLC ⁇ are part of the transducisome.
  • Transducisomes also contribute to the nature of unitary (i.e. single photon) responses in the eye.
  • a quantum bump represents the coordinated activation of a few hundred light-
  • the organization of INAD complexes into a supramolecular complex represents the structural basis of a quantum bump, insuring both reliability and coordinated signaling.
  • Transducisomes can be identified and selected for screens and assays described herein, by analyzing the structural basis of, and the interaction between, PDZ domains and their targets. For instance, the X-ray crystallographic structure of the third PDZ domain from the synaptic protein PSD-95, alone or in complex with its peptide ligand has been 27 28 determined and can be used as a model .
  • peptide libraries can be generated to display different target sites that bind PDZ domains and show preferences
  • PDZ domains has been identified in mammalian cells and can be involved in functioning as a signaling scaffold, organizing specific transduction complexes at the synapse.
  • Current models of PDZ-target interaction involve a binding site composed of
  • PDZ binding regions of targets can be located at other sites, such as located within 120 amino acid of the n-terminus of the target.
  • all three INAD targets lack such a motif (position -4 from the C-terminus in TRP, positions -122 and -507 in eye-PKC and several sites PLCs, the closest at residue -26).
  • transducisome proteins can function as modular proteins by maintaining separate binding functions in different regions.
  • INAD functions as a modular protein and is demonstrated by the fact that eliminating one target does not prevent INAD's ability to interact with the others.
  • transduction complexes By using the methods described herein, it is possible to custom design transduction complexes by manipulating the number, orientation and distribution of PDZ domains in of transducisome proteins.
  • mutant transducisomes such as null inaD mutant, makes assays based on such compositions now possible.
  • PDZ domains provide a framework for recruiting target molecules into membrane-bound macromolecular complexes.
  • PSD-95 has been shown to mediate the clustering of both NMD A receptors ' and K channels .
  • members of the PSD-95/93 family form synaptic complexes ' , and a
  • 26 PDZ domain in FAP1 binds to the FAS membrane receptor .
  • PDZ-PDZ interactions can mediate of protein-protein interactions .
  • PDZ domains have been found in more than 50 proteins, including many involved in cell
  • 27-29 signaling and can be used as a source of signal transduction proteins.
  • One aspect of the invention includes a method of identifying modulators of signal transduction.
  • the method comprises contacting a first cell with a test chemical, wherein the first cell comprises at least one signal transduction protein and a polynucleotide encoding a transducisome protein.
  • the transducisome protein functionally binds to the signal transduction protein to permit or enhance signal transduction.
  • the method can include activating signal transduction in the first cell either with the test chemical or with another chemical.
  • the method includes the step of detecting signal transduction from the first cell with a signal transduction detection system. Different types of cells are described herein and mammalian cells, particularly human cells, are preferred.
  • Signals from the first cell can be compared with signals from a second cell assayed under different conditions, for instance under control conditions.
  • the method includes contacting a second cell with the test chemical, wherein the second cell comprises the signal transduction protein and a polynucleotide encoding a defective transducisome protein.
  • the defective transducisome protein fails to functionally bind at least one signal transduction protein to permit or enhance signal transduction.
  • second cell may be used that fails to express the transducisome protein to permit the transducisome protein to functionally bind to at least one signal transduction protein to permit or enhance signal transduction.
  • the method can include the step of activating signal transduction in the second cell with a test chemical or another chemical and detecting signal transduction from the second cell with a signal transduction detection system.
  • the method includes the step of comparing signal transduction from the first cell with signal transduction from the second cell.
  • the second cell is the same type of cell as the first cell.
  • the second cell can also comprise an amino acid mutation in a PDZ domain of the defective transducisome protein that prevents functional binding of a signal transduction protein, as described herein.
  • Another aspect of the invention includes a method of identifying modulators of signal transduction by overexpressing the transducisome protein to enhance or permit signal transduction.
  • the method comprises contacting a cell with a test chemical, wherein the first cell comprises at least one signal transduction protein and a polynucleotide encoding a transducisome protein, the polynucleotide permits increased expression of the transducisome protein and the transducisome protein functionally binds to the signal transduction protein to permit or enhance signal transduction compared to the absence of increased expression of the transducisome protein.
  • the method includes optionally activating the signal transduction with a signal that increases or activates the signal transduction in the cell, and detecting signal transduction from the first cell with a signal transduction detection system.
  • the method can also include contacting a second cell with the test chemical.
  • the second cell comprises the signal transduction protein and a polynucleotide encoding a defective transducisome protein.
  • the defective transducisome protein fails to functionally bind the signal transduction protein to permit or enhance signal transduction or the second cells fails to express the transducisome protein to permit the transducisome protein to functionally bind to the signal transduction protein to permit or enhance signal transduction.
  • Signal transduction from the second cell is detected with a signal transduction detection system, and compared to signal transduction from the first cell.
  • the method can include contacting a second cell with the test chemical, wherein the second cell lacks the signal transduction protein to permit the transducisome protein to functionally bind to the signal transduction protein to permit or enhance signal transduction.
  • the signal for signal transduction can be any signal compatible with the assay being used.
  • the signal may be a test chemical itself or a known activator of signal transduction.
  • the activating step can include activating signal transduction with a signal selected from the group consisting of a chemical signal found in blood, a chemical signal found in a synaptic cleft, a chemical signal found in interstitial fluid, light or a chemical signal found in air or other signals known the art or developed in the future.
  • the signal transduction protein is heterologously expressed and is selected from the group consisting of a kinase, a phosphatase, a GPCR, a tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion channel, a G-protein, a phospholipase and a calcium binding protein.
  • Another aspect of the invention is a method of identifying modulators of a cell surface receptor using transducisome proteins.
  • the method comprises contacting a cell with a test chemical, wherein the cell comprises at least one cell surface receptor and a polynucleotide encoding a transducisome protein.
  • the polynucleotide permits increased expression of the transducisome protein and the transducisome protein functionally binds to the cell surface receptor to permit or enhance signal transduction compared to the absence of increased expression of the transducisome protein.
  • Signals can be detected associated with the cell surface receptor in the presence and absence of the test chemical or known ligand of the receptor.
  • Various cells and receptors described herein or known in the art can be used.
  • the invention also provides a method of identifying modulators of a GPCR.
  • the method includes contacting a cell with a test chemical, wherein the cell comprises a polynucleotide encoding a GPCR comprising one or more regions that bind a PDZ domain(s) and a polynucleotide encoding a heterologous protein comprising a PDZ domain that binds the GPCR.
  • the method includes detecting a signal associated with the activity the GPCR.
  • Various methods may be employed with the present invention concerning the screening of GPCRs, including the use of response elements and promiscuous G proteins, as described in commonly owned U.S. applications by Negulescu et al, 60/020,234, filed June 21, 1996 and serial number not yet available, filed June 19, 1997, herein incorporated by reference.
  • Such embodiments are particularly well suited for identifying modulators to prevent or reduce the association of specific signal transduction proteins with a transducisome.
  • modulators alter signal transduction by reducing the amount of complexed transducisomes.
  • Such modulations can often reduce signal transduction without completely eliminating signal transduction, which can enable to the cell to perform a basal level of activity. This can be an advantage compared to a antagonist that completely inactivates the receptor.
  • the invention also includes a method of identifying modulators of an ion channel using transducisome proteins.
  • the method comprises contacting a cell with a test chemical, wherein the cell comprises at least one ion channel and a polynucleotide encoding a transducisome protein, the polynucleotide permits increased expression of the transducisome protein.
  • the transducisome protein functionally binds to the ion channel to permit or enhance signal transduction compared to the absence of increased expression of the transducisome protein.
  • the method includes detecting a signal associated with the activity the ion channel.
