EP1105734A2 - Proteines a interaction de la famille homer - Google Patents

Proteines a interaction de la famille homer

Info

Publication number
EP1105734A2
EP1105734A2 EP99945113A EP99945113A EP1105734A2 EP 1105734 A2 EP1105734 A2 EP 1105734A2 EP 99945113 A EP99945113 A EP 99945113A EP 99945113 A EP99945113 A EP 99945113A EP 1105734 A2 EP1105734 A2 EP 1105734A2
Authority
EP
European Patent Office
Prior art keywords
homer
cell
protein
compound
receptor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99945113A
Other languages
German (de)
English (en)
Other versions
EP1105734A4 (fr
Inventor
Paul F. Worley
Jian Cheng Tu
Bo The Johns Hopkins University XIAO
Daniel Leahy
Jutta Beneken
Anthony A. Lanahan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Johns Hopkins University
School of Medicine of Johns Hopkins University
Original Assignee
Johns Hopkins University
School of Medicine of Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Johns Hopkins University, School of Medicine of Johns Hopkins University filed Critical Johns Hopkins University
Priority to EP06003521A priority Critical patent/EP1662255A3/fr
Publication of EP1105734A2 publication Critical patent/EP1105734A2/fr
Publication of EP1105734A4 publication Critical patent/EP1105734A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to protein-protein interactions and more specifically to molecules involved in mediating receptor-activated or ion channel- mediated intracellular calcium mobilization or concentration.
  • the mature central nervous system exhibits the capacity to alter cellular interactions as a function of the activity of specific neuronal circuits. This capacity is believed to underlie learning and memory storage, age-related memory loss, tolerance to and dependence on drugs of abuse, recovery from brain injury, epilepsy as well as aspects of postnatal development of the brain (Schatz, C, Neuron, 5:745, 1990).
  • activity-dependent synaptic plasticity is best understood in the context of learning and memory.
  • Cellular mechanisms underlying activity-dependent plasticity are known to be initiated by rapid, transmitter-induced changes in membrane conductance properties and activation of intracellular signaling pathways (Bliss and Collingridge, Nature,
  • Immediate early genes are rapidly induced in neurons by neurotransmitter stimulation and synaptic activity and are hypothesized to be part of the macromolecular response required for long-term plasticity (Goelet, et al, supra; Sheng and Greenberg, Neuron, 4:477, 1990; Silva and Giese, Neurobiology, 4:413, 1994).
  • differential cloning techniques have been used to identify genes that are rapidly induced by depolarizing stimuli (Nedivi, et al, Nature, 2(_3:713, 1993; Qian, et al, Nature, 261:453, 1993; Yamagata, et al, Neuron, 11:371, 1993; Yamagata, et al, Learning and Memory 1:140, 1994; Yamagata, et al, Journal of Biological Chemistry, 262:16333, 1994; Andreasson and Worley, Neuroscience, 69:781, 1995; Lyford, et al, Neuron, 14:433, 1995).
  • IEGs In contrast to the earlier focus on transcription factors, many of the newly characterized IEGs represent molecules that can directly modify the function of cells and include growth factors (Nedivi, et al, supra; Andreasson and Worley, supra ), secreted enzymes that can modify the extracellular matrix, such as tissue plasminogen activator (Qian, et al, supra), enzymes involved in intracellular signaling, such as prostaglandin synthase (Yamagata, et al, supra), and a novel homolog of H-Ras, termed Rheb (Yamagata, et al, supra), as well as a novel cytoskeleton-associated protein, termed Arc (Lyford, et al, supra).
  • growth factors Nedivi, et al, supra; Andreasson and Worley, supra
  • secreted enzymes that can modify the extracellular matrix such as tissue plasminogen activator (Qian, et al, supra)
  • Cellular mechanisms that modify important intracellular signals can involve changes in intracellular calcium. This type of mechanism is used in brain neurons to adapt to changes in intercellular signaling, and is demonstrated to exert powerful effects on cellular responses induced by glutamate. Similar, though distinct, cellular mechanism may be used to modulate intracellular calcium signals in other tissues including heart, lung, liver and skeletal muscle. Compounds that can modify this mechanism can modulate natural transmitter signals and may exert therapeutic effects.
  • Second messenger pathways include the phosphoinositide pathway, which regulates intracellular calcium; the adenylate cyclase pathway, which regulates levels of cyclic AMP; the guanylate cyclase pathway, which regulates levels of cGMP; and the nitric oxide pathway which regulates NO.
  • the regulated release of intracellular calcium is essential to the function of all tissues. Each tissue possesses a distinct physiology that is dependent on receptor/transmitter-regulated release of intracellular calcium. For example, synaptic function is modulated in brain neurons by glutamate receptor regulated release of intracellular calcium. Contractility of cardiac and smooth muscle is also regulated by intracellular calcium. Recent reviews of the role of calcium signaling in cellular responses include: Berridge, Nature 386:759 (1997); Berridge, J. Physiol. (London)
  • a multi-PDZ containing protein was identified in Drosophila (termed InaD) that couples the membrane-associated, light-activated ion channel with its effector enzymes (Tsunoda et al, Nature 388:243 (1997)).
  • Drosophila (termed InaD) that couples the membrane-associated, light-activated ion channel with its effector enzymes (Tsunoda et al, Nature 388:243 (1997)).
  • the biochemical consequence of this clustering is that the local concentrations of molecules that convey the signals between proteins are as high as possible. Consequently, signaling takes place efficiently.
  • the clustering activity of these proteins is essential to normal function of the signaling cascade (Lester and Scott, supra 1997; Tsunoda et al, supra 1997). Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly, Accordingly,
  • Homer proteins the products of neuronal immediate early genes, selectively bind the carboxy-termini of certain cell-surface receptors (e.g., group 1 metabotropic receptors), certain intracellular receptors and binding proteins (e.g., inositol trisphosphate receptors, ryanodine receptor, Shank proteins, 142).
  • cell-surface receptors e.g., group 1 metabotropic receptors
  • binding proteins e.g., inositol trisphosphate receptors, ryanodine receptor, Shank proteins, 142).
  • Many forms of Homer proteins contain a "coiled-coil" structure in the carboxy-terminal domain which mediates homo- and heteromultimerization between Homer proteins.
  • the present invention is based on the seminal discovery that Homer plays a significant role in mediating receptor-activated calcium mobilization from internal stores and that Homer proteins regulate aspects of receptor clustering
  • a method for identifying a compound that modulates a cellular response mediated by a cell-surface receptor.
  • the method includes incubating a test compound and a cell expressing a cell-surface receptor and a Homer protein under conditions sufficient to permit the compound to interact with the cell, and exposing the cell to a cell-surface receptor ligand.
  • a cellular response to the ligand by the cell incubated with the compound is compared with a cellular response of the cell not incubated with the compound wherein a difference in cellular response identify a compound that modulates a Homer-associated cellular response.
  • a method for identifying a compound that modulates a cellular response mediated by an intracellular receptor.
  • the method includes incubating the compound, and a cell expressing an intracellular receptor and a Homer protein under conditions sufficient to permit the compound to interact with the cell and exposing the cell to conditions that activate the intracellular receptor.
  • a cellular response by a cell incubated with the compound is compared with a cellular response of a cell not incubated with the compound wherein a difference in a cellular response identifies a compound that modulates a Homer-associated cellular response.
  • a method for identifying a compound that modulates receptor activated calcium mobilization in a cell.
  • the method includes incubating the compound and a cell expressing a Homer protein under conditions sufficient to permit the compound to interact with the cell and exposing the cell to conditions sufficient to activate calcium mobilization.
  • the receptor-activated calcium mobilization of a cell incubated with said the compound is compared with the receptor-activated calcium mobilization of a cell not incubated with the compound wherein a difference in calcium mobilization is indicative of an effect of the compound on Homer-associated calcium mobilization.
  • a method for modulating receptor- mediated calcium mobilization. The method includes exposing a cell expressing Homer protein to a compound in a sufficient amount to modulate the calcium mobilization that typically occurs when a cell is exposed to an amount of ligand sufficient to activate an intercellualr signaling pathway that includes Homer protein.
  • a method for identifying a compound that inhibits Homer protein activity.
  • the method includes identifying an inhibitor of Homer binding or crosslinking activity and identifying an inhibitor of Homer protein activity that forms covalent or non-covalent bonds with amino acids in a Homer protein binding site, based upon the crystal structure coordinates of Homer protein binding domain, and synthesizing the inhibitor.
  • a method for identifying a compound that affects the formation of cell surface receptors into clusters.
  • the method includes incubating the compound and a cell expressing a Homer protein and a Homer interacting protein, e.g., a Shank protein, under conditions sufficient to allow the compound to interact with the cell and determining the effect of the compound on the formation of cell-surface receptors into clusters.
  • the formation of cell-surface receptors into clusters of a cell contacted with the compound is compared to the formation of cell-surface receptors into clusters of a cell not contacted with the compound, wherein a difference in the formation of clusters is indicative of a compound that affects formation of cell surface receptors into clusters.
  • a method for treating a disorder associated with glutamate receptors, including metabotropic and NMDA-type glutamate receptors, in a subject.
  • the method includes administering to a subject in need, a therapeutically effective amount of a compound that modulates Homer protein activity.
  • a method for treating a disorder associated with Homer protein activity including administering to a subject in need a therapeutically effective amount of a compound that modulates Homer protein activity.
  • the compound may be identified by a method of the invention described herein.
  • an isolated nucleic acid encoding Homer protein lb having the nucleotide sequence as set forth in SEQ ID NO:3 as well as an isolated Homer protein having substantially the same amino acid sequence as set forth in SEQ ID NO:4.
  • an isolated nucleic acid encoding Homer protein lc having the nucleotide sequence as set forth in SEQ ID NO: 5 as well as an isolated Homer protein having substantially the same amino acid sequence as set forth in SEQ ID NO:6.
  • an isolated nucleic acid encoding Homer protein 2a having the nucleotide sequence as set forth in SEQ ID NO: 7 as well as an isolated Homer protein having substantially the same amino acid sequence as set forth in SEQ ID NO: 8.
  • Homer protein 2b having the nucleotide sequence as set forth in SEQ ID NO:9 as well as an isolated Homer protein having substantially the same amino acid sequence as set forth in SEQ ID NO: 10.
  • an isolated nucleic acid encoding Homer protein 3 having the nucleotide sequence as set forth in SEQ ID NO: 11 as well as an isolated Homer protein having substantially the same amino acid sequence as set forth in SEQ ID NO: 12.
  • an isolated peptide having the amino acid sequence set forth in SEQ ID NO: 13 an isolated peptide having the amino acid sequence set forth in SEQ ID NO: 14.
  • an isolated nucleic acid encoding Homer Interacting Protein having the nucleotide sequence as set forth in SEQ ID NO: 15 or 17 with a deduced amino acid sequence as set forth in SEQ ID NO: 16 or 18, respectively.
  • an isolated Homer Interacting Protein having substantially the same amino acid sequence as set forth in SEQ ID NO: 19.
  • an isolated Homer Interacting Protein having substantially the same amino acid sequence as set forth in SEQ ID NO:20.
  • a substantially purified polypeptide containing a proline rich region that is specifically capable of specifically binding to polypeptides of the Homer family.
  • transgenic non-human animal having a transgene that expresses a Homer protein, e.g., Homer la, chromosomally integrated into the germ cells of the animal.
  • a Homer protein e.g., Homer la
  • FIG. 1 Schematic Representation of EVH1 Domain-containing Proteins.
  • EVH1 domains are found at or near the N-termini of Homer, Ena, Mena, VASP, and WASP proteins.
  • Homer lb/2/3 encode a CC domain which mediates multimerization between various Homer proteins.
  • Mena, VASP, WASP, and N-WASP the EVH1 domain is followed by a central proline rich region of variable length. The proteins are drawn to the scale shown, and the respective amino acid lengths are shown at the right.
  • Mutations in the EVH1 domain of the WASP gene are indicated in lower case letters below the WASP amino acid sequence. Mutations that are associated with the severe WAS phenotype are show in bold letters (Zhu et al, 1997). Sites mutated to more than one residue are indicated by asterisks. Bold asterisks indicate residues that, when mutated, affect the interaction of WASP with WIP (Stewart et al., 1999). Residues of Homer, ⁇ -spectrin, and IRS-1 that align well following structural superposition and were used to calculate rms differences in C ⁇ positions between these domains are underlined in the IRS-1 sequence.
  • Gaps are indicated by dashes while continued sequences at amino- and carboxy-termini are indicated by periods. Residue numbering for Homer la is shown above its amino acid sequence. The number of the last included residue of each protein is shown at the end of each row.
  • FIG. 1 Ribbon Diagram of the Homer la EVH1 Domain. The amino and carboxy termini are indicated, and elements of secondary structure are labeled to correspond to homologous structures in PH and PTB domains. An additional short region of ⁇ -strand between ⁇ l and ⁇ 2 has been labeled ⁇ i.
  • FIG. 4 Structural Comparison of EVH1, PH, and PTB Domains. Ribbon diagrams (A)-(C) and surface representations (D)-(F) of the Homer 1 EVH1, ⁇ - spectrin PH, and IRS-1 PTB domains, respectively, are shown. All molecules are shown in a similar orientation, which is rotated about 45° about the vertical axis from orientations shown in Figure 3.
  • the ⁇ -spectrin PH domain is shown with bound inositol trisphosphate (Hyvonen et al, 1995).
  • the IRS-1 domain is shown complexed to a phosphotyrosine-containing peptide derived from the insulin receptor (ECk et al, 1996).
  • FIG. 5 Versatile Ligand Recognition by PH-Like Domain. Sterodiagram of a backbone trace of Homer 1 EVHl doamin showing the relative positions of IP3 as bound by the ⁇ -spectrin and PLC- ⁇ PH domains, as well as the peptide ligands for the IRS-1 and Numb PTB domains is shown. The orientations of the EVHl domain is similar to that in Figure 4. Ligand positions were determined by superimposing the backbone traces of the EVHl, PH and PTB domains in the program ) (Jones et al., 1991).
  • EVHl Surface (A) and (B) Surface representations of the Homer 1 EVHl doamin with sites homologous to positions of WASP mutations (in parentheses) colored according to solvent accessibility. Solvent exposed residues are shown in magenta, and buried or partially buried residues are shown in blue. Residue assignments are based on the sequence shown in Figure 2. WASP EVHl mutations are listed in Table 2. Surface representations of Homer 1 EVHl domain showing the location of residues targeted by site-directed mutagenesis. Mutations that disrupt binding of Homer EVHl to ligands in an in vitro binding assay are shown in red, while those that have no effect on binding are shown in light blue (see Table 3). The orientation of the EVHl domain in panels A and C is identical to that in Figure 4A and D. IN panesl B and D,. the moleucle is rotated about 180 degrees about the vertical axis.
  • Homer represents a family of proteins that selectively binds the carboxy- terminus of group 1 metabotropic receptors and is enriched at excitatory synapses (Brakeman et al, 1977). In the adult brain, Homer is rapidly and transiently induced by physiological synaptic stimuli that evoke ion-term potentiation in the hippocampus (Brakeman et al, 1997; Kato et al, 1997), and is also induced in the striatum by dopaminetic drugs of addiction (Brakeman et al, 1997). The first Homer gene identified, now termed Homer la (Brakeman et al, Nature 38(5:2284-288 (1997); GenBank Accession No.
  • U92079 is a member of a family of closely related Homer proteins that are constitutively expressed in brain (Kato et al, 1998; Sun et al, 1998; Xiao et al, 1998). There are now three mammalian genes identified and at least six distinct transcripts expressed in brain (Xiao et al, 1998). All Homer family members, including Homer la, contain an amino-terminal region of about 110 amino acids that binds metabotropic glutamate receptors la and 5 (mGluRla and mGluR5) (Xiao et al, 1998).
  • EHl domain The region of Homer that interacts with mGluRla or 5 is termed "EVHl domain", based on homology to similar domains in a family of proteins that include Drosophila Enabled (Gertler et al, 1996), mammalian VASP (Haffner et al, 1995) and the Wescott-Aldrige protein (WASP) (Ponting and Phillips, 1997; Symons et al, 1996).
  • Drosophila Enabled Gertler et al, 1996)
  • mammalian VASP Haaffner et al, 1995
  • WASP Wescott-Aldrige protein
  • the EVHl domain of Homer is conserved at a level of about 80% between Drosophila, rodent and human (Xiao et al, 1998)
  • the Homer family EVHl domain also can bind to intracellular receptors such as the inositol trisphosphate receptor and dyamin III. Binding of Homer proteins in the EVHl region is mediated by an amino acid sequence motif that is rich in proline residues.
  • a consensus for binding was determined to be PPXXFR, consistent with the observation that mutation of either of the proline residues or the phenylalanine, or a change in their relative position, interrupted binding.
  • the arginine in the last position was preferred over other tested amino acids, but is not essential. Mutations were identically effective in interrupting binding to each of the Homer family members including Homer la, Vole, 2a/b, 3 and an EVHl fragment (110 amino acids) of Homer 1. Thus, it was concluded that the interaction with mGluR5 was mediated by the Homer EVHl domain.
  • 10-mer peptides with either the wild type, or F-to-R mutation were prepared.
  • the wild type peptide blocked binding of mGluRla or mGluR5 to each of the Homer family members (Tu et al, 1998). Approximately half of the binding was blocked at a peptide concentration of 3.4 micromolar. By contrast, the F-to-R mutant peptide did not alter binding at concentrations as high as 340 micromolar.