  • the invention provides another a method of identifying modulators of an ion channel.
  • the method comprises contacting a cell with a test chemical, wherein the cell comprises a polynucleotide encoding an ion channel comprising one or more regions that bind PDZ domains and a polynucleotide encoding a heterologous protein comprising a PDZ domain that binds the ion channel.
  • the method includes detecting a signal associated with the activity the ion channel.
  • the screening assay system comprises an isolated, non- natural ly occurring cell comprising at least one signal transduction protein and a polynucleotide encoding a transducisome protein.
  • the polynucleotide permits increased expression (including inducible or constitutive expression) of the transducisome protein.
  • the transducisome protein functionally binds to the signal transduction protein to permit or enhance signal transduction compared to the absence of increased expression of the transducisome protein.
  • the screening assay system can include a signal transduction detection system for signal transduction in the isolated, non-naturally occurring cell.
  • the assay system can further include at least one test chemical. Often such chemicals will be tested in arrays enabling high throughput screening of at least 10,000 chemicals per day.
  • the invention also includes a screening assay system for detecting protein-protein interactions.
  • This screening assay system comprises a recombinant protein comprising at least one PDZ domain, a PDZ binding protein, and at least one test chemical.
  • the recombinant protein can be either in solution or membrane associated.
  • the PDZ binding protein can also be either in solution or membrane associated.
  • the test chemical is in solution.
  • the recombinant protein or the PDZ binding protein are membrane bound and the test chemical is in solution.
  • the recombinant protein or the PDZ binding protein or both have a label to facilitate detecting specific binding.
  • the recombinant protein has a donor and the PDZ binding protein has a quencher, wherein the donor and quencher are energy transfer partners, as described herein.
  • the PDZ binding protein can be selected from the group of a kinase, a phosphatase, a GPCR, a tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion channel, a G-protein, a phospholipase and calcium binding protein. More preferably the energy transfer partners are a GFP or modified GFP FRET partner pair.
  • the invention provides for isolated, non-naturally occurring cells.
  • Such cells can be used with the methods described herein and comprise 1) a heterologously expressed transducisome protein comprising one or more PDZ domains and 2) an expressed protein comprising a signal transduction protein that binds to one or more the PDZ domains.
  • the invention provides for isolated, non-naturally occurring cells, comprising: a cell capable of expressing: a) a non-naturally occurring polynucleotide comprising a coding region for a transducisome protein comprising one or more PDZ domains and b) a non-naturally occurring polynucleotide comprising a coding region for a heterologous protein comprising a signal transduction protein.
  • the signal transduction protein can be selected from the group of a kinase, a phosphatase, a GPCR, a tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion channel, a G-protein, a phospholipase and a calcium binding protein.
  • a kinase a phosphatase, a GPCR, a tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion channel, a G-protein, a phospholipase and a calcium binding protein.
  • a test chemical For cell-based assays such cells will typically be contacted with a test chemical.
  • the cell may also optionally include a signal transduction detection system.
  • the invention also provides for chimeric transducisome proteins.
  • Such chimerics typically comprise at least two PDZ binding domains not found in naturally occurring proteins.
  • Such proteins can be used to generate signal transduction systems with chimeric properties.
  • Such system can be useful for screening for modulators of signal transduction proteins. Often such systems will have enhanced signal transduction.
  • a chimeric can include a PDZ domain that binds a phospholipase and a GPCR, where neither the phospholipase nor the GPCR normally bind to a signal PDZ containing protein. By binding to the chimeric, coupling of signal transduction will be increased between the phospholipase and the GPCR.
  • Chmierics can comprise at least one first PDZ domain that binds a first signal transduction protein and at least one second PDZ domain binds a second signal transduction protein, wherein said chimeric transducisome protein is not a naturally occurring protein.
  • the chimeric transducisome protein can bind a first signal transduction protein(s) selected from the group of a kinase, a phosphatase, a GPCR, a tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion channel, a G-protein, a phospholipase and a calcium binding protein. More preferably, The chimeric transducisome protein can bind second signal transduction protein is selected from the group consisting of PKC, TRP, and PLC ⁇ .
  • transducisome proteins with altered function can be used to determine the specificity of signals.
  • a transducisome protein that normally binds four different signal transduction proteins can be mutated to only bind signal transduction proteins A, B and C but not D.
  • Such a transducisome protein can be used as a control for binding of signal transduction protein D.
  • Phenotypes related to transducisomes can also be ascertained using mutant transducisome proteins.
  • the invention provides for animals, such as mammals (particularly mice) or insects with mutant transducisome proteins.
  • the invention includes a fly comprising an amino acid mutation in a transducisome protein that prevents functional binding of a signal transduction protein, wherein the amino acid mutation is not a naturally occurring mutation of inaD.
  • the amino acid mutation results from a mutation selected from the group consisting of inaD 2 and inaD'.
  • the mutation may be in a human homolog of inaD.
  • the invention provides for an isolated cell comprising a polynucleotide encoding a transducisome protein with an amino acid mutation that prevents functional binding of a signal transduction protein, wherein the amino acid mutation is a naturally occurring mutation of inaD.
  • the cell is a insect cell or a mammalian cell, particularly a human cell.
  • the amino acid mutation results from a mutation selected from the group consisting of inaD 2 and inaD' .
  • the invention provides for an isolated polynucleotide comprising a coding region for a transducisome protein with an amino acid mutation in a PDZ domain that prevents functional binding of a signal transduction protein.
  • the amino acid mutation is not a naturally occurring mutation inaD 215 .
  • the transducisome protein is INAD or a human homolog thereof.
  • the amino acid mutation results from a mutation selected from the group consisting of inaD 2 and inaD 1 .
  • the invention also provides for an isolated protein comprising a polypeptide of SEQ ID NO.: 1 with an amino acid mutation in a PDZ domain that prevents functional binding of a signal transduction protein.
  • the invention also includes chemicals identified by the methods described herein.
  • the invention includes chemicals identified by preventing the binding of a transducisome protein with a signal transduction protein.
  • the invention includes chemicals identified by the modulation of signal transduction in a either: 1) a cell comprising: a) a heterologously expressed transducisome protein comprising one or more PDZ domains and b) an expressed protein comprising a signal transduction protein that binds to one or more the PDZ domains, or 2) a cell capable of expressing: a) a non-naturally occurring polynucleotide comprising a coding region for a transducisome protein comprising one or more PDZ domains and b) a non-naturally occurring polynucleotide comprising a coding region for a heterologous protein comprising a signal transduction protein.
  • the invention also includes a method of treating a transducisome related disease.
  • the method comprises administering a therapeutically effective amount of a chemical to modulate the association of a transducisome and at least one PDZ binding protein.
  • the invention also includes a method of modulating a signal transduction in a cell, comprising contacting a cell with a chemical to modulate the association of a transducisome and at least one PDZ binding protein.
  • the signal transduction is typically selected from the group consisting of G-protein coupled, ion channels, kinases and phospholipases.
  • Such methods can use a therapeutic compound for treating a transducisome related disease, comprising a chemical to modulate the association of a transducisome and at least one PDZ binding protein.
  • transducisome protein interactions with signal transduction proteins can be monitored and used for identifying modulators of such interactions.
  • the transducisome protein, a signal transduction protein, or both include a label that permits an assay for specific binding of the transducisome protein to the signal transduction protein.
  • a PDZ domain containing fragment of the transducisome protein, a fragment of a signal transduction protein that specifically binds a PDZ domain of the transducisome protein or both include a label that permits an assay for specific binding of the transducisome protein fragment to the signal transduction protein fragment.
  • Specific binding is determined by established methods for binding assays and can include comparison to binding in the presence of control chemicals, amino acid mutations that prevent binding and other conditions as known in the art or developed in the future or described herein.