  • Homer protein encodes a carboxy-terminal domain with a "coiled-coil" structure. This coiled-coil domain mediates homo- and heteromultermization between Homer proteins (Kato et al, 1998; Xiao et al, 1998) and such multimers can be identified in normal brain tissue (Xiao et al, 1998).
  • Homer proteins are enriched in brain tissue fractions from postsynaptic densities and are localized at the ultrastructural level to postsynaptic densities.
  • Homer la differs from the other members of the Homer family in that Homer la is not constitutively expressed and it does not contain a carboxy terminal coiled-coil domain. Experimental data showing that Homer proteins interact with cell-surface receptors and with intracellular receptors, and form multimeric complexes with other Homer proteins indicates an important role for Homer proteins in intracellular signaling.
  • polynucleotide encoding a Homer protein
  • polynucleotide refers to a polymeric form of nucleotides at least 10 bases in length.
  • isolated polynucleotide is meant a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.
  • the nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
  • a polynucleotide encoding Homer includes "degenerate variants", sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 is functionally unchanged.
  • a nucleic acid molecule encoding Homer includes sequences encoding functional Homer polypeptides as well as functional fragments thereof.
  • functional polypeptide refers to a polypeptide which possesses biological function or activity which is identified through a defined functional assay (e.g., EXAMPLE 3), and which is associated with a particular biologic, morphologic, or phenotypic alteration in the cell.
  • functional fragments of Homer polypeptide refers to fragments of a Homer polypeptide that retain a Homer activity, e.g., the ability to interact with cell-surface or intracellular receptors or mediate intracellular calcium mobilization, and the like.
  • functional Homer fragments may act as competitive inhibitors of Homer binding, for example, biologically functional fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
  • a functional Homer polypeptide includes a polypeptide as set forth in SEQ ID NO:2 and conservative variations thereof.
  • the terms "conservative variation” and “substantially similar” as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like.
  • Homer nucleic acid and amino acid sequences including Homer lb (SEQ ID NOS:3 and 4); Homer lc (SEQ ID NOS:5 and 6); Homer 2a (SEQ ID NOS:7 and 8); Homer 2b (SEQ ID NOS:9 and 10); Homer 3 (SEQ ID NOS:l l and l2).
  • Cell-surface receptors are important intermediaries in intercellular signaling.
  • a "cell-surface receptor” is a protein, usually having at least one binding domain on the outer surface of a cell where specific molecules may bind to, activate, or block the cell surface receptor.
  • Cell surface receptors usually have at least one extracellular domain, a membrane spanning region ("transmembrane") and an intracellular domain. Activation of a cell-surface receptor can lead to changes in the levels of various molecules inside the cell.
  • ligand-gated receptors ligand-gated channels, voltage-activated receptors, voltage-activated channels, ion channels and the like.
  • EAA receptors excitatory amino acid receptors
  • EAA receptors are membrane spanning proteins that mediate the stimulatory actions of glutamate and possibly other endogenous acidic amino acids. EAA are crucial for fast excitatory neurotransmission and they have been implicated in a variety of diseases including Alzheimer's disease, stroke schizophrenia, head trauma and epilepsy. EAA have also been implicated in the process of aging In addition, EAA are integral to the processes of long-term potentiation, one of the synaptic mechanisms underlying learning and memory. There are three main subtypes of EAA receptors: (1) the metabotropic or trans ACPD receptors; (2) the ionotropic NMDA receptors; and (3) the non-NMDA receptors, which include the AMPA receptors and kainate receptors.
  • Ionotropic glutamate receptors are generally divided into two classes: the
  • AMPA ⁇ -amino -3-hydroxy-5-methylisoxazole-4-propionic acid
  • AMPA ⁇ -amino -3-hydroxy-5-methylisoxazole-4-propionic acid
  • GluR5-7 are termed kainate receptors as these are preferentially sensitive to kainic acid.
  • AMPA receptor is a non-NMDA receptor that can be activated by AMPA.
  • AMPA receptors include the GluRl-4 family, which form homo-oligomeric and hetero-oligomeric complexes which display different current-voltage relations and Ca 2+ permeability.
  • Polypeptides encoded by GluRl-4 nucleic acid sequences can form functional ligand-gated ion channels.
  • An AMPA receptor includes a receptor having a GluRl, GluR2, GluR3 or GluR4 subunit.
  • NMDA receptor subtypes include class NR2B and NR2D, for example.
  • Metabotropic glutamate receptors are divided into three groups based on amino acid sequence homology, transduction mechanism and binding selectivity: Group I, Group II and Group III.
  • Each Group of receptors contains one or more types of receptors.
  • Group I includes metabotropic glutamate receptors 1 and 5 (mGluRl and mGluR5)
  • Group II includes metabotropic glutamate receptors 2 and 3 (mGluR2 and mGluR3)
  • Group III includes metabotropic glutamate receptors 4, 6, 7 and 8 (mGluR4, mGluR ⁇ , mGluR7 and mGluR8).
  • Each mGluR type may be found in several subtypes.
  • subtypes of mGluRl include mGluRla, mGluRlb and mGluRlc.
  • Group I metabotropic glutamate receptors represent a family of seven membrane spanning proteins that couple to G-proteins and activate phospholipase C (Nakanishi, 1994). Members of the family include mGluRl and mGluR5. Activation of these receptors results in the hydrolysis of memberane phosphatidylinositol bisphosphate to diacylglycerol, which activates protein kinase C. and inositol trisphosphate, which in turn activates the inositol trisphosphate receptor to release intracellular calcium. (Aramori and Nakanishi, 1992; Joly et al, 1995 Kawabata et al, 1998)
  • a “cellular response” is an event or sequence of events that singly or together are a direct or indirect response by a cell to activation of a cell surface receptor.
  • a “cellular response” is also the blockade or activation of selective and non-selective cation channels and potentiation or inhibition of other cell-surface receptor responses.
  • a “cellular response” may be the activation of an intracellular signaling pathway, including the activation of all steps or any one step in an intracellular signaling pathway.
  • intracellular signaling pathway is a sequence of events that transduces information about an extracellular event into a signal to intracellular receptors or effector molecules such as enzymes.
  • One type of intracellular signaling pathway is a second messenger signaling pathway. It may begin with the activation of receptors on the cell surface, which activation evokes changes in the level of specific, diffusible molecules inside the cell. The regulated production of these molecules serves to signal events to the intracellular receptors and is therefore termed a second messenger signaling pathway.
  • Major second messenger pathways include the adenylate cyclase pathway, which regulates levels of cyclic AMP, the phosphoinositide pathway, which regulates intracellular calcium, guanylate cyclase, which regulates levels of cGMP, and the nitric oxide pathway, which regulates nitric oxide.
  • a cellular response mediated by cell surface receptors can also include calcium mobilization.
  • a compound can modulate cellular responses mediated by cell surface receptors by inhibiting or potentiating the release of calcium from intracellular stores.
  • a compound increases calcium mobilization by increasing the release of calcium from intracellular stores.
  • a compound decreases calcium mobilization by inhibiting of the release of calcium from intracellular stores.
  • Cell-surface receptors are known to mediate cellular responses. Methods for demonstrating cellular responses are well known in the art (e.g. electrophysiological and biochemical methods). (See Examples section for additional methodology).
  • a method is provided for identifying a compound that modulates a cellular response mediated by a cell-surface receptor. The method includes incubating the compound and a cell expressing a cell-surface receptor and a Homer protein under conditions sufficient to permit the compound to interact with the cell.
  • the cell may be any cell of interest, including but not limited to neuronal cells, glial cells, cardiac cells, bronchial cells, uterine cells, testicular cells, liver cells, renal cells, intestinal cells, cells from the thymus and spleen, placental cells, endothelial cells, endocrine cells including thyroid, parathyroid, pituitary and the like, smooth muscle cells and skeletal muscle cells.
  • the cell is exposed to a cell-surface receptor ligand.
  • a "cell surface receptor ligand" is a compound that binds to the binding site of the cell-surface receptor thereby initiating a sequence of events that singly or together embrace a "cellular response".
  • a suitable control includes, but is not limited to, a cellular response of a cell not contacted with the compound.
  • the term "incubating” includes conditions which allow contact between the test compound and the cell of interest. "Contacting" may include in solution or in solid phase.
  • Compounds which modulate a cellular response can include peptides, peptidomimetics, polypeptides, pharmaceuticals, chemical compounds and biological agents, for example.
  • Antibodies, neurotropic agents, anti-epileptic compounds and combinatorial compound libraries can also be tested using the method of the invention.
  • One class of organic molecules preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • the test agent may also be a combinatorial library for screening a plurality of compounds.
  • Compounds such as peptides identified in the method of the invention can be further cloned, sequenced, and the like, either in solution of after binding to a solid support, by any method usually applied to the isolation of a specific DNA sequence Molecular techniques for DNA analysis (Landegren et al, Science 242:229- 237, 1988) and cloning have been reviewed (Sambrook et al, Molecular Cloning: a Laboratory Manual. 2nd Ed.; Cold Spring Harbor Laboratory Press, Plainview, NY, 1998, herein incorporated by reference).
  • Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs.
  • Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • agents may be included in the screening assay. These include agents like salts, neutral proteins, e.g. , albumin, detergents, etc. that are used to facilitate optimal protein-protein binding and/or reduce nonspecific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents and the like may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 10 h will be sufficient.
  • a method for identifying a compound that modulates a cellular response mediated by an intracellular receptor.
  • An "intracellular receptor” is a protein that binds particular intracellular molecules. Intracelluar receptors include ryanodine receptors and inositol trisphosphate receptors, for example, an "inositol trisphosphate receptor” is a receptor that binds the compound inositol 1,4,5 trisphosphate, which is an important intracellular second messenger.
  • Inositol 1,4,5 trisphosphate is released from phosphatidyl inositol bisphosphate by the action of a specific phospholipase C enzyme (PLC) and binds to and activates a calcium channel in the endoplasmic reticulum (ER).
  • PLC phospholipase C enzyme
  • a compound can modulate a cellular response mediated by an intracellular receptor by inhibiting or potentiating the release of calcium from intracellular stores, for example, a compound increases calcium mobilization by increasing the release of calcium from intracellular stores. A compound decreases calcium mobilization by inhibiting of the release of calcium from intracellular stores.
  • the method of the invention includes incubating the compound and a cell expressing an intracellular receptor and a Homer protein under conditions sufficient to permit the compound to interact with the cell, exposing the cell to conditions that activate said intracellular receptor, and comparing a cellular response in a cell incubated with said compound with the response of a cell not incubated with said compound.
  • Methods for determining cellular responses mediated by intracellular signals are well known to one of skill in the art (e.g., biochemical assays) and provided in the Examples as well.
  • a method for identifying a compound that modulates receptor-activated calcium mobilization means a change in the amount or concentration of free calcium (Ca +2 ) sequestered in the endoplasmic reticulum, sarcoplasmic reticulum or mitochondria of a cell.
  • the method includes incubating the compound and a cell expressing a Homer protein under conditions sufficient to permit the compound to interact with the cell and exposing the cell to conditions sufficient to activate calcium mobilization. Then, the cellular response of the cell exposed to the compound is compared to the cellular response of a cell not exposed to the compound. A difference in a cellular response is indicative of a compound that modulates receptor-activated calcium mobilization in a cell.
  • a method for modulating receptor-mediated calcium mobilization in a cell including exposing a cell to a compound in a sufficient amount to modulate the calcium mobilization that normally occurs when a cell is exposed to an amount of ligand sufficient to activate an intracellular signaling pathway.
  • the calcium mobilization that normally occurs depends on the cell type and on the ligand activating the intracellular pathway (Berridge, 1997 supra; Berridge, 1998 supra; Bootman, 1997 supra).
  • Methods of measuring free calcium flux are well known in the art (e.g., imaging methodology using calcium-sensitive dyes such as fura-2 and the like).
  • a ligand which activates the intracellular signaling pathway may be an agonist or antagonist of metabotropic glutamate receptors.
  • agonist and “antagonist” are meant to include compounds that bind to the receptor and, respectively, activate or block activation of the receptor.
  • Known agonists of metabotropic glutamate receptors include glutamate, quisqualate, Ibotenate, homocysteine sulfmate and the neurotoxin ⁇ -N-methylamino-L-alanine.
  • Antagonists of metabotropic glutamate receptors include MCPG.
  • Known agonists of the NMDA type glutamate receptor include glutamate and NMDA and known antagonists include MK-801 and APV.
  • Another embodiment of the invention includes a method of identifying a compound that inhibits Homer protein activity.
  • the method relies on functional properties of the Homer EVHl and coiled-coil binding domains that can be used to establish high-throughput screens for molecules that influence these and other functional properties of Homer family members.
  • Homer protein activity may be blocked, partially or completely, by interfering with a protein or other molecule in the intracellular signaling pathway though which Homer proteins act.
  • Homer activity can be modulated, for example, by modulating Homer protein expression, by modifying the activity of the Homer EVHl domain, by modification of the activity of the Homer CC domain, by modification of Homer crosslinking activity, and the like.
  • Homer activity can also be modulated with by interfering with the expression or activity of Homer Interacting Protein 142, Homer Interacting Protein 130, NR2D, ACK-2, Shank proteins, ryanodine, inositol trisphosphate, and hlnaD, and the like
  • Homer proteins function as a regulated adapter network that cross-links interacting proteins.
  • Cross-linking is determined by the binding properties of the Homer EVHl domain, which recognize a unique pro line-rich ligand with a core sequence consensus of PXXF. This Homer ligand is present in all identified proteins that naturally associate with Homer, and the ability of Homer proteins to bind can be disrupted by single amino acid changes in this motif.
  • Cross-linking activity of Homer proteins has demonstrated effects on glutamate receptor signaling and this action is due to the formation of signaling complexes that link cell-surface receptors with intracellular receptors.
  • Cross-linking by Homer proteins may also have consequences on receptor trafficking or other cellular functions of the interacting proteins.
  • the method includes designing inhibitors of Homer protein that form non-covalent bonds with amino acids in the Homer binding sites based upon the crystal structure co-ordinates of Homer protein binding domain; synthesizing the inhibitor; and determining whether the inhibitor inhibits the activity of Homer protein.
  • the "Homer protein binding domain” is a conserved sequence of amino acids in the amino-terminal region of the that interacts with other proteins. All Homer proteins possess a conserved region of about 175 amino acids at their amino-termini.
  • the 110 terminal amino acids in this region interact with the carboxy-termini of other proteins, for example metabotropic glutamate receptors, inositol trisphosphate receptors, Shank, and the like.
  • the carboxy-termini region of the proteins to which the Homer protein binding domain may bind usually contains an amino acid sequence that contains a high number of proline residues.
  • One aspect of the invention resides in the obtaining of crystals of Homer protein of sufficient quality to determine the three dimensional (tertiary) structure of the protein by X-ray diffraction methods.
  • the knowledge obtained concerning Homer proteins may be used in the determination of the three dimensional structure of the binding domain of Homer proteins.
  • the binding domain can also be predicted by various computer models.
  • small molecules which mimic the functional binding of Homer protein to its ligands can be designed and synthesized This is the method of "rational" drug design.
  • Another approach to "rational" drug design is based on a lead compound that is discovered using high thoughput screens; the lead compound is further modified based on a crystal stucture of the binding regions of the molecule in question.
  • another aspect of the invention is to provide material which is a starting material in the rational design of drugs which mimic or prevents the action of Homer proteins.
  • crystal structure coordinates refers to mathematical coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of a Homer protein molecule in crystal form.
  • the diffraction data are used to calculate an electron density map of the repeating unit of the crystal.
  • the electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal.
  • the coordinates of the Homer protein binding domain can also be obtained by means of computational analysis.
  • the term "selenomethione substitution” refers to the method of producing a chemically modified form of the crystal of Homer.
  • the Homer protein is expressed by bacterial in meida that is depleted in methionine and supplement in selenomethionine. Selenium is thereby incorporated into the crystal in place of methionine sulfurs..
  • the location(s) of selenium are determined by X-ray diffraction analysis of the crystal. This information is used to generate the phase information used to construct three-dimensional structure of the protein.
  • the term "heavy atom derivatization” refers to the method of producing a chemically modified form of the crystal of Homer.
  • a crystal is soaked in a solution containing heavy metal atom salts or organometalhc compounds, which can diffuse through the crystal and bind to the surface of the protein.
  • the location(s) of the bound heavy metal atom(s) are determined by X-ray diffraction analysis of the soaked crystal. This information is used to generate the phase information used to construct three-dimensional structure of the protein.
  • unit cell refers to the basic parallelipiped shaped block.
  • the entire volume of a crystal may be constructed by regular assembly of such blocks.
  • space group refers to the arrangement of symmetry elements of a crystal.
  • molecular replacement refers to a method that involves generating a preliminary model of an Homer crystal whose structure coordinates are not known, by orienting and positioning a molecule whose structure coordinates are known.
  • Phases are then calculated from this model and combined with observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are known.
  • the crystal structure coordinates of Homer protein may be used to design compounds that bind to the protein and alter its physical or physiological properties in a variety of ways.
  • the structure coordinates of the protein may also be used to computationally screen small molecule data bases for compounds that bind to the protein.
  • the structure coordinates of Homer mutants e.g., missense mutations, deletion mutations, and the like, obtained by site-directed mutagenesis, by exposure to mutagenic agents, through selection of naturally occurring mutants, etc.
  • a potential inhibitor is designed to form hydrogen bonds with tryptophan 24 , phenylalanine 74 , threonine 66 , threonine 68 , glutamine 76 , alanine 78 , threonine 70 , and valine 85 of the Homer binding domain.
  • a method for identifying a compound that affects the formation of cell surface receptors into clusters includes incubating the compound and a cell expressing a Homer protein and a Homer Interacting protein, such as a Shank protein, a Homer Interacting Protein, and the like, under conditions sufficient to allow the compound to interact with the cell, determining the effect of the compound on the formation of cell-surface receptors into clusters, and comparing the formation of cell-surface receptors into clusters in cells contacted with the compound with the formation of cell surface receptors into clusters in cells not contacted with the compound.
  • a Homer protein and a Homer Interacting protein such as a Shank protein, a Homer Interacting Protein, and the like
  • Shank proteins are a novel family of proteins found at the postsynaptic density (PSD) and which are capable of binding to other proteins.
  • Shank proteins contain multiple protein interaction domains, including ankyrin repeats, SH3 domain, PDZ domain, at least one proline rich domain and at least one SAM domain.
  • the PDZ domain of Shank mediates binding to the carboxy-terminus of guanylate kinase associated protein (GKAP), and this interaction is important in neuronal cells for the synaptic localization of Shank proteins.
  • Shank proteins also interact with Homer proteins and therefore Shank and Homer may serve as a protein bridge that links specific proteins that bind to Homer and specific proteins that bind to Shank.
  • Exemplary Shank proteins include Shank la, Shank lb and Shank 3, and cortactin binding protein, and the like.
  • a compound can affect the formation of cell-surface receptors into clusters by either stimulating the formation of cell-surface receptors into clusters or by inhibiting the recruitment of cell-surface receptors into clusters.
  • the effect is “inhibition”
  • cell-surface clustering is decreased as compared with the level in the absence of the test compound.
  • the effect is “stimulation”
  • cell-surface clustering is increased as compared to a control in the absence of the test compound.
  • a method is further provided for treating a subject with a disorder associated with metabotropic receptors or ion channel receptors comprising administering to the subject a therapeutically effective amount of a compound that modulates Homer protein activity.
  • a method is provided for treating a subject with a disorder associated with Homer protein activity, comprising administering to the subject a therapeutically effective amount of a compound that modulates Homer protein activity.
  • any disorder that is etiologically linked to a glutamate receptor, an inositol trisphosphate receptor, a ryanodine receptor, a Shank protein , 142 (or other Homer interacting proteins) or to a Homer protein could be considered susceptible to treatment with an agent that modulates Homer protein activity.
  • the disorder may be a neuronal cell disorder. Examples of neuronal cell disorders include but are not limited to Alzheimer's disease, Parkinson's disease, stroke, epilepsy, neurodegenerative disease, Huntington's disease, and brain or spinal cord injury/damage, including ischemic injury.
  • the disorder may also be a disorder of a cardiac disorder, a disorder of musculature, a renal disorder, a uterine disorder or a disorder of bronchial tissue.
  • the disorder may be epilepsy, glutamate toxicity, a disorder of memory, a disorder of learning or a disorder of brain development.
  • Detection of altered (decreased or increased) levels of "Homer protein activity” can be accomplished by hybridization of nucleic acids isolated from a cell of interest with a Homer polynucleotide of the invention. Analysis, such as Northern Blot analysis, are utilized to quantitate expression of Homer, such as to measure Homer transcripts. Other standard nucleic acid detection techniques will be known to those of skill in the art. Detection of altered levels of Homer can also accomplished using assays designed to detect Homer polypeptide. For example, antibodies or petides that specifically bind a Homer polypeptide can be utilized. Analyses, such as radioimmune assay or immunohistochemistry, are then used to measure Homer, such as to measure protein concentration qualitatively or quantitatively.
  • Treatment can include modulation of Homer activity by administration of a therapeutically effective amount of a compound that modulates Homer or Homer protein activity.
  • modulate envisions the suppression of Homer activity or expression when Homer is overexpressed or has an increased activity as compared to a control.
  • modulate also includes the augmentation of the expression of Homer when it is underexpressed or has a decreased activity as compared to a control.
  • compound as used herein describes any molecule, e.g., protein, nucleic acid, or pharmaceutical, with the capability of altering the expression of Homer polynucleotide or activity of Homer polypeptide.
  • Treatment may inhibit the interaction of the EVHl domain of Homer with its target protein , may increase the avidity of this interaction by means of allosteric effects, may block the binding activity of the coiled-coil doamin of Homer or influence other functional properties of Homer proteins .
  • Candidate agents include nucleic acids encoding a Homer, or that interfere with expression of Homer, such as an antisense nucleic acid, ribozymes, and the like. Candidate agents also encompass numerous chemical classes wherein the agent modulates Homer expression or activity.
  • nucleic acid sequences that interfere with the expression of Homer can be used. In this manner, the coupling of cell-surface and intracellular receptors can be inhibited.
  • This approach also utilizes, for example, antisense nucleic acid, ribozymes, or triplex agents to block transcription or translation of Homer mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving it with a ribozyme in disorders associated with increased Homer.
  • a dominant negative form of Homer polypeptide could be administered.
  • candidate agents include antisense nucleic acid sequences.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American, 262:40). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, 1988, Anal.Biochem., 172:289).
  • triplex strategy Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al, 1991, Antisense Res. and Dev., 1(3):227; Helene, C,
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J.Amer.Med. Assn., 260:3030). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • ribozymes There are two basic types of ribozymes namely, tetrahymena-type
  • Tetrahymena-type ribozymes recognize sequences which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
  • nucleic acid sequences that encode Homer can be used.
  • An agent which modulates Homer expression includes a polynucleotide encoding a polypeptide of SEQ ID NO:2, 4, 6, 8, 10 or 12, or a conservative variant thereof.
  • an agent of use with the subject invention includes agents that increase the expression of a polynucleotide encoding Homer or an agent that increases the activity of Homer polypeptide.
  • a transgenic non- human animal having a transgene that expresses Homer la chromosomally integrated into the germ cells of the animal.
  • transgenic Animals are referred to as “transgenic” when such animal has had a heterologous DNA sequence, or one or more additional DNA sequences normally endogenous to the animal (collectively referred to herein as “transgenes”) chromosomally integrated into the germ cells of the animal.
  • transgenic animal including its progeny will also have the transgene fortuitously integrated into the chromosomes of somatic cells.
  • transgenic animals of the subject invention can be employed. Generally speaking, three such methods may be employed. In one such method, an embryo at the pronuclear stage (a "one cell embryo") is harvested from a female and the transgene is microinj ected into the embryo, in which case the transgene will be chromosomally integrated into both the germ cells and somatic cells of the resulting mature animal. In another such method, embryonic stem cells are isolated and the transgene incorporated therein by electroporation, plasmid transfection or microinjection, followed by reintroduction of the stem cells into the embryo where they colonize and contribute to the germ line. Methods for microinjection of mammalian species is described in United States Patent No. 4,873,191.
  • embryonic cells are infected with a retrovirus containing the transgene whereby the germ cells of the embryo have the transgene chromosomally integrated therein.
  • the animals to be made transgenic are avian, because avian fertilized ova generally go through cell division for the first twenty h in the oviduct, microinjection into the pronucleus of the fertilized egg is problematic due to the inaccessibility of the pronucleus. Therefore, of the methods to make transgenic animals described generally above, retrovirus infection is preferred for avian species, for example as described in U.S. Patent No. 5,162,215.
  • microinjection is to be used with avian species, however, a recently published procedure by Love et al, (Biotechnology, 12, Jan 1994) can be utilized whereby the embryo is obtained from a sacrificed hen approximately two and one-half h after the laying of the previous laid egg, the transgene is microinj ected into the cytoplasm of the germinal disc and the embryo is cultured in a host shell until maturity.
  • the animals to be made transgenic are bovine or porcine
  • microinjection can be hampered by the opacity of the ova thereby making the nuclei difficult to identify by traditional differential interference-contrast microscopy.
  • the ova can first be centrifuged to segregate the pronuclei for better visualization.
  • the “non-human animals” of the invention are murine typically (e.g., mouse).
  • the “transgenic non-human animals” of the invention are produced by introducing "transgenes" into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell.
  • the zygote is the best target for microinjection.
  • the use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al, Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
  • transgenic is used to describe an animal which includes exogenous genetic material within all of its cells.
  • a “transgenic” animal can be produced by cross-breeding two chimeric animals which include exogenous genetic material within cells used in reproduction. Twenty- five percent of the resulting offspring will be transgenic i.e., animals which include the exogenous genetic material within all of their cells in both alleles. 50% of the resulting animals will include the exogenous genetic material within one allele and 25% will include no exogenous genetic material.
  • the transgene is digested and purified free from any vector DNA e.g. by gel electrophoresis. It is preferred that the transgene include an operatively associated promoter which interacts with cellular proteins involved in transcription, ultimately resulting in constitutive expression. Promoters useful in this regard include those from cytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus, as well as those from the genes encoding metallothionin, skeletal actin, P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, and thymidine kinase.
  • CMV cytomegalovirus
  • MMV Moloney leukemia virus
  • PEPK P-enolpyruvate carboxylase
  • PGK phosphoglycerate
  • DHFR thymidine kinase
  • Promoters for viral long terminal repeats such as Rous Sarcoma Virus can also be employed.
  • Constructs useful in plasmid transfection of embryonic stem cells will employ additional regulatory elements well known in the art such as enhancer elements to stimulate transcription, splice acceptors, termination and polyadenylation signals, and ribosome binding sites to permit translation.
  • Retroviral infection can also be used to introduce transgene into a non-human animal, as described above.
  • the developing non-human embryo can be cultured in vitro to the blastocyst stage.
  • the blastomeres can be targets for retro viral infection (Jaenich, R., Proc. Natl. Acad. Sci USA 73:1260-1264, 1976).
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y).
  • the viral vector system used to introduce the transgene is typically a replication-defective retro virus carrying the transgene (Jahner, et al, Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der Putten, et al, Proc. Natl Acad. Sci USA £2:6148-6152, 1985).
  • Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al, EMBO J. (5:383-388, 1987).
  • infection can be performed at a later stage.
  • Virus or virus-producing cells can be injected into the blastocoele (D.
  • transgenes are mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic nonhuman animal. Further, the founder may contain various retro viral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (D. Jahner et al, supra).
  • a third type of target cell for transgene introduction is the embryonal stem cell
  • ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al. Nature 222:154-156, 1981; M.O. Bradley et al, Nature 302: 255-258, 1984; Gossler, et al, Proc. Natl Acad. Sci USA 83: 9065-9069,
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. (For review see Jaenisch, R., Science 240: 1468-1474, 1988).
  • Transformed means a cell into which (or into an ancestor of which) has been introduced, by means of recombinant nucleic acid techniques, a heterologous nucleic acid molecule.
  • Heterologous refers to a nucleic acid sequence that either originates from another species or is modified from either its original form or the form primarily expressed in the cell.
  • Transgene means any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism (i.e., either stably integrated or as a stable extrachromosomal element) which develops from that cell.
  • a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. Included within this definition is a transgene created by the providing of an RNA sequence which is transcribed into DNA and then incorporated into the genome.
  • transgenes of the invention include DNA sequences which encode Homer protein-sense and antisense polynucleotides, which may be expressed in a transgenic non-human animal.
  • transgenic as used herein additionally includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene knockout.
  • transgenic includes any transgenic technology familiar to those in the art which can produce an organism carrying an introduced transgene or one in which an endogenous gene has been rendered nonfunctional or "knocked out”.
  • Antibodies of the invention may bind to Homer proteins or Homer interacting proteins provided by the invention to prevent normal interactions of the Homer proteins and Homer Interacting proteins. Binding of antibodies to Homer proteins or Homer Interacting Proteins can interfere with cell-signaling by interfering with an intracellular signaling pathway. Binding of antibodies can interfere with Homer protein binding to extracellular receptors, e.g., to NMDA receptors, to metabotropic receptors, and the like. Binding of antibodies can interfere with Homer protein binding to intracellular receptors, e.g., inositol trisphosphate receptors, and the like. Furthermore, binding to Homer proteins or to Homer Interacting Proteins can interfere with cell-surface receptor clustering mediated by Homer family proteins.
  • the antibodies of the invention can be used in any subject in which it is desirable to administer in vitro or in vivo immunodiagnosis or immunotherapy.
  • the antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • the antibodies in these immunoassays can be detectably labeled in various ways.
  • types of immunoassays which can utilize antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay.
  • Detection of the antigens using the antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
  • antibody as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which are capable of binding to an epitopic determinant present in an invention polypeptide. Such antibody fragments retain some ability to selectively bind with its antigen or receptor.
  • Antibodies which bind to an invention polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. For example, it may be desirable to produce antibodies that specifically bind to the N- or C-terminal domains of an invention polypeptide.
  • the polypeptide or peptide used to immunize an animal is derived from translated cDNA or chemically synthesized and can be conjugated to a carrier protein, if desired.
  • Commonly used carrier proteins which may be chemically coupled to the immunizing peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), tetanus toxoid, and the like.
  • polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See, for example, Coligan, et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated by reference).
  • the antibodies of the invention can be bound to many different carriers and used to detect the presence of an antigen comprising the polypeptides of the invention.
  • carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention . Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to ascertain such, using routine experimentation.
  • labels and methods of labeling known to those of ordinary skill in the art.
  • Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and biolumi- nescent compounds.
  • Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain such, using routine experimentation.
  • Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyi, puridoxal, and fluorescein, which can react with specific antihapten antibodies.