  • transducisome protein interactions with signal transduction proteins are monitored using energy transfer moieties and methods.
  • a transducisome protein can include a first energy transfer moiety (e.g., donor) and a signal transduction protein can include a second energy transfer moiety (e.g., quencher).
  • the transducisome protein has at least one, preferably two, specific PDZ domains that separately and functionally bind at least one, preferably two, signal transduction proteins.
  • the first and second energy transfer moieties may be on signal transduction proteins to permit energy transfer between signal transduction proteins.
  • the first energy transfer moiety and the second energy transfer moiety are suitably matched to permit to energy transfer when one is a donor and the other is an acceptor or quencher.
  • the energy transfer moieties are FRET (fluorescence resonance energy transfer) pairs and more preferably fluorescent proteins that are FRET pairs or partners.
  • FRET fluorescence resonance energy transfer
  • other types of donor or acceptors can be used, , as described herein and in PCT Application WO 96/30540(Tsien et al) and PCT Application WO 96/41166 (Tsien et al), particularly with labeling techniques.
  • Fluorescent protein FRET pairs are chosen such that the excitation spectrum of one of the moieties (the acceptor fluorescent protein moiety) overlaps with the emission spectrum of the excited protein moiety (the donor fluorescent protein moiety).
  • the donor and acceptor fluorescent protein moieties are attached to either a transducisome protein or signal transduction protein (e.g, fusion proteins). Binding of the signal transduction protein to the transducisome protein creates a change in relative position (e.g., distance) and orientation of the donor and acceptor fluorescent protein moieties compared to the respective proteins in the unbound state (e.g., free in solution).
  • Binding of the signal transduction protein to the transducisome protein alters the relative amounts of fluorescence, due to energy transfer from the two fluorescent protein moieties when the donor is excited by irradiation.
  • binding of the signal transduction protein to the transducisome protein changes the ratio of the amount of light emitted by the donor and acceptor fluorescent protein moieties at a particular excitation wavelength. The ratio between the two emission wavelengths provides a measure of the binding of the signal transduction protein to the transducisome protein in the sample. Affinities related to the binding of the signal transduction protein to the transducisome protein can be readily measured, as well as reduced or increased by binding in the presence of test chemicals.
  • FIG. IC shows, the donor fluorescent protein moiety is covalently linked to a first region (e.g., the amino terminus) of the transducisome protein, and the acceptor fluorescent protein moiety is covalently linked a first region of to the signal transduction protein (e.g., the carboxy terminus).
  • Functional binding of the signal transduction protein to the transducisome protein decreases the distance between the donor and acceptor moieties.
  • Both the signal transduction protein and the transducisome protein can be free in solution, membrane bound or both depending on the signal transduction system. Alternatively, the donor and acceptor moieties can move farther apart upon disassociation.
  • a linker moiety can be added between the fluorescent protein and the transducisome protein or the signal transduction protein to enhance energy transfer.
  • the linker moiety is flexible enough to permit binding of the signal transduction protein to the transducisome protein and allow closer association of the fluorescent moieties to facilitate a form of pseudodimerization.
  • the fluorescent moieties themselves can be selected from dimerizing proteins to enhance energy transfer. Linking moieties are described, for example, in Huston, J.S., et al, PNAS 85:5879-5883 (1988), Whitlow, M., et al, Protein Engineering 6:989-995 (1993), and Newton, D.L., et al, Biochemistry 35:545-553 (1996).
  • the donor moiety is excited by light of appropriate intensity within the excitation spectrum of the donor moiety ( ⁇ exc ⁇ tatl0n ).
  • the donor moiety emits the absorbed energy as fluorescent light ( ⁇ em ⁇ sslon ,).
  • the acceptor fluorescent protein moiety is positioned to quench the donor moiety in the excited state, the fluorescence energy is transferred to the acceptor moiety that can emit fluorescent light ( ⁇ em ⁇ ssIon 2 ).
  • FRET can be manifested as a reduction in the intensity of the fluorescent signal from the donor moiety ( ⁇ em ⁇ sslon ), reduction in the lifetime of the excited state of the donor moiety, or emission of fluorescent light at the longer wavelengths (lower energies) characteristic of the acceptor moiety ( ⁇ e ⁇ mss ⁇ on 2 ).
  • the fluorescent protein moieties come closer, and FRET is increased.
  • the efficiency of FRET depends on the separation distance and the orientation of the donor and acceptor fluorescent protein moieties.
  • the Forster equation describes the efficiency of excited state energy transfer, based in part on the fluorescence quantum yield of the donor moiety and the energetic overlap with the acceptor moiety.
  • the Forster equation is:
  • E is the efficiency of FRET
  • F and F 0 are the fluorescence intensities of the donor moiety in the presence and absence of the acceptor, respectively
  • R is the distance between the donor moiety and the acceptor moiety.
  • the characteristic distance RQ at which FRET is 50% efficient depends on the quantum yield of the donor moiety (i.e., the shorter-wavelength fluorophore), the extinction coefficient of the acceptor moiety (i.e., the longer- wavelength fluorophore), and the overlap between the emission spectrum of the donor moiety and the excitation spectrum of the acceptor moiety.
  • Ro is given (in A) by: where K 2 is an orientation factor having an average value close to 0.67 for freely mobile donors and acceptors, Q is the quantum yield of the unquenched donor moiety, n is the refractive index of the medium separating the donor moiety and the acceptor moiety, and J is the overlap integral. J can be quantitatively expressed as the degree of spectral overlap between the donor moiety and the acceptor moiety according to the equation:
  • FRET is a nondestructive spectroscopic method that can monitor proximity and relative angular orientation of fluorophores in living cells.
  • the emission spectrum of the donor fluorescent protein moiety should overlap as much as possible with the excitation spectrum of the acceptor fluorescent protein moiety to maximize the overlap integral J. Also, the quantum yield of the donor fluorescent protein moiety and the extinction coefficient of the acceptor fluorescent protein moiety should be as large as possible to maximize R Q .
  • the excitation spectra of the donor and acceptor moieties should overlap as little as possible so that a wavelength region can be found at which the donor moiety can be excited selectively and efficiently without directly exciting the acceptor moiety.
  • Direct excitation of the acceptor moiety should be avoided since it can be difficult to distinguish direct emission from fluorescence arising from FRET.
  • the emission spectra of the donor and acceptor moieties should have minimal overlap so that the two emissions can be distinguished.
  • High fluorescence quantum yield of the acceptor moiety is desirable if the emission from the acceptor moiety is to be monitored to determine the amount of its labeled protein in a sample.
  • the donor fluorescent protein moiety is excited by ultraviolet ( ⁇ 400 nm) and emits blue light ( ⁇ 500 nm), and the acceptor fluorescent protein moiety is efficiently excited by blue but not by ultraviolet light and emits green light (>500 nm), for example, P4-3 and S65T, respectively.
  • the donor fluorescent moiety is excited by violet (400- 430 nm) and emits blue-green (450-500 nm) and the acceptor fluorescent moiety is efficiently excited by blue-green (450-500 nm) and emits yellow-green light (?520 nm), for example WIB and 10C respectively.
  • the amount of a labeled protein in a sample can be determined by determining the degree of FRET in the sample.
  • Labeled protein concentration can be determined by monitoring FRET at different concentrations to establish a calibration curve.
  • the degree of FRET can be determined by any spectral or fluorescence lifetime characteristic of the excited donor moiety. For example, intensity of the fluorescent signal from the donor, the intensity of fluorescent signal from the acceptor, the ratio of the fluorescence amplitudes near the acceptor's emission maxima to the fluorescence amplitudes near the donor's emission maximum, or the excited state lifetime of the donor can be monitored.
  • changes in the degree of FRET are determined as a function of the change in the ratio of the amount of fluorescence from the donor and acceptor moieties; a process referred to as "ratioing.”
  • ratioing Changes in the absolute amount of indicator, excitation intensity, and turbidity or other background absorbances in the sample at the excitation wavelength affect the intensities of fluorescence from both the donor and acceptor approximately in parallel. Therefore the ratio of the two emission intensities is a more robust and preferred measure of cleavage than either intensity alone.
  • Fluorescence in a sample is measured using a fluorometer.
  • excitation radiation from an excitation source having a first wavelength, passes through excitation optics.
  • the excitation optics cause the excitation radiation to excite the sample.
  • fluorescent proteins in the sample emit radiation which has a wavelength that is different from the excitation wavelength.
  • Collection optics then collect the emission from the sample.
  • the device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned.
  • a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed.
  • the multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer.
  • the computer also can transform the data collected during the assay into another format for presentation.
  • Methods of performing assays on fluorescent materials are well known in the art and are described in, e.g., Lakowicz, J.R., Principles of Fluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D.L. & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219- 243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.
  • the excited state lifetime of the donor moiety is, likewise, independent of the absolute amount of substrate, excitation intensity, or turbidity or other background absorbances. Its measurement requires equipment with nanosecond time resolution.
  • Quantum yields of wild-type GFP, S65T, and P4-1 mutants can be estimated by comparison with fluorescein in 0.1 N NaOH as a standard of quantum yield 0.91. J.N. Miller, ed., Standards in Fluorescence Spectrometry, New York: Chapman and Hall (1981). Mutants P4 and P4-3 were likewise compared to 9-aminoacridine in water (quantum yield 0.98).
  • any fluorescent protein can be used in the invention, including proteins that fluoresce due intramolecular rearrangements or the addition of cofactors that promote fluorescence.
  • green fluorescent proteins of cnidarians which act as their energy-transfer acceptors in bioluminescence, are suitable fluorescent proteins for use in the fluorescent indicators.
  • a green fluorescent protein (“GFP”) is a protein that emits green light
  • BFP blue fluorescent protein
  • GFPs have been isolated from the Pacific Northwest jellyfish, Aequorea victoria, the sea pansy, Renilla reniformis, and Phialidium gregarium. See, Ward, W.W., et al, Photochem.
  • Suitable pairs for example a blue-shifted GFP mutant P4-3 (Y66H/Y145F) and an improved green mutant S65T can respectively serve as a donor and an acceptor for fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • a fluorescent protein is an Aequorea- ⁇ elated fluorescent protein if any contiguous sequence of 150 amino acids of the fluorescent protein has at least 85% sequence identity with an amino acid sequence, either contiguous or non-contiguous, from the wild type Aequore ⁇ green fluorescent protein.
  • a fluorescent protein is an Aequore ⁇ -related fluorescent protein if any contiguous sequence of 200 amino acids of the fluorescent protein has at least 95% sequence identity with an amino acid sequence, either contiguous or non-contiguous, from the wild type Aequore ⁇ green fluorescent protein.
  • the fluorescent protein can be related to Renill ⁇ or Phi ⁇ lidium wild-type fluorescent proteins using the same standards.
  • T203I Other fluorescent proteins can be used in the proteins of the invention , such as, for example, yellow fluorescent protein from Vibrio fischeri strain Y-l, Peridinin- chlorophyll a binding protein from the dinoflagellate Symbiodinium sp.phycobiliproteins from marine cyanobacteria such as Synechococcus, e.g., phycoerythrin and phycocyanin, or oat phytochromes from oat reconstructed with phycoerythrobilin.
  • yellow fluorescent protein from Vibrio fischeri strain Y-l Peridinin- chlorophyll a binding protein from the dinoflagellate Symbiodinium sp.phycobiliproteins from marine cyanobacteria such as Synechococcus, e.g., phycoerythrin and phycocyanin, or oat phytochromes from oat reconstructed with phycoerythrobilin.
  • a localization sequence may be used to enhance targeting the transducisome or signal transduction protein to a predetermined cellular location.
  • Localization sequences can be targeting sequences which are described, for example, in "Protein Targeting", chapter 35 of Stryer, L., Biochemistry (4th ed.). W.H. Freeman, 1995.
  • the localization sequence can also be a localized protein.
  • Some important localization sequences include those targeting the nucleus (KKKRK), mitochondrion (amino terminal MLRTSSLFTRRVQPSLFRMLRLQST-), endoplasmic reticulum (KDEL at C-terminus, assuming a signal sequence present at N-terminus), peroxisome (SKF at C-terminus), prenylation or insertion into plasma membrane (CaaX, CC, CXC, or CCXX at C- terminus), cytoplasmic side of plasma membrane (fusion to SNAP-25), or the Golgi apparatus (fusion to furin).
  • nucleic acids encoding transducisome proteins can be obtained by methods known in the art.
  • a nucleic acid encoding other transducisome proteins can be isolated by polymerase chain reaction of cDNA from various tissues and organism using primers based on the DNA sequences of proteins containing PDZ domains or using cDNA probes from the same. PCR methods are described in, for example, U.S.
  • Mutant versions of transducisomes can be made by site-specific mutagenesis of nucleic acids encoding transducisome proteins (particularly PDZ domains), or by random mutagenesis caused by increasing the error rate of PCR of the original polynucleotide with 0.1 mM MnCl 2 and unbalanced nucleotide concentrations.
  • Other mutant screening methods as known in the art, developed in the future or described herein can be used. Such mutants can be readily tested for their ability to decrease binding to signal transduction proteins.
  • Such defective transducisome proteins can be used as controls in the assays described herein, both as controls in biochemical assays and in cell-based assays (e.g., cell lines with defective transducisomes as described herein). New fluorescent proteins can also be made by mutagensis.
  • nucleotide sequence coding for expression of a polypeptide refers to a sequence that, upon transcription and translation of mRNA, produces the polypeptide. This can include sequences containing, e.g., introns.
  • expression control sequences refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of the mRNA, and stop codons.
  • start codon i.e., ATG
  • Methods which are well known to those skilled in the art can be used to construct expression vectors comprising a transducisome protein or signal transduction protein coding sequence, or both, and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. (See, for example, the techniques described in Maniatis, et al, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989).
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method by procedures well known in the art.
  • CaCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
  • Eukaryotic cells can also be cotransfected with DNA sequences encoding the fusion polypeptide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Techniques for the isolation and purification of either microbially or eukaryotically expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies or antigen.
  • a variety of host-expression vector systems may be utilized to express transducisome protein or signal transduction protein coding sequence or both. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a protein of the invention coding sequence; yeast transformed with recombinant yeast expression vectors containing the a protein of the invention coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a protein of the invention coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing a transducisome protein or signal transduction protein coding sequence, or both; or animal cell systems infected with recombinant virus expression vectors (e.g.
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see, e.g., Bitter, etal, Methods in Enzymology 153:516-544, 1987).
  • inducible promoters such as pL of bacteriophage ?, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted transducisome protein or signal transduction protein coding sequence, or both.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the transducisome protein or signal transduction protein coding sequence expressed. For example, when large quantities of the transducisome protein or signal transduction protein are to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Those which are engineered to contain a cleavage site to aid in recovering transducisome protein or signal transduction protein coding sequence are preferred. In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel, et al, Greene Publish. Assoc. & Wiley Interscience, Ch.
  • yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol.l 1, A Practical Approach, Ed. DM Glover, IRL Press, Wash., D.C., 1986).
  • vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • transducisome protein or signal transduction protein coding sequence may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson, et al, Nature 310:511-514, 1984), or the coat protein promoter to TMV (Takamatsu, et al, EMBO J. 3:17-311, 1987) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi, et al, 1984, EMBOJ.