  • the detectably labeled antibody is given a dose which is diagnostically effective.
  • diagnostically effective means that the amount of detectably labeled antibody is administered in sufficient quantity to enable detection of the site having the antigen comprising a polypeptide of the invention for which the antibodies are specific.
  • the concentration of detectably labeled antibody which is administered should be sufficient such that the binding to those cells having the polypeptide is detectable compared to the background. Further, it is desirable that the detectably labeled antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.
  • the dosage of detectably labeled antibody for in vivo treatment or diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. Such dosages may vary, for example, depending on whether multiple injections are given, antigenic burden, and other factors known to those of skill in the art.
  • Homer la is an IEG and is the original member of a family of proteins that function together as a regulated adapter system that is hypothesized to control the coupling of membrane receptors to intracellular pools of releasable calcium.
  • Homer proteins function at excitatory synapses to couple membrane group 1 metabotropic glutamate receptors (mGluR) to endoplasmic reticulum-associated inositol trisphosphate receptors (IP3R) (Brakeman et al, 1997; Tu et al, 1998; Xiao et al, 1998). Current studies suggest a broader role for Homer proteins in calcium signaling and receptor trafficking.
  • mGluR metabotropic glutamate receptors
  • IP3R endoplasmic reticulum-associated inositol trisphosphate receptors
  • Shank family of proteins was identified based on their association with Homer (Naisbitt et al, 1999; Tu et al, 1999). Shank, together with Homer, appears to be part of both the NMDA and group 1 mGluR signaling complexes. By virtue of its interaction with Shank, Homer provides a mechanism to couple NMDA Ca2+ influx to intracellular Ca2+-induced Ca2+ release pools.
  • the inventors have identified additional Homer-interacting proteins that provide insight into the role of Homer in trafficking of group 1 mGluR (e.g., SEQ ID NOS: 16, 18, 20). Because these Homer-dependent cellular processes are regulated by the IEG form of Homer (Homer la), mechanisms by which Homer proteins can modulate Ca2+ dynamics of mGluR and NMDA receptors, as well as regulate receptor trafficking are defined.
  • Homer family proteins possess an N-terminal EVHl domain that mediates interactions with mGluRs, IP3R, Shank and other novel proteins.
  • the EVHl domain has been determined to bind the proline rich motif PPXXFR (Tu et al, 1998).
  • the present invention provides the crystal structure of the Homer EVHl domain.
  • genetic approaches were used to identify critical residues in both the EVHl domain and the ligand that modulate the affinity of the Homer- mGluR (and other Homer-interacting proteins) interaction. This information is essential to an understanding of the integrative cellular actions of Homer proteins,. Together, these studies define the molecular basis of specificity of EVHl interaction with its ligands, and provide insight into how the EVHl interaction is regulated.
  • This patent application includes a description of several Homer-interacting proteins that are part of the signaling network that is controlled by Homer (e.g., SEQ ID NOS: 16, 18, 20).
  • Yeast two-hybrid screens and searches of NCBI protein data bases identified a set of known and novel candidate interacting proteins for Homer include the ryanodine receptor, NMDA receptor subunit NR2D, human InaD and novel interacting proteins termed 142 and 130.
  • current data indicate that agents can be developed that specifically modulate the crosslinking activity of Homer for these various receptors and thereby provide novel theraputics that regulate the output of these receptors on cellular function.
  • Homer acts in several ways to regulate cellular function.
  • Homer and Homer- related proteins function as an adapter system to couple membrane receptors to intracellular pools of releasable Ca. This "signaling" function of Homer is documented in Xiao et (1998), Tu et al (1998 and 1999) Naisbett et al (1999), as well as by studies of the novel Homer-interacting protein termed 142 (see below).
  • Homer proteins play a role in synaptogenesis and spatial targeting/trafficking of GluRs to other postsynaptic structural proteins. This function of Homer is supported by observations in Tu et al (1999) and Naisbett et al (1999).
  • Homer selectively binds group 1 metabotropic receptors and is enriched at synapses
  • a yeast 2-hybrid technique (Chevray and Nathans, 1992; Fields and Song, 1989) was used to screen a cDNA library prepared from rat hippocampus and cortex. The full length Homer IEG was used as bait.
  • -30 confirmed interacting cDNAs one encoded the C-terminal 250 aa of mGluR5.
  • HEK293 cells heterologous cells
  • mGluRla also bound to mGluRla, but not mGluR2, 3, 4, or 7. This was an interesting clue to the function of Homer since mGluRl and mGluR5 (termed group 1 metabotropic receptors) couple to phospholipase C and active hydrolysis of phosphoinositides to generate inositol trisphosphate and diacylglycerol (Nakanishi et al, 1994). mGluRla and 5 also share sequence similarity in their long, cytosolically disposed C-terminus. Other metabotropic glutamate receptors (termed group 2 and 3) inhibit adenylate cyclase activity, and have short C-termini that lack homology to group 1 receptors.
  • metabotropic glutamate receptors (termed group 2 and 3) inhibit adenylate cyclase activity, and have short C-termini that lack homology to group 1 receptors.
  • Homer is a member of a family of closely related proteins that are enriched at the excitatory synapse
  • Homer la and splice forms that encode CC domains termed Homer lb and lc.
  • the lb and lc splice forms differ in their inclusion of an approximately 10 amino acid sequence located between the EVHl and CC domains.
  • Homer family members that encode CC domains are also referred to as CC-Homers to distinguish them from Homer la, which lacks a CC domain.
  • Homer 2 encodes two CC-Homer splice forms termed Homer 2a and 2b, which also differ by a short internal sequence between EVHl and CC domains.
  • Homer 3 encodes a single form.
  • the CC domains are less conserved than the EVHl domain (-40% identity between rat Homer 1, 2 and 3) but they are able to specifically bind to themselves and to CC-domains of other Homer family members (Xiao et al, 1998).
  • Homer CC domains do not interact with other representative CC-domain proteins in GST pulldown assays, and a yeast 2- hybrid screen of brain cDNA with the CC-domain of Homer 1 identified multiple copies of Homer 1, Homer 2 and Homer 3, but not other CC domains (Xiao et al, 1998).
  • Homer la in the same material. mRNA and protein expression of Homer Vole, Homer 2 and Homer 3 are unchanged in hippocampus following a seizure while Homer la mRNA and protein are induced at least 10 fold.
  • Antibodies were generated that specifically recognize each of the CC-Homers. Antibodies were raised against synthetic C-terminal peptide sequences. Because Homer lb and lc possess identical C-termini, the C-terminal antibodies recognize both splice forms. Similarly, C-terminal Homer 2 antibodies recognize both Homer 2a and 2b. Accordingly, when using these antibodies to detect Homer proteins, we refer to the immunoreactivity as Homer lb/c or Homer 2a/b. We used these antibodies to determine that Homer lb/c and 3 are enriched in a detergent resistant fraction of the postsynaptic density (PSD) (Xiao et al, 1998).
  • PSD postsynaptic density
  • Homer 2a/b is also enriched in synaptic fractions, but is relatively more soluble than Homer lb/c and Homer 3.
  • each of the CC-Homers co-immunoprecipitates with group 1 mGluRs from brain (Xiao et al, 1998).
  • Immunogold electron microscopy (EM) demonstrated that Homer lb/c and Homer 3 are ultrastructurally localized at the PSD (Xiao et al, 1998).
  • Homer la functions as a natural dominant negative protein.
  • the level of transgene expression in the hippocampus was similar to natural Homer 1 a expression induced by a seizure. In contract to the natural Homer 1 a, however, the transgene was constitutively expressed in the unstimulated mouse.
  • a prediction of the dominant negative hypothesis is that the ability to co- immunoprecipitate mGluR with Homer lb/c or Homer 3 antibodies should be diminished in the transgenic mouse.
  • Homer binds a proline rich sequence that is -50 aa from the C-terminus of group 1 mGluRs.
  • IP3R co- immunoprecipitates with each of the Homer lb/c, 2a/b and 3 from detergent extracts of cerebellum [ (Tu et al, 1998) ].
  • Homer appears to be associated with a substantial portion of IP3R in the cerebellum, since a cocktail of the three Homer antibodies is able to specifically (compared to a cocktail of preimmune serums) co- immunoprecipitate -50% of the total IP3R in detergent extracts (CHAPS).
  • CC-Homers function to link mGluR5 and IP3R in a signaling complex.
  • IP3R is part of the signaling network that is activated upon glutamate stimulation of mGluRl/5.
  • Signaling complexes had previously been described including; AKAP proteins which function as scaffolds for specific kinases and their substrates [Lester, 1997 #149], and the Drosophila protein InaD which couples the membrane light activated channel with its down stream effector enzyme, phospholipase C [Tsunoda, 1997 #147].
  • Homer would need to form a bridge between receptors in two different membranes.
  • ER and plasma membranes can come in close apposition in neurons
  • Dr. Kristin Harris Harvard
  • SER smooth ER
  • the SER forms close appositions with the plasma membrane that were uniquely localized to the lateral margin of the PSD.
  • the IP3R is present in spines of cerebellar Purkinje neurons where it is associated with the spine apparatus (Satoh et al, 1990). (Interestingly, in hippocampal neurons, the RYR is present in the spine apparatus while the IP3R appears to be restricted to the dendritic shaft [reviewed in (Narasimhan et al, 1998)]. Homer lb/c and 3 are also enriched in the cytosol at the lateral margin of the PSD [ (Xiao et al, 1998)]. Thus, available anatomic evidence supported the notion that synaptic mGluRs come in close apposition with SER-associated IP3Rs at sites that are enriched for CC-Homers.
  • Plasmids expressing Homer la or Homer lb were transfected along with green fluorescent protein (gene gun) and identified Purkinje neurons were stimulated with quisqualate.
  • a patch electrode containing the Ca2+ detector Fura-2 was attached to the soma and a holding potential of -60 mV was applied.
  • Tetrodotoxin and picrotoxin were included in the bath to block synaptic input and EDTA/MgCl was included to assure that measured Ca2+ increases in the cell were generated from intracellular stores.
  • CC-Homers alter trafficking of mGluRla/5 in heterologous cells.
  • mGluR5 should contain immature, high mannose carbohydrates which are sensitive to digestion with the enzyme Endoglycosidase H (Endo H).
  • Endoglycosidase H Endoglycosidase H
  • Mature carbohydrates would be anticipated if mGluR5 was on the cell surface or if it was sequestered in a post-Golgi intracellular compartment such as endosomes.
  • the subcellular localization of the group II metabotropic glutamate receptor mGluR2 was the same whether expressed alone or with Hlb.
  • mGluR5 P1125L which does not bind to Homer in vitro (Tu et al, 1998), was not retained in the ER when co-expressed with Hlb.
  • IP3Rs in Purkinje neurons are associated with dense stacks of ER (Satoh et al, 1990) and this stacking morphology has been shown to be regulated by neural activity (Takei et al, 1994). Since a substantial portion of IP3R in cerebellum is associated with CC-Homers, it is possible that the ability of CC-Homer to crosslink interacting proteins on two adjacent membranes plays a regulatory role in ER morphology and function. Experiments in Aims 2 and 3 will examine this hypothesis.
  • Group 1 mGluRs modulate ionic currents by activating pertussis toxin- sensitive and -insensitive G proteins (Naisbitt et al, 1999). Modulation of Ca2+ currents by heterologously expressed group 1 mGluRs in superior cervical ganglion (SCG) neurons proceeds through multiple pathways involving both the a and ⁇ g - subunits of G proteins.
  • SCG superior cervical ganglion
  • CC-Homers including lb, 2b and 3 produced a similar reduction of the effect of group 1 mGluRs (Kim et al, 1997; Naisbitt et al, 1999; Naisbitt et al, 1997; Takeuchi et al, 1997).
  • Homer la or an engineered short form of Homer 2 did not block group 1 mGluR effects, but were able to partially reverse the effect of the CC-Homers.
  • Shank proteins appear to be recruited to excitatory synapses by virtue of their interaction with GKAP, a synaptic protein that binds to the guanylate kinase domain of PSD-95 (Kim et al, 1997; Naisbitt et al, 1999; Naisbitt et al, 1997; Takeuchi et al, 1997).
  • GKAP GKAP
  • Shank contains domains that mediate self- multimerization and interaction with cortactin (Golshani et al, 1998). Shank also directly interacts with Homer [ (Lujan et al., 1997)].
  • Shank-Homer interaction is mediated by the EVHl domain of Homer which binds to a single Homer-ligand site present in the proline-rich domain of Shank proteins [ (Tu et al, 1999)].
  • a quaternary complex of Homer/Shank/GKAP/PSD-95 is assembled in heterologous cells, with Homer and PSD-95 co-localizing in large clusters [ (Berridge, 1998)].
  • Shank provides a molecular bridge that links the NMDA receptor complex with Homer and its associated proteins.
  • the Homer-Shank interaction also produces clustering of group 1 mGluRs [ (Satoh et al, 1990; Villa et al, 1992)]. Clustering molecules have previously been identified for a variety of receptors and ion channels (Selig et al, 1995), but Shank- Homer are the first clustering proteins for group 1 mGluR. It is notable that the mechanism of clustering involves a linkage of mGluRs with the previously defined NMDA receptor scaffold. Thus the Shank-Homer interaction could be relevant to synaptogenesis, by docking mGluRs to a preestablished "core" of NMDA receptors.
  • NMDA receptors appear to precede the emergence of metabotropic receptors in the hippocampus and cerebellum (Xiao et al, 1998).
  • Homer proteins in association with Shank, could function to localize and cluster the mGluRs in proximity to NMDARs, and may contribute to the perisynaptic localization of group 1 metabotropic receptors (Lujan et al, 1997).
  • Shank-Homer interaction might also contribute to examples of glutamate receptor crosstalk for which physical proximity of molecules may be important, such as activation of phospholipase C (Beneken et al, submitted) or protein kinase C (Aniksztejn et al, 1991; Ben-Ari et al, 1992).
  • the Homer/Shank/GKAP/PSD-95 assembly may mediate physical association (and perhaps functional coupling) of the NMDAR with IP3R/RYR and intracellular Ca2+ stores.
  • the monovalent Homer la IEG product appears to function to uncouple proteins that are linked via the constitutively expressed CC- Homer multimers, and thereby dynamically regulate the assembly of this postsynaptic network.
  • Cocaine-induced increases in Homer la may thus modulate both mGluR and NMDA Ca2+ responses in spines.
  • the F is the single most critical side chain for the interaction when tested with larger peptides for both EVHl domains (Niebuhr et al, 1997; Tu et al, 1998).
  • the F side chain is not placed in a clear hydrophobic pocket (the ring appears to coordinate an arginine) and superposition of the ligand coordinates in Homer EVHl is even less obviously stabilized.
  • a total of 30 missense mutants of the Homer EVHl domain were expressed in HEK293 cells and assayed for binding to either mGluRla or Shank3 using GST pulldown assays.
  • Surface-exposed mutations within the region homologous to the peptide binding site of PTB domains had no affect on peptide binding.
  • mutations based on the WASP data were also ineffective in disrupting binding.
  • certain of the mutants based the Mena ligand site did disrupt Homer EVHl binding.
  • 142 (SEQ ID NOS: 17 and 18) encodes a novel protein that was first identified in a Y2H screen of brain cDNA with the Homer EVHl domain. Current information indicates that 142 functions with Homer at the excitatory synapse. We have generated 142 specific antisera and can demonstrate robust co-immunoprecipitation of 142 with Homer from brain. ImmunoEM analysis demonstrates that 142 is localized to the postsynaptic density. The predicted domain structure of 142 indicates that it shares certain properties with Shank including a N-terminal structural domain (a band 4.1 domain in 142), a single PDZ domain, and a central proline rich domain with a single Homer-ligand site. Additionally, there is a C-terminal type 1 PDZ ligand motif. We have identified a related sequence in the data base (KIAA sequence has several errors with frame shifts) suggesting that 142 may represent a gene family.
  • ⁇ -Pix also termed Cool-1 (Allen et al, 1998).
  • GEF guanine nucleotide exchange factor
  • This interaction appears robust using GST pulldown assays and we have recently confirmed the interaction using co- immunoprecipitation assays from brain.
  • Biochemical assays indicate that the PDZ domain of 142 binds its own C-terminus (may be intra or inter molecular). Based on these observations, 142 functions as a scaffold/cytoskeletal regulatory protein that responds to specific signals and may link between mGluR activation and Rac- dependent cytoskeletal remodeling.
  • the RYR encodes a potential Homer binding site near the N-terminus (Bhat et al, 1999) and using GST pulldown assays we observe that GSTHomer binds to the relevant fragment of RYRl. Importantly, we have demonstrated that the RYR co- immunoprecipitates with Homer from detergent extracts of skeletal muscle. The interaction between RYR and Homer is understood to be consistent with the function of Homer proteins to regulate the coupling of membrane receptors with intracellular calcium pools. Glutamate mediates an inhibitory postsynaptic potential in dopamine neurons of the midbrain and this is mediated by mGluRl release of intracellular Ca2+ from RYR sensitive CICR pools (Bhat et al, 1999).
  • RYR have recently been implicated as an important source of NMDAR-induced calcium rise in the post synaptic spine (Emptage et al, 1999). Since Shank is part of the NMDA receptor signaling complex (Naisbitt et al, 1999) and binds Homer, it is compelling to evaluate the possible interaction between RYR and Homer.
  • NMDA Receptor Type 2D (NR2D) and Homer.
  • NR2D NMDA receptor type 2D
  • NR2D has not been as extensively studied as NR2B but is expressed in developing cerebellum and interneurons in the forebrain (Dunah et al, 1998; Goebel and Poosch, 1999).
  • NMDAR that include the NR2DR have slower channel properties (Cull-Candy et al, 1998; Okabe et al, 1998; Vicini et al, 1998).
  • the C-terminus of NR2D is highly proline rich consistent with our observation that Homer binds a specific proline rich sequence. Thus, in the case of NR2D, Homer proteins form a direct coupling to CICR pools.
  • 130 is a novel member of the family of abl binding proteins. Related proteins function as adaptor proteins that regulate cell growth [Ziemnicka-Kotula, 1998 #392; Biesova, 1997 #393] and are hypothesized . 130 encodes a SH3 domain and a Homer binding site. Accordingly, Homer is anticipated to link this protein to other Homer- interacting proteins including metabotropic glutamate receptors and IP3R. (See SEQ ID NOS: 15, 16, 19 and 20).