  • An alternative expression system which could be used to express a transducisome protein or signal transduction protein coding sequence is an insect system.
  • Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the transducisome protein or signal transduction protein coding sequences may be cloned into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • transducisome protein or signal transduction protein coding sequence coding sequence will result in inactivation of the polyhedrin gene and production of non- occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, see Smith, et al, J. Viol. 46:584, 1983; Smith, U.S. Patent No. 4,215,051.
  • Eukaryotic systems and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur.
  • Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and, advantageously secretion of the gene product should be used as host cells for the expression of proteins of the invention.
  • host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38.
  • Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression may be engineered.
  • the transducisome protein or signal transduction protein coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
  • Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing the proteins of the invention in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81 : 3655-3659, 1984).
  • the vaccinia virus 7.5K promoter may be used, (e.g., see, Mackett, et al, Proc. Natl. Acad. Sci. USA ,79: 7415-7419, 1982; Mackett, et al, J. Virol. 49: 857-864, 1984; Panicali, et al, Proc. Natl.
  • vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver, et al. , Mol. Cell. Biol. 1 : 486, 1981). Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression.
  • These vectors can be used for stable expression by including a selectable marker in the plasmid, such as the neo gene.
  • the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the fluorescent indicator gene in host cells (Co ⁇ & Mulligan, Proc. Natl. Acad. Sci. USA, 81 :6349-6353, 1984). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters.
  • transducisome protein or signal transduction protein cDNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • engineered cells may be allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al, Cell, 11 : 223, 1977), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci.
  • adenine phosphoribosyltransferase genes can be employed in tk " , hgprt or aprt cells respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al, Proc. Natl. Acad. Sci. USA, 11: 3567, 1980; O'Hare, et al, Proc. Natl. Acad. Sci. USA, 8: 1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc.
  • ODC ornithine decarboxylase
  • DNA sequences encoding the transducisome protein or signal transduction protein coding sequence polypeptide of the invention can be expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” are cells in which a vector can be propagated and its DNA expressed.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, in other words when the foreign DNA is continuously maintained in the host, are known in the art.
  • Recombinant transducisome protein or signal transduction protein coding sequence can be produced by expression of nucleic acid encoding the protein in prokaryotes, such as E. coli or in eukaryotes, such as yeast cells or mammalian cells.
  • the construct can also contain a tag to simplify isolation of the transducisome protein or signal transduction protein coding sequence.
  • a polyhistidine tag of, e.g., six histidine residues, can be incorporated at the amino terminal end of the fluorescent protein.
  • the polyhistidine tag allows convenient isolation of the protein in a single step by nickel-chelate chromatography.
  • the transducisome protein or signal transduction protein coding sequence is a fusion protein with a fluorescent protein produced by recombinant DNA technology in which a single polypeptide includes a donor moiety, a peptide linker moiety or an acceptor moiety and a peptide linker moiety.
  • the donor moiety can be positioned at the ammo-terminus relative to the acceptor moiety in the polypeptide.
  • Such a fusion protein has the generalized structure: (amino terminus) donor or acceptor fluorescent protein moiety -peptide linker moiety -transducisome protein or signal transduction protein (carboxy terminus).
  • the donor moiety can be positioned at the carboxy-terminus of the transducisome protein or signal transduction protein.
  • all combinations such as N- donor-C-N-transducisome protein-C/N-acceptor-C-N-signal transduction protein-C, N- transducisome protein-C-N-donor-C/N-acceptor-C-N-signal transduction protein-C, N- donor-C-N-transducisome protein-C/N-signal transduction protein-C-N-acceptor-C, N- transducisome protein-C-N-donor-C/N-signal transduction protein-C-N-acceptor-C, N- acceptor-C-N-transducisome-C/N-donor-C-N-signal transduction protein-C, and N- transducisome-C-N-acceptor-C/N-donor-C-C-C-
  • Tranducisome protein fragments and signal transduction fragments can be made this way as well.
  • the invention also envisions fusion proteins that contain extra amino acid sequences at the amino and/or carboxy termini, for example, polyhistidine tags.
  • fluorescent transducisome and signal transduction proteins or fragments thereof can also be used as non-FRET proteins in fluorescent binding assays for screening compounds.
  • FRET binding pair of transducisome protein and signal transduction proteins can be encoded by a recombinant nucleic acid sequences.
  • the elements are selected so that upon expression into fusion proteins, the donor and acceptor moieties exhibit FRET when the donor moiety is excited and binding occurs.
  • the recombinant nucleic acid can be incorporated into an expression vector comprising expression control sequences operatively linked to the recombinant nucleic acid.
  • the expression vector can be adapted for function in prokaryotes or eukaryotes by inclusion of appropriate promoters, replication sequences, markers, etc.
  • the expression vector can be transfected into a host cell for expression of the recombinant nucleic acid.
  • Host cells can be selected for high levels of expression in order to purify the fluorescent binding pair of transducisome protein and signal transduction proteins.
  • E. coli is useful for this purpose, especially if larger quantities of full-length proteins or functionally fragments are desired for biocehmical assays.
  • the host cell can be a prokaryotic or eukaryotic cell selected to study the activity of an enzyme produced by the cell.
  • the linker peptide is selected to include an amino acid sequence recognized by the protease.
  • the cell can be, e.g., a cultured cell or a cell in vivo.
  • proteins of the invention are prepared by normal protein biosynthesis, thus completely avoiding organic synthesis and the requirement for customized unnatural amino acid analogs.
  • the constructs can be expressed in E. coli in large scale for in vitro assays. Purification from bacteria is simplified when the sequences include polyhistidine tags for one-step purification by nickel-chelate chromatography. Alternatively, the substrates can be expressed directly in a desired host cell for assays in situ, as described herein for screening assays. Cells and Targets
  • Cells endogenously expressing proteins of the invention are suitable for many embodiments, as well as proteins expressed from heterologous nucleic acids.
  • cells may be transfected with a suitable vector encoding one or more targets that are known to those of skill in the art or may be identified by those of skill in the art.
  • a suitable vector encoding one or more targets that are known to those of skill in the art or may be identified by those of skill in the art.
  • receptors or channels it is preferred to use cells transformed or transfected with heterologous DNAs encoding such ion channels and/or receptors so as to express predominantly a single type of ion channel or receptor.
  • Many cells that may be genetically engineered to express a heterologous cell surface protein are known.
  • Exemplary membrane proteins include, but are not limited to, surface receptors and ion channels.
  • targets for identifying chemicals that are useful in modulating the activity of a target in the presence of a transducisome protein.
  • the target can be any biological entity, such as a protein, sugar, nucleic acid or lipid.
  • targets will be proteins such as cell surface proteins or enzymes.
  • Targets can be assayed in either biochemical assays (targets free of cells), or cell based assays (targets associated with a cell).
  • cells expressing transducisome proteins may be loaded with ion or voltage sensitive dyes to report receptor or ion channel activity, such as calcium channels or N- methyl-D-aspartate (NMDA) receptors, GABA receptors, kainate/AMPA receptors, nicotinic acetylcholine receptors, sodium channels, calcium channels, potassium channels excitatory amino acid (EAA) receptors, and nicotinic acetylcholine receptors.
  • Assays for determining activity of such receptors can also use agonists and antagonists to use as negative or positive controls to assess activity of tested chemicals.
  • IP3 inositol triphosphate
  • G-protein-coupled receptors are muscarinic acetylcholine receptors (mAChR), adrenergic receptors, serotonin receptors, dopamine receptors, angiotensin receptors, adenosine receptors, bradykinin receptors, metabotropic excitatory amino acid receptors and the like.
  • mAChR muscarinic acetylcholine receptors
  • adrenergic receptors serotonin receptors
  • dopamine receptors dopamine receptors
  • angiotensin receptors adenosine receptors
  • bradykinin receptors metabotropic excitatory amino acid receptors and the like.
  • Cells expressing such G-protein-coupled receptors may exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it may be desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a
  • cyclic nucleotide-gated ion channels e.g., rod photoreceptor cell channels and olfactory neuron channels (see, Altenhofen, W. et al. (1991) Proc. Natl.
  • Cells for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-gated ion channel, transducisome protein and DNA encoding a receptor (e.g., certain metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like), which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.
  • a receptor e.g., certain metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like
  • Ion channels can also be used with the invention and include, but are not limited to, calcium channels comprised of the human calcium channel ⁇ 2 ⁇ and/or ⁇ -subunits (see also, WO89/09834; human neuronal ⁇ 2 subunit); rabbit skeletal muscle al subunit (Tanabe, et al. (1987) Nature 328, pp. 313-E318); rabbit skeletal muscle ⁇ 2 subunit (Ellis, et al. (1988) Science 241, pp. 1661-1664); rabbit skeletal muscle p subunit (Ruth, et al. (1989) Science 245, pp. 1115-1118); rabbit skeletal muscle ⁇ subunit (Jay, et al. (1990) Science 248, pp.
  • potassium ion channels e.g., rat brain (BK2) (McKinnon, D. (1989) J. Biol Chem. 264, pp. 9230-8236); mouse brain (BK1) (Tempel, et al. (1988) Nature 332, pp. 837-839); and the like; sodium ion channels, e.g., rat brain I and ⁇ (Noda, et al. (1986) Nature 320, pp. 188-192); rat brain III (Kayano, et al. (1988) FEBS Lett. 228, pp. 187-1.94); human II (ATCC No.
  • GPCRs that can be used with the invention include, but are not limited to, muscarinic receptors, e.g., human M2 (GenBank accession #M 16404); rat M3 (GenBank accession #M 16407); human M4 (GenBank accession #M16405); human M5 (Bonner, et al., (1988) Neuron l, pp. 403- 410); and the like; neuronal nicotinic acetylcholine receptors, e.g., the human ⁇ 2 , ⁇ 3 , and ⁇ 2 , subtypes.
  • the human ⁇ 5 subtype Choini, et al. (1992) Proc. Natl. Acad. Sci.
  • bovine X , and X , subunits (Levitan, et al. (1988) Nature 335, pp. 76-79); the ⁇ -subunit (Pritchett, et al. (1989) Nature 338, pp. 582-585); the ⁇ 2 , and ⁇ 3 , subunits (Ymer, et al. (1989) EMBO J. 8, pp. 1665-1670); the 8 subunit (Shivers, B.D. (1989) Neuron 3, pp. 327-337); and the like; glutamate receptors, e.g., rat GluRl receptor (Hollman, et al. (1989) Nature 342, pp.
  • glutamate receptors e.g., rat GluRl receptor (Hollman, et al. (1989) Nature 342, pp.
  • rat GluR2 and GluR3 receptors (Boulter et al. (1990) Science 249:1033-1037; rat GluR4 receptor (Keinanen et al. (1990) Science 249: 556-560 ); rat GluR5 receptor (Bettler et al. (1990) Neuron 5: 583-595); rat GluR6 receptor (Egebjerg et al. (1991) Nature 351 : 745- 748); rat GluR7 receptor (Bettler et al. (1992) neuron 8:257- 265); rat NMDARl receptor (Moriyoshi et al.
  • rat metabotropic mGluR5 receptor (Abe et al. (1992) I. Biol. Chem. 267: 13361- 13368); and the like; adrenergic receptors, e.g., human ⁇ l (Frielle, et al. (1987) Proc. Natl. Acad. Sci. 84, pp. 7920-7924); human ⁇ 2 (Kobilka, et al. (1987) Science 238, pp. 650-656); hamster 2 ⁇ (Dixon, et al. (1986) Nature 321, pp.
  • dopamine receptors e.g., human D2 (Stormann, et al. (1990) Molec. Pharm. 37, pp. 1-6); mammalian dopamine D2 receptor (U.S. Patent No. 5,128,254); rat (Bunzow, et al. (1988) Nature 336, pp. 783- 787); and the like; and the like; and the like; serotonin receptors, e.g., human 5HTla (Kobilka, et al. (1987) Nature 329, pp. 75-79); serotonin 5HT1C receptor (U.S. Patent No. 4,985,352); human 5HT1D (U.S. Patent No.
  • ion channels PCT publication WO 93/13423
  • cell surface receptors U.S. Patents 5,401,629, and 5,436,128 and PCT Application WO 93/13423 (Akong et al)
  • intracellular receptors PCT publication WO 96/41013,U.S. Patent 5,548,063, U.S. Patent 5,171,671, U.S. Patent 5,274,077, U.S. Patent 4,981,784, EP 0 540 065 Al, U.S. Patent 5,071,773, and U.S. 5,298,429). All of the foregoing references are herein incorporated by reference in their entirety.
  • a cell(s) of the invention can packaged into a container that is packaged within a kit.
  • a kit may also contain any of the various isolated nucleic acids, antibodies, proteins, signal transduction detection systems, substrates, and/or drugs described herein, known in the art or developed in the future.
  • a typical kit also includes a set of instructions for any or all of the methods described herein.
  • T7-fusion protein consisting of the last 300 residues of the protein. All antibodies were checked for specificity and affinity using wild type, mutant, and transgenic controls.
  • the INAD antibody was diluted 1 :500 in phosphate buffered saline, 1% BSA, 0.1% saponin (PBS-S); the TRP antibody was first preabsorbed with a homogenate of trp mutant heads to reduce background staining and used at a final dilution of 1:100.
  • Rhodopsin (1:300), eye-PKC (1:50), PLC (1 : 1000), TRPL (1 :100), and DGq (1 :200) were detected using polyclonal antibodies as previously described ' ' .
  • Frozen heads (500-1,000) were homogenized in 2 to 3 mL of buffer A (50 mM Tris-HCl (pH 7.5), 50 mM NaCI, 1 mM EDTA and protease inhibitors) using a glass-glass homogenizer. The homogenate was centrifuged at 4,000 x g for one minute to remove chitinous material. Membranes were isolated by centrifugation at 100,000 x g for 30 minutes at 4°C, and resuspended in 0.8 to 1 ml of buffer A to determine protein concentration.
  • buffer A 50 mM Tris-HCl (pH 7.5), 50 mM NaCI, 1 mM EDTA and protease inhibitors
  • Photoreceptors were isolated from adult flies ( ⁇ 6 hr after eclosion) and whole-cell, patch-
  • Photoreceptors were stimulated by a 75 W Xenon source connected to the epifluorescence port of an inverted
  • Fluovert FS Leitz
  • light was bandpass-filtered (Jl ⁇ S ⁇ O ⁇ 10 nm) and focused onto the photoreceptor cells with a 0.5 numerical aperture, 40X objective.
  • the membrane of the photoreceptors was voltage-clamped at a holding potential of -40 mV. Traces were low-pass filtered at 2 kHz (Bessel filter) and digitized at 2 kHz, unless stated otherwise. Measured series resistance, 16 M ⁇ on average, was 80% compensated.
  • the bath solution contained (in mM): 124 NaCI, 4 KCL, 10 HEPES, 5 proline, 25 sucrose, 1.5 CaCl 2 , 1 MgCl 2 , pH 7.15.
  • Pipette solution contained 95 KGluconate, 40 KC1, 10 mM HEPES, 2 MgCl 2 , 2 EGTA, pH 7.15.
  • photoreceptors were clamped at -70 mV, and stimulated with a dim light flash to generate quantum bumps around 50% of the time. Signals were lowpass-filtered at 1 kHz and digitized at 2 kHz.