  • Cdc42-associated tyrosine Kinase- 2 (ACK-2) interaction with Homer ACK-2 is a non-receptor tyrosine kinase that is regulated by the Rho-related GTP-binding protein Cdc42 [Yang, 1999 #391].
  • ACK-2 is activated by signals that result from cell adhesion, by for example activation of the integrin receptor.
  • One cellular consequence of ACK-2 activation is down stream activation of c-Jun kinase.
  • Our observation that ACK-2 interacts with Homer indicates that this signaling pathway can be linked to other membrane receptors by Homer, and identifies another signaling cascade that can be manipulated by agents that alter Homer crosslinking function.
  • EST sequence (ID#442801) which is about 73% homologous to a portion of 5' coding region of Homer-I cDNA sequence.
  • RT-PCR Forward: 5'-GAC AGC AGA GCC AAC ACC GTG-3'; Reverse: 5'-GTC TGC AGC TCC ATC TCC CAC-3'
  • the PCR products (-330 bp) consisted of two different sequences, one of which contains an additional insertion of 33 bp. A mixture of these two cDNA fragments were used as probes to screen an adult mouse brain cDNA library.
  • a search of the EST Database allowed the identification of several human EST's corresponding to mouse and rat Homer- lb, Homer-2a and 2b cDNA sequences.
  • RT-PCR was used to clone the human Homer- lb and Homer 2a and 2b coding regions.
  • a 5' degenerate primer (5'-ATG GG(A/G/C) GA(A/G) CA(A G) CC(T/C/G) AT(T/C) TTC-3') was designed based on an amino-terminal seven residue amino acid sequence (MGEQPIF) that is conserved among human EST clone #HCE003, mouse, rat, and Drosophila Homer homologue sequences.
  • the 3' primers (5 '-GAG GGT AGC CAG TTC AGC CTC-3') for human Homer-1 and human Homer-2 (5'-GTT GAT CTC ACT GCA TTG TTC-3') were made from the sequences of human EST clones #562862 and #HIBAB15 respectively.
  • Human Homer-lb and Homer-2a and 2b were amplified from new born human frontal cortex.
  • the sequences of human Homer lb, Homer 2a and Homer 2b were derived from sequencing several PCR clones and EST clones and are shown in SEQ ID NO's:3, 7 and 9.
  • Human and mouse Homer-3 were identified by searching EST Database, using Homer-1 and Homer-2 sequences. Two full-length human Homer-3 clones were identified (Clone ID #284002 and #38753) and sequenced. Numerous mouse Homer-3 clones were found and one of them (Clone ID #1162828 ) contains an almost full-length coding region. Also identified were several Drosophila EST sequences exhibiting significant homology at the amino acid level to the N-terminal region of Homer family members. The sequence presented in SEQ ID NO: 11 is derived from Clone #LD3829.
  • Mammalian expression constructs were made by cloning cDNA into Sail and Notl sites of pRK5 (Genentech), so that the cDNA was fused in-frame to an N terminal c-Myc tag.
  • GST-fusion constructs were made by cloning Homer cDNA into the Sail and Notl sites of pGEX4T-2 (Pharmacia).
  • the full-length coding regions of mouse Homer-lb, rat Homer-lc, mouse Homer-2b and human Homer-3 were engineered with Sail and Notl sites at the 5' and 3' ends by PCR using high fidelity DNA polymerase Pfu (Stratagene).
  • the sequence of Homer la was used to screen cDNA libraries prepared from rat and mouse brain for related gene products.
  • Homer la sequence was also used to search GenBank data bases. Several related rodent and human sequences were identified.
  • cDNAs that are most closely related to Homer la appear to represent alternative splice forms. This inference is based on nucleotide sequence identity of their 5'UTRs and the first 175 amino acids of the open reading frames (ORF).
  • the presumptive novel splice variants, termed Homer lb and lc, are completely divergent from Homer la after residue 175 of the ORF and they possess entirely distinct 3'UTRs. comparison at the point of sequence divergence indicates that Homer la encodes a unique eleven amino acid carboxy terminus of the ORF and about 5 kb 3' UTR region. The unique eleven amino acid carboxy-terminal sequence of Homer la does not possess a recognizable motif.
  • Homer lb and lc appear to be formed by a splicing event that substitutes a relatively long and unique carboxy-terminus of the ORF and shorter 3'UTR sequence that lacks the characteristic IEG motif.
  • Multiple independent isolates of rat and mouse Homer lb and lc were identified and sequenced to confirm their natural expression in brain.
  • cDNA sequences that appear to represent two additional Homer genes termed Homer 2 and Homer 3.
  • the sequences of two splice forms of Homer 2 and one Homer 3 sequence is presented (See Figures section).
  • the predicted size of the protein products and general domain structure are similar to Homer lb and lc.
  • each of the Homer 2 and Homer 3 proteins contain about 120 amino acids at the amino-terminal that is highly similar to the amino-terminal domain of Homer la.
  • the degree of amino acid identity in these regions is about 88% between Homer 1 and Homer 2 and about 86% between Homer 1 and Homer 3. Many of the amino acid differences are conservative.
  • the carboxy-terminal regions of Homer 2 and 3 are only about 22% identical to Homer lb, but like Homer lb and lc are predicted to possess a CC secondary structure.
  • the CC domains of all Homer family members exhibit significant homology (about 40- 45% amino acid similarity) to the CC regions of myosin heavy chain (Strehler et al, J Mol Biol 120:291 (1986)), kinesin heavy chain (Yang et al, Cell 56:879 (1989)) and dynactin (Gill et al, J Cell Biol 115:1639 (1991)).
  • Homer 2a and Homer 2b The distinct splice forms of Homer 2, termed Homer 2a and Homer 2b, are differentiated by an eleven amino acid insertion at residue 131 in Homer 2b.
  • Human Homer 1, 2 and 3 are mapped to chromosomes 5, 15 and 19, respectively by the Human Genome Project.
  • Drosophila Homer possess the basic domain structure of mammalian Homers.
  • the amino-terminus is highly homologous to that of mammalian Homer and the carboxy terminus is predicted to form a CC secondary structure.
  • Detergent (2% SDS) extracts from rat cortex, hippocampus, and cerebellum were separated on 8% SDS-PAGE gels and transferred to nitrocellulose membranes. Blots was probed with polyclonal anti-Homer sera. Specificity was tested by incubating the antiserum with 10 ⁇ g/ml of relevant peptide at room temperature for 10 m prior to use. Rabbit polyclonal antiserum was also generated against the full length GST-Homer la fusion protein, as described previously (Brakeman, et al, Cell 87:227 1997). This antiserum recognizes all Homer 1 isoforms.
  • Unpurified antibodies were tested for their sensitivity and specificity in detecting heterologously expressed, full length Homer proteins with amino-terminal c-myc tags. Each Homer protein was selectively detected on Western blot by the appropriate Homer antibody in soluble extracts of transfected HEK293 cells. The myc-tagged Homer proteins migrated with an apparent molecular mass of 50 kDa. There was no cross reactivity between antibodies for one Homer form and other family members.
  • HEK293 cells were transiently transfected (using calcium phosphate) with full length mGluRla and mGluR5 constructs in pRK5 (Brakeman et al, 1997). Cell lysates were made 24-48 h post-transfection.
  • GST fusion proteins bound to glutathione agarose were prepared of Homer la, Homer lc, Homer 2b, Homer 3 and two amino terminal fragments of Homer 2 according to the following procedure.
  • GST fusion constructs were prepared by polymerase chain reaction with specific primers that included Sail and Notl sequences and subcloned into pGEX4T-2 vector (Pharmacia Biotech, Uppsala, Sweden). Constructs were confirmed by sequencing.
  • GST-fusion proteins were expressed in BL21 bacterial strains. Bacteria were harvested and lysed in PBS, 1% Triton XI 00, 2 mM phenylmethylsulfonyl fluoride (PMSF) and pelleted at 13,000 rpm (Sorvall SS-34) at 4°C for 5 m Proteins were purified by incubating 1 ml bed volume glutathione-sepharose (GST) beads (Sigma USA) with bacterial supernatant at 4°C for 10 m, washing twice with PBS and PBS plus 1% Triton X-100. Protein was eluted with 10 mM glutathione and dialyzed against PBS at 4°C.
  • GST glutathione-sepharose
  • Protein concentrations were measured by BCA (Pierce, Illinois). Cell lysates of the transfected cells were incubated with equivalent amounts of various Homer-GST fusion proteins at 4 °C for 2 h, washed with PBS and 1% Triton X-100. Proteins were eluted in 2% SDS sample buffer and separated on 8% or 2.5% SDS-PAGE gels and probed with appropriate antibody.
  • mGluR5 also bound to all full length Homer constructs and to a Homer 2 amino-terminal fragment of about 141 residues but not to GST alone. The relative binding in the three assays were comparable for each of the three Homer types. A Homer 2 deletion mutant that includes only the amino-terminal 92 residues did not bind mGluR5. Similar binding of Homer proteins to mGluRl was also observed. EXAMPLE 4
  • mGluRl ⁇ monoclonal antibody was obtained from PharMingen (San Diego CA). Rabbit polyclonal mGluR5 antibody was a gift from Dr. Richard Huganir, Johns Hopkins School of Medicine.
  • Homer family members are naturally associated with group I metabotropic receptors in brain. This analysis was performed using cerebellum since all three Homer family members are expressed in this tissue. Detergent extracts of whole adult rat cerebellum were incubated with antibodies to Homer lb/c. Homer 2a/b or Homer 3 and immunopreciptates were blotted with a mouse monoclonal antibody to mGluRl ⁇ . mGluRl ⁇ co-immunoprecipitates with each of the antisera directed against Homer proteins. The predominate band after electrophoreses corresponded to the monomer form of mGluRl ⁇ (about 150 kDA) and other bands corresponding to multimers of mGluRl ⁇ are also observed.
  • Embryonic mouse cerebellar cultures were prepared and maintained according to the method of Schilling et al. (Schilling et al, Neuron 7:891 1991). At 4-5 DIV, cultures were transfected with plasmids coding for E-GFP (Clontech) and either full-length Homer lb or an IEG form of Homer 1. The IEG form of Homer 1 was a 186 amino acid amino-terminal fragment of Homer lb. Plasmids were purified by cesium banding. Three combinations of the plasmids were transfected.
  • Group I control
  • group II 20 ⁇ g of E-GFP and 40 ⁇ g of pRK5 vector
  • group II 20 ⁇ g of E-GFP and 40 ⁇ g of ⁇ RK5 Homer 1 IEG
  • group III 20 ⁇ g of E-GFP and 40 ⁇ g of pRK5 Homer lb.
  • Plasmid DNA was mixed with gold particles (0.6 micron), and coated onto plastic tubing. DNA was then ballistically transfected into cells according to the manufacturer's protocol (Helios Gene Gun System, BIO-RAD). After transfection, cultures were returned to the incubator and maintained for an additional 2 days for a total of 7-8 DIV at the time of use for imaging experiments.
  • Patch electrodes were attached to the somata of GFP-expressing Purkinje cells and a holding potential of -60 mV was applied.
  • Micropressure electrodes (l ⁇ m tip diameter) were filled with quisqualate (100 ⁇ m in external saline) and were positioned -20 ⁇ m away from large-caliber dendrites.
  • Test pulses were delivered using positive pressure (6 psi, 1 sec).
  • Cells were bathed in a solution that contained (in mM) NaCl (140), KC1 (5), EGTA (0.2), MgCl 2 (0.8), HEPES (10), glucose (10), tetrodotoxin (0.005), and picrotoxin (O.l), adjusted to pH 7.35 with NaOH, which flowed at a rate of 0.5 ml/m
  • the recording electrode contained CsCl (135), HEPES (10), fura-2 K 5 salt (0.2), and Na 2 -ATP (4), adjusted to pH 7.35 with C s OH. Patch electrodes yielded a resistance of 3-5 M ⁇ when measured with the internal and external salines described above.
  • Fura-2 ratio imaging of intracellular free Ca 2 + was accomplished by measuring the background corrected fluorescence ratio at 340 and 380 nm excitation using a cooled CCD camera system, as previously described (Linden et al, JNeurosci 15:5098 1995). Exposure times were 200 msec per single wavelength image. Experiments were conducted at room temperature. Enhanced GFP is weakly excited by illumination in the 380-400 nm spectrum.
  • the decay phase of the Ca +2 response appeared somewhat slower in neurons transfected with the truncated form. While the total Ca +2 flux appeared similar in cells transfected with truncated and complete Homer proteins and in empty vector controls, the measurement could not be made because the tail of the Ca +2 response was abbreviated due to the constraints of the image buffer capacity.
  • Residues 1-120 of rat Homer la were expressed in Escherichia coli BL21 cells as a C-terminal fusion to glutathione-S-transferase (GST-laENH) as previously described (Tu et al, Neuron 21:717 1998).
  • GST-laENH glutathione-S-transferase
  • GST-laEVH was prepared by expression in the methionine auxotrophic strain B834 (DE3) ( ⁇ ovagen). 5 mL of an overnight culture grown at 37°C in LB media supplemented with 100 ⁇ g/mL ampicillin (Sigma) was added to 4L M9 minimal media (Gibco BRL) supplemented with 100 ⁇ g/mL ampicillin, 0.05 mg/mL alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, L-selenomethione , 1 ⁇ /mL thiamine (Sigma), 2mM MgSO 4 , 1% glucose, 100 ⁇ M CaCl 2 .
  • the column was washed in succession with twenty column volumes of lx PBS/1% Triton, twenty column volumes of lx PBS, and ten column volumes of cleavage buffer (50 mM Tris [7.4], 150 mM NaCl, 2.5 mM CaCl 2 ,. 50 mM ⁇ -mercaptoethanol). All buffers were degassed.
  • a 50% slurry of glutathione-agarose beads loaded with fusion protein was incubated with 20 U of biotinylated Thrombin (Novagen) for 16 h at room temperature.
  • the released cleavage product (la-EVH) was collected, and the biotinylated Thrombin was removed with streptavidin-agarose beads (Novagen).
  • la-EVH was further purified by cation-exchange chromatography using a Resource S column (Amersham-Pharmacia).
  • Crystals of native and SeMet protein were grown in hanging drops by the method of vapor diffusion (Wlodawer et al, Proc Natl Acad Sci USA 12:711 1975). l ⁇ l of a 9 mg/mL native or SeMet protein solution was mixed with a 1:1 dilution of reservoir buffer (30% PEG 3350, 87 mM MgSO 4 , 50 mM HEPES, pH 7.3) with distilled water and equilibrated over I mL of reservoir buffer. All crystallization trials for the SeMet protein were set up under anaerobic conditions to minimize potential problems due to oxidation. Two different crystal forms were observed for both the native and the SeMet protein.
  • Crystals in the orthorhombic space group P2 1 2 1 2 1 typically grew to a size of 0.5 mm x 0.03 mm x 0.03 mm.
  • Point mutants of N- terminally myc-tagged, full-length Homer lb and lc and Homer 1 EVHl were made using the QuikChange TM Site-Directed Mutagenesis Kit (Stratagene). Expression constructs were transiently transfected into HEK293 cells using calcium phosphate methods. About 24-48 h post-transfection, cell lysates were prepared in lx PBS/1% Triton X-100 (Sigma) and protease inhibitors.
  • GST pull-down assays were performed by mixing 100 ⁇ l of cell lysate with GST-mGluR5 or GST-Shank3 (residues 1143-1408) (Tu et al, in press) bound to glutathione-agarose, incubating at 4°C for 2 h, and washing with lx PBS and lx PBS/1% Triton X-100. Bound products were eluted with 100 ⁇ l 2x SDS loading buffer and detected by SDS-PAGE and immunoblot using anti-myc antibody 9EI0 (Invitrogen) and ECL reagents (Amersham).
  • Rat Homer la was cloned based on its rapid upregulation in hippocampal granule cell neurons following electrically-induced seizure (MECS; see Brakeman et al, Nature 186:284 1997) The expression of other members of the Homer family was examined in the brain following seizure.
  • Radio-labeled riboprobes were prepared using unique sequences for Homer la, Homer lb, Homer lc, Homer 2a, Homer 2b and Homer 3. Probes used did not distinguish between the splice forms of Homer lb and lc or Homer 2 and 2b.
  • Anti-sense and sense cRNA probes were generated from each mouse Homer plasmid by in vitro transcription in the presence of [ S]UTP, as previously described (Lyford et al, Neuron 14:433 1995).
  • Probe for Homer-la (Xiao, 1998; GenBank # ) was derived from nucleotides 1342 to 2140, for Homer lb/c (Xiao, 1998; GenBank # ) from nucleotides 785 to 1396, for Homer-2a/b (Xiao, 1998; GenBank # ) from nucleotides 486 to 1561, and for Homer-3 (GenBank # ) from nucleotides 371 to 2123.
  • Probe (about 10 6 cpm in 75 ⁇ l hybridization buffer) was applied to each slide. Coverslipped slides were then incubated in humidified chambers overnight at 56°C. Following completion of wash steps, slides were air dried and exposed to Kodak Biomax MR film for 2-3 days.
  • Homer la is similar to the expression of Homer lb and Homer lc. High levels of expression of Homer la are observed in the hippocampus, striatum and cortex. In the cortex, there is laminar expression with the highest levels in the superficial and deep layers. Expression of Homer 2a and 2b is enriched in the thalamus, olfactory bulb and principle neurons of the hippocampus in contrast to the cortex where low levels of expression of Homer 2a and 2b are observed. Homer 3 is expressed primarily in the cerebellum and hippocampus.
  • CC secondary structure is implicated in protein-protein interactions (Lupas, 1996 supra). Therefore, the possibility that this domain might confer the ability to form homo- or hetero-multimers between Homer family members was examined.
  • myc-tagged Homer- lc and Homer-2b were transfected into HEK293 cells and cell extracts were made 2-3 days post-transfection. Cell lysates were treated as described above.
  • CC domains of these proteins show modest sequence to Homer family CC domains and bind to the CC domain of dynactin (Gill, 1991 supra). None of the myc-tagged Homer family members bound to either dynein IC-la or dynein IC-2c.
  • rats were deeply anesthetized with sevoflurane and perfused through the aorta with 250 ml of saline followed by 400 cc each of 4% paraformaldehyde in 0.1 % phosphate buffer (pH 6.5) and 4% paraformaldehyde in 0.1% phosphate buffer (pH 8.5).
  • the rat was allowed to postfix for 1 hr. at room temperature and then prefused with 15% sucrose in 0.1 % phosphate buffer (pH 7.4).
  • the brain was removed and sectioned at 40 ⁇ m on a freezing sliding block microtome and collected in PBS. Tissue was stained with an immunoperoxidase technique, as follows.
  • Immunohistochemistry was performed to determine the cellular localization of Homer lb/c and Homer 2a/b and Homer 3 in rat brain. Light microscopic examinations indicated that all three Homer proteins are enriched in Purkinje neurons. Immunoreactivity is present in the cytoplasmic region of the soma and extends prominently into the dendritic arbor. The nucleus is not stained. Little or no staining is detected in the contiguous granule cell layer. A similar light microscopic pattern of cellular localization was detected for Homer 3. Homer 2 immunostaining in cerebellum also showed staining in Purkinje neurons, but appeared technically less differentiated.
  • a postembedding immunogold method as described previously (Wang, et al, J Neurosci 18:1148 1998) was used and modified from the method of (Matsubara, et al, J Neurosci 16:4457 1996). Briefly, male Sprague-Dawley rats were perfused with 4% paraformaldehyde plus 0.5% glutaraldehyde in 0.1 M phosphate buffer. Two hundred micrometer parasagittal sections of the rostral cerebellum (folia III-V) were cryoprotected in 30% glycerol and frozen in liquid propane in a Leica EM CPC.
  • Frozen sections were immersed in 1.5% uranyl acetate in methanol at -90°C in a Leica AFS freeze-substitution instrument, infiltrated with Lowicryl HM 20 resin at -45°C, and polymerized with UV light. Thin sections were incubated in 0.1% sodium borohydride plus 50 mM glycine in Tris-buffered saline/0.1% Triton X-100 (TBST), followed by 10% normal goat serum (NGS) in TBST, primary antibody in 1% NGS/TBST, lO nm immunogold (Amersham) in 1% NGS/TBST plus 0.5% polyethylene glycol, and finally staining in uranyl acetate and lead citrate. Primary antibodies were used at dilutions of 1 :500 for Homer lb and 1:100-1:400 for Homer 3.
  • Peripheral Tissues are expressed in peripheral tissues. In detergent extracts of heart and kidney, a single band at 47 kDa immunoreactive to Homer lb and lc is detected. In extracts of liver, a complex of three bands ranging from about 44 to 47 kDa is detected. In heart, liver, skeletal muscle and intestine, bands immunoreactive to Homer 2a and 2b are detected. Homer 3 immunoreactive bands are detected in extracts of lung and thymus.
  • N-terminal myc-tagged full-length Homer la ORF was cloned into the expression vector pT2 (Gordon, et al, Cell 50:445 1987; Aigner, et al, Cell 83:269 1995).
  • Transgenic mice were generated at the University of Alabama Transgenic Facility. Expression of the transgene protein was assayed by western blot with rabbit polyclonal antisera that recognizes all Homer 1 isoforms (pan-Homer 1 antibody) and myc antibody.
  • Homer la is unique within the family of Homer related proteins in that it is dynamically regulated and it lacks the CC domain. Accordingly, it was hypothesized that the IEG would bind to group 1 metabotropic receptors and disrupt the formation of multivalent complexes of Homer and mGluR. To examine this hypothesis, a transgenic mouse was generated that expresses Homer la under the control of a modified Thy-1 promoter (Gordon et al, 1987, supra), which drives neuron-specific expression in postnatal brain (Aigner et al, 1995, supra). Transgenic mice expressed Homer la at high levels in cortex, hippocampus, cerebellum and thalamus/brainstem relative to levels in wild type litter mate controls.
  • Homer 3 is less highly expressed in forebrain than Homer lb/c or Homer 2a/b and co-immunoprecipitates of mGluR5 with Homer 3 antibody were less clean. Accordingly, it could not be determined in these experiments whether Homer la also competes with Homer 3. Identical results were obtained in tow independent mouse lines that express Homer la transgene. The Homer la expressing transgenic mice have not been behaviorally characterized but appear normal in size and gross motor activity.
  • a novel family of proteins was identified based on its ability to interact with Homer family proteins in a yeast two-hybrid screen of a brain cDNA library.
  • Homer la was subcloned into pPC97 (Chevray and Nathans, Proc. Natl. Acad. Sci. U.S.A., .8.9:5789 (1992)) and used to screen a random primed cDNA library prepared from seizure-stimulated rat hippocampus and cortex cloned in pPC86 (Chevray and Nathans, 1992, id.) as described previously (Brakeman et al, Nature, 386:284 (1997)).
  • mGluR5 constructs included a wild type C-terminal 241 amino acid fragment and a four amino acid carboxy-terminal deletion of the same fragment.
  • Shank 1 and 3 are closely related to a previously described protein, termed Cortactin Binding protein (CortBP-1; Du et al, Mol. Cell Biol, 18:5838 (1998)).
  • Cortactin Binding protein CortBP-1; Du et al, Mol. Cell Biol, 18:5838 (1998).
  • Shank cDNAs isolated from the yeast two-hybrid screen were expressed in HEK293 for GST pulldown assays with GST-Homer la.
  • the interaction between Homer and Shank proteins was further characterized by co- immunoprecipitation assays.
  • Shank expression constructs were prepared as described (Naisbitt et al, in press). Site directed point mutants of Shank were generated using Quik Change (Stratagene).
  • GST fusion constructs were prepared by polymerase chain reaction (PCR) using Pfu Polymerase (Stratagene) with specific primers that included Sail and Notl sequences. After digestion with Sall/Notl, PCR products were subcloned into pGEX4T-2 vector (Pharmacia Biotech, Uppsala, Sweden) or N-myc-tagged pRK 5 vector (modified from Genentech). All constructs were confirmed by sequencing. GST- fusion proteins were expressed in BL21 E. coli strains (GIBCO, BRL).
  • Expression constructs were transiently transfected into HEK293 cells using the calcium phosphate method.
  • Cells were lysed 24-48 h post-transfection with PBS plus 1 % Triton X-100.
  • GST pull down assays were performed by mixing 100 ⁇ l cell lysates with beads charged with GST fusion proteins (1-3 ⁇ g/50 ⁇ l bed vol.) at 4°C for 2 h followed by washing once with PBS, once with PBS plus 1% Triton X-100. Bound proteins were eluted with 100 ⁇ l 2 x SDS loading buffer and detected by SDS- PAGE and immunoblotting using ECL reagents (Amersham).
  • GST pull down assays of mGluRla and mGluR5 from brain lysates were performed by sonicating rat cerebellum or cortex in 50mM Tris, ImM EDTA, 1% CHAPS (Sigma), 0.5% deoxycholic acid (Sigma) and proteinase inhibitors with GST-proteins and these tissue extracts were then processed as above.
  • transfected cells were extracted in RIPA (see Naisbitt et al, in press). Soluble extracts were precipitated with 2 ⁇ g control non-immune IgG, Myc or Shank 1 (56/e) antibodies (Naisbitt et al, in press).
  • Extracts of forebrain crude synaptosomes for immunoprecipitation were prepared using deoxycholic acid as described previously (Dunah et al, Mol. Pharmacol. 53429 (1998)).
  • Forebrain P2 fraction was extracted in 1% deoxycholic acid, dialyzed over night into 0.1 % Triton X-100, 50 mM Tris, pH 7.4. Concurrently, 5 g of each antibody was pre-incubated overnight with 10 ⁇ l bed volume protein A- sepharose. After centrifugation at 100,000g for 1 h, 50 ⁇ g of extract was incubated with antibody-protein A in 100 ⁇ l 0.1% Triton X-100, 50 mM Tris, pH 7.4 for 2 h at 4°C. Pellets were washed 4 times with 1 ml incubation buffer, and bound proteins were analyzed by immunoblotting.
  • Shank antibodies were raised in rabbits immunized with GST- fusions of Shank 3 residues 1379-1740 and 1379-1675 (Covance, Denver, PA). Similar bands were seen on rat brain immunoblots with both antisera. GKAP, PSD 95 and Shank 1 (56/e) antibodies are described in (Naisbitt et al, in press). Homer antibodies are described above. Anti-mGluR la monoclonal antibody is from Pharmingen and rabbit polyclonal mGluR5 antiserum was obtained from Dr Richard Huganir (Johns Hopkins University).
  • Shank 1, 2, 3 and CortBP-1 are conserved in Shank 1, 2, 3 and CortBP-1.
  • Shank 3 three deletion fragments of Shank 3 that included, respectively, amino acid residues 559-908, amino acid residues 1143-1408, and amino acid residues 1379-1740 were testing for their ability to bind to Homer lb, Homer lc, Homer 2 and Homer 3 in GST pulldown assays. Similar binding specificity was detected with each of the Homer proteins. Only Shank3 fragment 1143-1408 bound to Homer.
  • Shankl contains the amino acid sequence that most closely resembles the Homer ligand peptide consensus (LVPPPEEFAN; residues 1307-1316).
  • Shankl A similar sequence is present in Shankl (PLPPPLEFSN 1563-1572; see Naisbitt et al, in press).
  • CortBP possesses two similar sites; (PLPPPLEFAN; residues 813-822) and (FLPPPESFDA residues 878-887).
  • Fragments of Shank3 containing amino acid residues located nearer the amino-terminal of the protein such as Shank 3 fragment 559-908 (which includes the PDZ domain and the first proline-rich motif) did not bind to Homer, but did bind to GKAP (Naisbitt et al, in press).
  • Shank3 fragment 1379-1740 which includes the carboxy-terminal proline-rich sequence and the SAM domain, did not bind to Homer, though it is capable of binding itself and cortactin (Naisbitt et al, in press). These studies identify the Homer binding site as being distinct from either the PDZ domain that binds GKAP, or the proline-rich binding site that binds cortactin and which is located nearer to the carboxy-terminal (Naisbitt et al, in press).
  • EVHl domain are necessary and sufficient for binding to Homer ligands (Brakeman et al, 1997, supra; Tu et al, 1998, supra). To confirm that the EVHl domain of
  • Shank proteins may link Homer proteins with components of a cell-surface clustering complex, such as the NMDA clustering complex.
  • COS7 cells were transfected using the Lipofectamine method (GIBCO-BRL) on poly-lysine coated coverslips for clustering experiments, as described in Naisbitt et al (in press) and Kim et al. (Neuron 17:103 1996).
  • Primary antibodies were used as follows: GKAP C9589, 1 ⁇ g/ml (Naisbitt et al, in press); Shank 56/e 0.5 ⁇ g/ml (Naisbitt et al, in press), PSD-95, 1:1000 diluted guinea pig serum (Kim et al, Neuron 378:85 1995).
  • Cy3 and (fluoroscein isothiocyanate conjugate (FITC)- conjugated secondary antibodies (Jackson Immunoresearch) were used at dilutions of 1 :500 and 1 : 100 respectively.
  • a yeast two-hybrid screen of the same rat brain cDNA library was performed using the PDZ domain of Shank3 as bait. From this screen, two identical clones of the carboxy-terminus of GKAP-3/SAPAP3 were isolated. In a reciprocal screen, Naisbitt et al. (in press) isolated multiple clones of Shankl, 2 and 3 using GKAP as bait. This result provides independent confirmation of the specificity of the interaction between the Shank and GKAP/SAPAP families of proteins.
  • the cDNA from the yeast two-hybrid screen encoding the carboxy-terminal 347 amino acids of GKAP-3 was expressed with an amino-terminal myc tag in HEK293 cells and tested for binding to GST fusion constructs of Shank3 and other PDZ containing proteins.
  • the GST fusion of Shank3 fragments containing just the PDZ domain was sufficient to bind GKAP3, while a Shank3 construct lacking the PDZ domain (residues 665-908) failed to bind.
  • PDZ domains of GRIP and SAP 102 failed to pull down GKAP3, demonstrating the specificity of the Shank-GKAP interaction.
  • Shank proteins may link Homer proteins with components of the NMDA clustering complex
  • co-clustering of these proteins in transfected COS cells was assessed.
  • both proteins showed a diffuse distribution in the cytoplasm. This is not surprising, since Homer and PSD-95 do not interact directly.
  • co-clustering of Homer and PSD-95 was not observed following co-transfection of Homer and PSD-95 with either Shankl or GKAP alone.
  • Shank and GKAP may mediate the formation of a quaternary protein complex containing PSD-95 and Homer (see also Naisbitt et al, in press).
  • Other types of macromolecular complexes may also form when Homer and Shank proteins interact.
  • Cells expressing Homer lb and Shank 1 (without GKAP or PSD-95) exhibited a redistribution of Homer lb into a reticular filamentous pattern, as well as into clusters; in both kinds of structures Shank and Homer immunoreactivities were co-localized.
  • GKAP can mediate the co-clustering of Homer and PSD-95 Shank may mediate clustering of group 1 metabotropic glutamate receptors (mGluRs).
  • mGluRs metabotropic glutamate receptors
  • Shankl and mGluR5 in COS cells did not result in obvious clustering of either protein. Similarly, Homer and mGluR5 do not form co-clusters. Co-expression of the three proteins Homer, Shank 1, and mGluR5, however, resulted in conspicuous co- clustering of mGluR5 with Shank 1. Clustering of mGluR5 in these triply transfected cells was dependent on the ability of Homer to bind the receptor since a point mutant of mGluR5 that does not interact with Homer failed to co-cluster with Shank. Thus, both Homer and Shank are required to mediate the clustering of mGluR5.
  • the Shank 3 PDZ Domain Binds the Carboxy-Terminus of Group 1 Metabotropic Receptors Directly at a Site Distinct from the Homer Binding Site
  • the Shank PDZ domain shows selective binding to the GKAP carboxy- terminus (Naisbitt et al, in press).
  • the carboxy-terminal sequence of GKAP (-QTRL) finds similarities with that of the group 1 mGluRs (mGluRla -SSSL; mGluR5 -SSTL) and therefore it was determined whether the PDZ domain of Shank can directly bind the carboxy-terminus of group 1 mGluRs.
  • GST-pulldown assays were performed using extracts from heterologous cells expressing a recombinant mGluR5 carboxy-terminal 241 amino acid peptide.
  • the mGluR5 carboxy-terminal tail bound two partially overlapping constructs of Shank 3 that included the PDZ domain (559-908; and 559-673), but not a construct from which the PDZ domain was deleted (amino acids 665-908). Binding of mGluR to the Shank3 PDZ domain was qualitatively similar to mGluR5 binding to Homer lc and Homer 2. Negative controls included absence of binding of mGluR to SAP102 PDZ1-3 and GRIP PDZ 4-6. Furthermore, a deletion mutant of the mGluR5 polypeptide that lacked the carboxy- terminal four amino acids failed to bind to the PDZ domain of Shank3.
  • Shank PDZ Identical interactions between Shank PDZ and mGluR5 C-terminal tail were detected in a yeast two-hybrid analysis. These studies indicate that the PDZ domain of Shank 3 can bind the carboxy-terminus of group 1 metabotropic receptors via a PDZ-mediated interaction with the carboxy-terminal sequence -S S/T L.
  • Shank3 PDZ domain can bind full length native mGluRs
  • GST pull down assays were performed with detergent extracts of forebrain or cerebellum.
  • the PDZ domain of Shank 3 bound specifically to mGluRla and mGluR5 from cerebellum and forebrain, respectively. (Cerebellum predominantly expresses mGluRl, while forebrain expresses predominantly mGluR5.) While it is possible that the Shank3 PDZ pulldown of mGluRs from brain extracts is indirect, via Shank PDZ pulling down a GKAP-Shank-Homer-mGluR complex, this extended complex is unlikely given the more modest ability of GST-GKAP to pull down Homer.
  • Shank may interact with the cytoplasmic tail of mGluRla/5 both directly, via its PDZ domain, and indirectly, via Homer.
  • the inability of Shank 1 to cluster mGluR5 in the absence of Homer indicates that the direct PDZ-dependent Shank-mGluR interaction is contingent upon a co-incident Homer interaction. Both modes of interaction with mGluR may be involved in mGluR clustering by Shank and contribute to physiological regulation.
  • Frozen sections were immersed in 1.5% uranyl acetate in methanol at -90 °C in a Leica AFS freeze-substitution instrument, infiltrated with Lowicryl HM 20 resin at -45 °C, and polymerized with UV light.
  • the first primary antibody e.g., Shank; Shank3 1379-1675 antigen
  • corresponding immunogold-conjugated antibody (10 nm gold) were applied, sections were exposed to paraformaldehyde vapors at 80°C for one hour, and the second primary (Homer lb and lc) and secondary (20 nm gold; Ted Pella/BBI International) antibodies were applied the following day.
  • Controls shown little or no gold labeling included absence of the primary antibody for single labeling and absence of the second primary antibody for double labeling.
  • Primary antibodies were used at dilutions of 1 :100-1:300 for Shank and 1:400 for Homer lb and lc.
  • Shank 3 An antibody generated against a carboxy-terminal region of Shank 3 (amino acids 1379-1675) was used to examine the ultrastructural distribution of the Shank proteins in brain. This antibody recognizes multiple bands on brain immunoblots, including major bands of -160-180 kD and -210 kD in forebrain and cerebellum, similar to those seen with other Shank antibodies (see Naisbitt et al, in press). The different size bands presumably derive from the multiple Shank genes and splice variants. All Shank immunoreactivity is blocked by incubation of the Shank antibody with the Shank fusion protein antigen.
  • PSD This distribution is similar to the distribution of NMDA receptors associated with the postsynaptic membrane (Petralia et al, 1999, supra) and distinct from the distribution of mGluR5 which are most prevalent in the perisynaptic membrane region just outside the PSD (Lujan et al, Eur J Neurosci 8:1488 1996). This spatial localization is consistent with the idea that Shank 3 and Homer interact with components of both the NMDA receptor and metabotropic receptor signaling complexes.
  • Shank This family of proteins that interact with Homer are identical to the Shank family of postsynaptic density (PSD) proteins that interact with GKAP and PSD-95 complex (Naisbitt et al, in press).
  • Shank uses distinct domains to bind to GKAP and to Homer, and thus can form a bridge between proteins of this family.
  • Shank/GKAP is also associated with NMDA receptors through the PSD-complex (Naisbitt et al, in press) and thus the Homer-Shank interaction indicates a molecular link between NMDA receptors and Homer-associated proteins such as mGlu receptors and inositol trisphosphate receptors. This linkage has important implications for the coupling of NMDA receptors to intracellular calcium release pools and for excitatory synapse assembly in general.
  • PSD postsynaptic density
  • mGluRl alpha metabotropic glutamate receptor
  • alphaPix stimulates p21- activated kinase activity through exchange factor-dependent and -independent mechanisms. J Biol Chem 274, 6047-50.
  • GRIP a synaptic PDZ domain-containing protein that interacts with AMPA receptors [see comments]. Nature 386, 279-84.
  • NMDA receptor subunit gene expression in the rat brain a quantitative analysis of endogenous mRNA levels of NRlCom, NR2A, NR2B, NR2C, NR2D and NR3A [In Process Citation].
  • Dendritic spines of CA 1 pyramidal cells in the rat hippocampus serial electron microscopy with reference to their biophysical characteristics. J Neurosci 9, 2982-97. Harris, K. M., and Stevens, J. K. (1988). Dendritic spines of rat cerebellar Purkinje cells: serial electron microscopy with reference to their biophysical characteristics. J Neurosci 8, 4455-69.
  • Hyvonen M., Macias, M. J., Nilges, M., Oschkinat, H., Sarastre, M., and Wilmanns, M. (1995). Structure of the binding site for inositol phosphates in a PH domain. EMBO J. 14, 4676-4685.
  • COX- 2 a synaptically induced enzyme, is expressed by excitatory neurons at postsynaptic sites in rat cerebral cortex. Proc Natl Acad Sci U S A 93 , 2317-21. Kim, E., Naisbitt, S., Hsueh, Y. P., Rao, A., Rothschild, A., Craig, A. M., and Sheng, M. (1997).
  • GKAP a novel synaptic protein that interacts with the guanylate kinase- like domain of the PSD-95/SAP90 family of channel clustering molecules.
  • Arc a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites. Neuron 14, 433-45.
  • Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol 140, 647-57.
  • Shank a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin. Neuron 23, 569-582.
  • a novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVHl domain, a protein module present in the Ena/VASP family. EMBO J 16, 5433-44. Nusser, Z., Mulvihill, E., Streit, P., and Somogyi, P. (1994).
  • Inositol 1,4,5-trisphosphate receptor causes formation of ER cisternal stacks in transfected fibroblasts and in cerebellar Purkinje cells. Neuron 12, 327-42.
  • SAPAPs A family of PSD-95/SAP90-associated proteins localized at postsynaptic density. J Biol Chem 272, 11943-51.
  • Rheb a growth factor and synaptic activity regulated gene, encodes a novel Ras-related protein. Journal of Biological Chemistry 269, 16333- 16339.
  • R value ⁇
  • the free R value was calculated from 10% of the data that was excluded from the refinement (Briinger,