  • INAD protein structure of INAD (SEQ. ID NO.: 1).
  • INAD was selected as a candidate transducisome because mutants in the gene, InaD, produce a dominant negative phenotype for photoreceptor activation.
  • the primary structure of INAD was analyzed using BLAST..
  • INAD is a modular protein composed of five closely linked PDZ domains (see FIG. 2). Each of these domains contains the structural hallmarks of a prototypical PDZ motif, including the conserved amino acid region target binding. Each PDZ domains, however, is different and displays sufficient differences in amino acid sequence to permit binding of different signal transduction proteins and to allow distinct protein-protein interactions. This finding was quite surprising, as previous studies had shown that INAD contains two PDZ domains instead of five.
  • FIG. 2 A shows a diagram of INAD protein with the locations of and size of each PDZ domain highlighted. Also shown is the relative location of the three inaD mutations related to photoreceptor activation.
  • FIG. 2B shows an amino acid alignment of PDZ domains from mammalian PSD-95 .
  • the circled residues refer to the site of point mutation in the three Drosophila inaD alleles.
  • FIG. 2C shows an immunoblot demonstrating the absence of INAD protein in inaD .
  • Another photoreceptor protein NinaA was used as a control in the blots. Note normal levels of NinaA in both lanes.
  • the inventors measured binding of signal transduction proteins to INAD.
  • the inventors assayed for binding of different phototransduction cascade proteins to INAD.
  • Immunoprecipitations and GST-INAD protein fusions were used to identify INAD binding entities or targets.
  • FIG. 3A shows that INAD antibodies co-immunoprecipitate TRP, eye-PKC and PLCn
  • Rhodopsin and Gq a failed to immunoprecipate despite the fact that both proteins are extremely abundant in photoreceptor cells.
  • Membranes prepared from the heads of wt flies (IP) or trp, norpA and inaC mutants were immunoprecipitated (100 ug) with anti-INAD antibody as described in Example 1.
  • the immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose and separately probed with antibodies specific for TRP, PLC, eye-PKC, G alpha subunit (G ⁇ ) and rhodopsin (Rhl). Heads refers to membranes before immunoprecipitation.
  • As a negative control for antibody and immunoprecipation specificity immunoprecipitations from inaD nulls did not precipitate TRP, PKC or PLC (data not shown).
  • FIG. 3B shows similar results obtained in vitro using a full length GST-INAD fusion protein.
  • Full length GST-INAD (N-GST— INAD-C) fusions associate with TRP, PLC ⁇ and PKC in cell extracts.
  • N-GST— INAD-C N-GST— INAD-C
  • the inventors dissected INAD. The inventors produced individual PDZ domains of INAD as GST- fusion proteins and assayed each domain for interaction with each of target proteins from whole retinal extracts.
  • FIG.3B also shows that the third PDZ domain of INAD is specific for TRP , while the fourth domain specifically interacts with eye-PKC and the fifth domain specifically interacts with PLC 9 .
  • Overexpression of each of the individual PDZ domains from INAD as GST-PDZ fusions (N-GST— PDZ 1, 2, 3, 4 or 5-C) produce highly preferred interactions in biochemical assays. PDZ 1, 2, 3, 4 or 5 are shown in FIG. 2.
  • transducisomes exist as PDZ domain containing proteins to mediate protein-protein interactions and the assembly of transduction complexes with multiple types of signal transduction proteins in the bound in the same complex.
  • Example 4 Transducisomes Permit or Enhance Signal Transduction and Transducisome Assembly In Vivo
  • the inventors isolated new inaD mutant alleles responsible for the dominant negative phenotype of photoreceptor deactivation and tested their corresponding proteins for the ability to assemble transducisomes and perform signal transduction in vivo.
  • the screening strategy was based on the loss of INAD antigen on immunoblots rather than on a hypothetical physiological or behavioral phenotype.
  • Fly stocks containing individual homozygous mutagenized second chromosomes (inaD maps to the second chromosome at position 59B1-2) were generated and each stock was then
  • inaD has an amber non-sense mutation at position 811, leading to premature termination of the polypeptide chain at amino acid residue 270 (see FIG. 2 and SEQUENCE ID LISTING).
  • inaD has a A->G change at nucleotide 1814, leading to the substitution of a conserved glycine for glutamic acid in the fifth PDZ domain (see
  • FIG.2 Nucleotide positions are based on the published nucleotide sequence of INAD
  • Immunofluorescent staining was performed on one micron thick cross-sections of wild type (showing localization), and inaD mutant (no localization of signal transduction proteins as described herein) photoreceptors for different transduction proteins. No INAD-immunoreactive material was found in inaD mutants.
  • transducisomes To investigate the ability of transducisomes to permit or enhance signal transduction of photoreceptors, the inventors performed electrophysiological recordings on photoreceptor cells from wildtype, inaD , and inaD flies. Wildtype cells, with a functional transducisome, showed enhanced signal transduction in response to light compared to inaD mutants. inaD mutants, compared to wildtype, are much less sensitive to light, responding only at the highest light intensities and with profoundly
  • InaD mutants compared to wildtype, are less sensitive to light, but more sensitive to light compared to inaD mutants, and have intermediate kinetics (see FIG.4).
  • the electroretinograms (ERG) recordings are from wild type (wt),
  • the inventors examined the instability of the signal transduction proteins.
  • the inventors assayed the steady-state levels of signal transduction proteins by immunoblot analysis at different stages post-eclosion.
  • the inventors assessed the affect of mutations of the INAD protein on signal transduction and signal transducton protein stability.
  • INAD interacts with multiple components of the phototransduction cascade and is essential for the assembly of signaling complexes, i.e. transducisomes.
  • the inventors therefore postulated that mutation of the PDZ domain for a particular target should prevent the recruitment of that protein into the transduction complexes. This should generate in vivo phenotypes that resemble mutations in the target proteins.
  • the original inaD allele, inaD has a missense mutation in the third PDZ domain .
  • TRP protein levels by immunoblot analysis, TRP subcellular localization by immunofluorescent staining of tissue sections and its function by performing whole-cell voltage-clamp recordings and electroretinograms (ERG).
  • TRP 215 inaD mutants.
  • TRP is present at significantly higher
  • TRP 215 levels and exclusively in the microvillar membranes of the rhabdomeres.
  • TRP levels are significantly reduced in the rhabdomeres (newly enclosed flies) and TRP prominently distributed throughout the plasma membrane.
  • inaD mutants display an ERG phenotype that approaches that of trp mutants, and does so on a time course similar to that of the decay of TRP protein seen in immunoblots.
  • inaD mutants were originally characterized as displaying slow deactivation kinetics in response to a flash of light (see FIG. 5A).
  • ERG recordings were from wildtype, trp , and inaD mutant eyes at 10 days after eclosion.
  • Light stimulus was a 30 sec. pulse of orange light (570 nm longpass filter).
  • Quantum bumps are the result of the activation of a single rhodopsin molecule and reflect the amplification of the entire
  • the quantum bumps from inaD flies display normal termination kinetics (wild type t9o% ⁇ 13.6 ⁇ 0.58 ms, in FIG. 5B, right panel), indicating that the macroscopic defect of inaD mutants cannot be due to an underlying defect in deactivation.
  • FIG. 5B shows whole-cell recordings (left traces) and
  • the mean latency times between stimulus and quantum bump generation were significantly altered in inaD mutants (47.6 ⁇ 1.3 ms in wild type vs. 67.0 ⁇ 3.2 ms in inaD ; FIG. 5C).
  • inaD photoreceptors do not have a defect in termination , nor do they display
  • Example 5 A GPCR Based Screen for Modulators of Signal Transduction Function
  • a screen for identifying modulators of signal transduction was designed using cells that can express a GPCR, a phospholipaseC , and a G protein that assemble into a transducisome.