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Psychiatry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Hospice & Palliative Care (AREA)
  • Pain & Pain Management (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Endocrinology (AREA)
  • Reproductive Health (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé relatif à l'identification d'un composé qui module une réponse cellulaire associée à la catégorie Homer et dont la médiation est assurée par un récepteur de surface ou intracellulaire. L'invention concerne en outre un procédé relatif à l'identification d'un composé qui module la mobilisation de calcium activée par récepteur associée à la catégorie Homer. L'invention concerne également un procédé relatif à l'identification d'un composé qui inhibe l'activité des protéines Homer sur la base des cordonnées de structure cristalline du domaine de liaison de ces protéines. L'invention concerne par ailleurs un procédé relatif à l'identification d'un composé qui affecte la formation en groupes des récepteurs de surface. L'invention concerne enfin des acides nucléiques codant les protéines Homer, des protéines Homer, et des protéines Homer à interaction.
EP99945113A 1998-08-18 1999-08-18 Proteines a interaction de la famille homer Withdrawn EP1105734A4 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP06003521A EP1662255A3 (fr) 1998-08-18 1999-08-18 Protéines interagissant avec Homer

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
US9733498P 1998-08-18 1998-08-18
US97334P 1998-08-18
US13849499P 1999-06-09 1999-06-09
US13842699P 1999-06-09 1999-06-09
US13849399P 1999-06-09 1999-06-09
US138493P 1999-06-09
US138426P 1999-06-09
US138494P 1999-06-10
PCT/US1999/018973 WO2000011204A2 (fr) 1998-08-18 1999-08-18 Proteines a interaction de la famille homer

Related Child Applications (1)

Application Number Title Priority Date Filing Date
EP06003521A Division EP1662255A3 (fr) 1998-08-18 1999-08-18 Protéines interagissant avec Homer

Publications (2)

Publication Number Publication Date
EP1105734A2 true EP1105734A2 (fr) 2001-06-13
EP1105734A4 EP1105734A4 (fr) 2005-04-13

Family

ID=27492912

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99945113A Withdrawn EP1105734A4 (fr) 1998-08-18 1999-08-18 Proteines a interaction de la famille homer

Country Status (4)

Country Link
EP (1) EP1105734A4 (fr)
JP (1) JP2002523056A (fr)
AU (1) AU772105B2 (fr)
WO (1) WO2000011204A2 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7033790B2 (en) 2001-04-03 2006-04-25 Curagen Corporation Proteins and nucleic acids encoding same
WO2003056329A2 (fr) * 2001-12-21 2003-07-10 7Tm Pharma A/S Utilisation d'un complexe de transduction de signal dans des processus de mise au point de medicaments
CA2510715A1 (fr) * 2002-12-20 2004-07-08 Enkam Pharmaceuticals A/S Methode de modulation de l'interaction d'un recepteur et d'un ligand
DE102006030366A1 (de) * 2006-06-27 2008-01-03 Universität Rostock Pharmazeutisches Mittel zur Behandlung des Parkinson-Syndroms und ein Verfahren zum Screening von Substanzen zur Verminderung der Parkinson-Symptomatik
EP2297325A4 (fr) * 2008-05-29 2011-10-05 Chum Méthodes de stratification, de pronostic et de diagnostic de la schizophrénie, molécules d acide nucléique mutantes et polypeptides
CN108004214B (zh) * 2017-11-29 2020-10-13 武汉大学 Homer2单克隆抗体及其应用
CN111141912A (zh) * 2020-03-02 2020-05-12 南通大学 Gabarap与optn结合在optn蓄积导致的神经元病变中的作用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997048724A2 (fr) * 1996-02-21 1997-12-24 Nps Pharmaceuticals, Inc. Nouveau recepteur humain du glutamate metabotrope
US5721107A (en) * 1990-12-12 1998-02-24 The Board Of Regents Of The University Of Washington Antibodies to G protein coupled glutamate receptors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5721107A (en) * 1990-12-12 1998-02-24 The Board Of Regents Of The University Of Washington Antibodies to G protein coupled glutamate receptors
WO1997048724A2 (fr) * 1996-02-21 1997-12-24 Nps Pharmaceuticals, Inc. Nouveau recepteur humain du glutamate metabotrope

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALLISON DANIEL W ET AL: "Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: Differential attachment of NMDA versus AMPA receptors" JOURNAL OF NEUROSCIENCE, vol. 18, no. 7, 1 April 1998 (1998-04-01), pages 2423-2436, XP002303914 ISSN: 0270-6474 *
BENEKEN JUTTA ET AL: "Structure of the Homer EVH1 domain-peptide complex reveals a new twist in polyproline recognition" NEURON, vol. 26, no. 1, April 2000 (2000-04), pages 143-154, XP002303917 ISSN: 0896-6273 *
BRAKEMAN P R ET AL: "HOMER: A PROTEIN THAT SELECTIVELY BINDS METABOTROPIC GLUTAMATE RECEPTORS" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 386, 20 March 1997 (1997-03-20), pages 284-288, XP002912002 ISSN: 0028-0836 *
See also references of WO0011204A2 *
TU JIAN CHENG ET AL: "Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors" NEURON, vol. 21, no. 4, October 1998 (1998-10), pages 717-726, XP002303916 ISSN: 0896-6273 *
XIAO BO ET AL: "Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of Homer-related, synaptic proteins" NEURON, vol. 21, no. 4, October 1998 (1998-10), pages 707-716, XP002303915 ISSN: 0896-6273 *

Also Published As

Publication number Publication date
AU772105B2 (en) 2004-04-08
EP1105734A4 (fr) 2005-04-13
AU5779899A (en) 2000-03-14
WO2000011204A2 (fr) 2000-03-02
WO2000011204A3 (fr) 2001-02-15
JP2002523056A (ja) 2002-07-30

Similar Documents

Publication Publication Date Title
US6864083B2 (en) Nucleic acid molecule encoding homer 2a protein
Fenster et al. Piccolo, a presynaptic zinc finger protein structurally related to bassoon
ES2298226T3 (es) Polipeptidos relacionados con wnt-1, y acidos nucleicos que los codifican.
JPH09502088A (ja) 細胞死レギュレータ
JP2010233570A (ja) Ｊｎｋ３調節物質およびその使用法
JP5053088B2 (ja) てんかんの診断および治療のための組成物ならびに方法
AU772105B2 (en) Homer interacting proteins
WO2001096359A1 (fr) Compositions, procedes et kits relatifs a des molecules du type resistine
US7217411B2 (en) CeSERT genes, proteins, and modulatory compounds
EP1272517B1 (fr) Matiere biologique et utilisations de celle-ci
EP1662255A2 (fr) Protéines interagissant avec Homer
GB2411403A (en) Transgenic animals with altered NCS-1 expression
CA2568201A1 (fr) Identification d'un transporteur d'ergothioneine et ses utilisations therapeutiques
WO1998041541A9 (fr) Utilisations therapeutiques de molecules de proteines grip et apparentees a grip
WO2003003013A1 (fr) Effets non nucleaires d'hormone thyroidienne
WO2000078921A2 (fr) Proteines de shank et leur procede d'utilisation
WO1999027088A2 (fr) Nouveaux gene et proteine exprimes dans les tissus nerveux et pancreatiques
KR20060129263A (ko) 분비된 신경 아폽토시스 억제 단백질
US20050233956A1 (en) Proteins involved in the regulation of energy homeostasis
WO2003027235A2 (fr) Sequences afap, polypeptides, anticorps et procedes associes
US20050119206A1 (en) Cg8327, cg10823, cg18418, cg15862, cg3768, cg11447 and cg16750 homologous proteins involved in the regulation of energy homeostasis
JP2005521424A (ja) 新規パンコルチン−パブロ蛋白相互作用およびそれらの使用方法
Krantz 7 Vesicular Neurotransmitter Transporters
US20040265918A1 (en) Non-nuclear effects of thyroid hormone
JP2005503793A (ja) 少なくとも視床下部、肝臓又は膵臓で発現する肥満に関する遺伝子

Legal Events

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

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010319

AK Designated contracting states

Kind code of ref document: A2

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

AX Request for extension of the european patent

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

RIC1 Information provided on ipc code assigned before grant

Ipc: 7C 07K 14/47 B

Ipc: 7C 12N 5/02 B

Ipc: 7C 12N 5/00 B

Ipc: 7G 01N 33/567 B

Ipc: 7G 01N 33/53 A

A4 Supplementary search report drawn up and despatched

Effective date: 20050224

17Q First examination report despatched

Effective date: 20050812

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

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

18D Application deemed to be withdrawn

Effective date: 20061214