  • a cell line containing Gq-type GPCR receptor that expresses ⁇ - lactamase in response to the addition of the agonist was produced by FACS selection.
  • the G ⁇ q protein was endogenously expressed.
  • the activation response is inhibited by an antagonist.
  • Jurkat clones expressing NFAT- ⁇ la as described in Negulescu et al filed June 19, 1997 (herein incorporated by reference) were transfected with expression vectors containing the Gq receptor and neomycin resistance gene (double transfection).
  • the transfected population was neo-selected and sorted by FACS for clones responding to the GPCR agonist.
  • Cells were stimulated for three hours with the indicated ligands.
  • Cells were then loaded with ⁇ -lactamase substrate CCF2/ac2AM for 1 hour, washed, dispensed into wells of a microtiter plate (100,000 cells/well) and the blue/green ratio was recorded by a plate reader.
  • transducisome protein A twenty-fold change in signal upon receptor activation with an agonist (saturating dose lOO ⁇ M) was observed.
  • Such controls include mutant or truncated transducisome protein that fails to functionally bind the signal transduction protein that is normally part of a transducisome complex or cells not expressing transducisome proteins from exogenous or mutated polynucleotides (e.g., non-induced polynucleotide having expression controlled by an inducable promoter, defective transducisome proteins due to a mutation or non-transfected cells).
  • Test compounds are added in the presence or absence of agonist, antagonist, inverse agonist or other known modulators.
  • Example 6 An Ion Channel Based Screen for Modulators of Signal Transduction Function
  • a screen for identifying modulators of signal transduction was designed using cells that can express rhodospin, an ion channel that binds a transducisome protein, and a phospholipase that binds a transducisome protein, a PKC that binds a transducisome protein and a transducisome protein.
  • Cells are maintained in the dark and exposed for a predetermined time to light at a wavelength that will activate rhodospin that in turn activates Gq ⁇ .
  • the G-protein in turn activates PLC ⁇ leading to the increase of inositol triphosphate and diacylgylcerol that lead to the activation of ion channel, TRP.
  • Ion channel activity is monitored with voltage sensitive dyes using a fluorimeter. Test compounds can be added before or after light activation.
  • Example 7 A FRET Based Screen for Modulators of Transducisome Protein binding to Signal Transduction Proteins
  • a screen for identifying modulators of transducisone binding was designed using cells that can express modified GFP FRET partners fused to transducisome fragments and modified GFP FRET partners fused to signal transduction proteins that bind to the corresponding transducisome fragment.
  • the resulting fusion protein is oriented as follows: N-GFP FRET- partner-C-N- PLC ⁇ protein-C; wherein N is the n-terminus of each respective fragment and C is the c-terminus of each respective fragment.
  • Expression of each modified GFP fusion protein is accomplished with use of a constitutive promoter and a vector suitable for expression in CHO or insect cells. After expression of the GFP fusion proteins, FRET is measured between the modified GFP FRET partners in the presence and absence of test chemicals using a fluorimeter.
  • the rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron 9, 929-942 (1992).
  • Light- Activated Conductance is Composed of the Two Channels TRP and TRPL.
  • Trp transient receptor potential protein
  • Hualing, D., et al GRIP a synaptic PDZ domain-containing protein that interacts with AMPA receptors. Nature 386, 279-284 (1997).
  • a method of modulating a signal transduction in a cell comprising contacting a cell with a chemical to modulate the association of a transducisome and at least one PDZ binding protein.
  • a therapeutic compound for treating a transducisome related disease comprising a chemical to modulate the association of a transducisome and at least one PDZ binding protein.

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Abstract

L'invention concerne des cellules et des procédés servant à identifier des modulateurs de transduction de signal en se basant sur des protéines transducisomes coordonnant et assemblant de nombreux types de protéines de transduction de signal. Un transducisome est une protéine contenant un domaine PDZ et se fixant à au moins une protéine de transduction de signal ou une protéine contenant un domaine PDZ et à laquelle est fixée au moins une protéine de transduction de signal. Des exemples de protéines transducisomes comprennent INAD, GRIP et d'autres protéines identifiées récemment et contenant des domaines PDZ multiples. Des exemples de protéines de transduction de signal comprennent des GPCR, des récepteurs de tyrosine kinase, des récepteurs de tyrosine phosphatase, des canaux ioniques, des phospholipases, des adénylate cyclases, des kinases et des G-protéines. Elle concerne également des procédés servant à identifier des modulateurs de transduction de signal, des protéines (et des polynucléotides codant ces dernières) correspondant à des transducisomes, des transducisomes modifiés ou des transducisomes défectueux à mettre en application dans des méthodes de détermination de transduction de signal, ainsi qu'une méthode de détection par criblage servant à détecter des interactions entre protéines.
PCT/US1998/014667 1997-07-15 1998-07-15 Compositions et procedes servant a identifier des modulateurs de transducisomes, nouvelle categorie de cibles therapeutiques WO1999003974A1 (fr)

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US7291458B2 (en) 1998-07-28 2007-11-06 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Leptin-mediated gene-induction
US6942981B1 (en) 1999-05-14 2005-09-13 Arbor Vita Corporation Method of determining interactions with PDZ-domain polypeptides
US7514224B2 (en) 1999-05-14 2009-04-07 Arbor Vita Corporation Arrays of PDZ domain polypeptides
US7432065B2 (en) 1999-05-14 2008-10-07 Arbor Vita Corporation Methods of identifying modulators of interaction between PDZ domain proteins and PL proteins
WO2001057240A3 (fr) * 2000-02-01 2002-04-25 Thorsten Melcher Interaction du recepteur de n-methyl-d-aspartate et de la tyrosine phosphatase
WO2001057240A2 (fr) * 2000-02-01 2001-08-09 Agy Therapeutics, Inc. Interaction du recepteur de n-methyl-d-aspartate et de la tyrosine phosphatase
WO2001079498A3 (fr) * 2000-04-18 2002-05-30 Millennium Pharm Inc 16051a and 16051b codant pour des membres de la famille pdz humains et leurs utilisations
WO2001079498A2 (fr) * 2000-04-18 2001-10-25 Millennium Pharmaceuticals, Inc. 16051a and 16051b codant pour des membres de la famille pdz humains et leurs utilisations
WO2001090188A3 (fr) * 2000-05-22 2002-06-13 Vlaams Interuniv Inst Biotech Piege a interaction base sur des recepteurs
EP1612221A3 (fr) * 2000-05-22 2008-07-23 Vlaams Interuniversitair Instituut voor Biotechnologie vzw. Piège à interaction basé sur un récepteur
WO2001090188A2 (fr) * 2000-05-22 2001-11-29 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Piege a interaction base sur des recepteurs
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US8003757B2 (en) 2000-05-22 2011-08-23 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Receptor-based interaction trap
USRE46905E1 (en) 2000-06-13 2018-06-19 Shared Spectrum Company System and method for reuse of communications spectrum for fixed and mobile applications with efficient method to mitigate interference
USRE47120E1 (en) 2000-06-13 2018-11-06 Shared Spectrum Company System and method for reuse of communications spectrum for fixed and mobile applications with efficient method to mitigate interference
US7691564B2 (en) 2000-10-16 2010-04-06 The Johns Hopkins University Heterotrimeric G-protein
US7575878B2 (en) 2004-11-18 2009-08-18 Vib Vzw Methods of inhibiting leptin-induced signaling with fibronectin III domain antibodies
WO2006119736A2 (fr) * 2005-05-09 2006-11-16 Combinature Biopharm Ag Modulateurs du domaine pdz
WO2006119736A3 (fr) * 2005-05-09 2007-09-20 Combinature Biopharm Ag Modulateurs du domaine pdz

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