MXPA01005030A - Peptides that modulate the interaction of b class ephrins and pdz domains - Google Patents

Abstract

The invention relates to complexes comprising a B class ephrin and a PDZ domain containing protein;peptides that interfere with the interaction of a B class ephrin with a PDZ domain binding site, and a PDZ domain containing protein;and, uses of the peptides and complexes. Methods for modulating the interaction of a B class ephrin and a PDZ domain containing protein, and methods for evaluating compounds for their ability to modulate the interaction are also described.

Description

PEPTIDES THAT MODULATE THE INTERACTION OF EFRINES OF CLASS B AND DOMAINS PDZ FIELD OF THE INVENTION The invention relates to complexes comprising an ephrin of class B with a binding site to the PDZ domain, and a protein containing the PDZ domain; peptides that interfere with the interaction of an ephrin of class B with a binding site to the PDZ domain, and a protein containing the PDZ domain; and, to the uses of peptides and complexes.

BACKGROUND OF THE INVENTION Among the large number of receptor tyrosine kinases (RTKs) identified in metazoan organisms, members of the Eph family are unusual in several aspects. Although only one Eph ERK that is encoded by the genome of Caenorhabdi tis elegans (the product of the vab-1 (2) gene) is known, vertebrates typically have up to 14 genes for Eph receptors, suggesting that these tyrosine kinases they can be important in the control of complex cellular interactions (3, 4). Consistent with this possibility, C. Elegans VAB-1 regulates Ref: 129789 morphogenetic cell motions during ventral closure in the embryo (2), while vertebrate Eph receptors have been implicated in the control of axon guidance and fasciculation, in the specification of topographic map formation within the nervous system central, in the organization of the movements of the cells of the neural crests during the development, in the direction of the fusion of the epithelial sheets in the closing of the palate, and in the angiogenesis (5-15). The first work on the expression patterns of EphB2 (formerly Nuk) suggested that this receptor accumulates in sites of the cell-cell junctions in the mesencephalon of the developing mouse and raised the possibility that Eph receptors can mediate the signals initiated by direct cell-cell interactions (5). Several lines of evidence support the notion that Eph receptors are normally activated by ligands that are physically associated with the surface of an adjacent cell. All known ligands for Eph receptors (called ephrins) are related in sequence, but can be divided into two groups based on their C-terminal portions. The class of E-ligands of ephrin become modified by a C-terminal glycosylphosphatidylinositol (GP1) portion, through which the ligand is anchored to the cell surface expressing the ligand (7, 9, 16). In contrast, type B ephrines possess a transmembrane element, and a highly conserved cytoplasmic tail, comprised of 82- 88 C-terminal residues (17-22). The Eph receptors can, in turn, be divided into subgroups A and B based on their sequential similarity and propensity to bind to soluble forms of either type A or type B ephrines, respectively (4, 23, 24). . However, although soluble ephrines are strongly bound to the relevant receptors, the consistent activation of the Eph tyrosine kinase activity requires either that the ligands are artificially accumulated in oligomers, or that the cells expressing the receptor are co-cultured with the cells that express the ephrins associated with the membranes (18). These data suggest that the ability of ephrins to aggregate and thereby activate Eph receptors depends on their coupling to the cell surface, consistent with the view that Eph receptor signaling involves cell-cell interactions. During embryonic development in the mouse, Eph receptors and their ligands are expressed in dynamic but complementary patterns, indicating that Eph receptors are probably activated at the boundaries where cells expressing Eph and Ephrin are directly juxtaposed to each other (23 , 25).

Genetic analysis of Eph receptor function in C. elegans and in the mouse has indicated that Eph receptors have kinase-dependent and kinase-independent signaling modes, and raise the possibility that Eph type B and ephrin receptors may mediate bidirectional cell-to-cell signaling (2, 6). Of interest, the binding of the Eph receptors to the ephrin Bl or ephrin B2 transmembranales, as well as the treatment of cells expressing ephrin B with platelet-derived growth factors (PDGF), leads to the phosphorylation of ephrins over the tyrosine residues within their highly conserved cytoplasmic tails (26, 27). In addition, expression of the cytoplasmic tail of a Xenopus ephrin B molecule leads to a surprising loss of cell adhesion in Xenopus embryos, an effect that is suppressed by treatment with fibroblast growth factor (28).

BRIEF DESCRIPTION OF THE INVENTION Ephrins of class B function as ligands for Eph receptor tyrosine kinases of class B and possess an intrinsic signaling function. The sequence at the carboxyl end of ephrines of type B it contains a PDZ binding site, providing a mechanism through which transmembrane ephrines interact with cytoplasmic proteins. A 10.5-day-old embryonic expression library was selected with a biotinylated peptide corresponding to the C-terminus of ephrin B3. Three of the positive cDNAs encoded the polypeptides with multiple PDZ domains, representing fragments of the GRIP molecule, the protein synthetin and PHIP, a novel protein containing the PDZ domain related to Caenorhabdi tis elegan ts PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library procedure (1) identified tyrosine phosphatase FAP-1 as a potential binding partner for Ephrin B. The in vitro studies demonstrated that the fifth PDZ domain of FAP-1 and full-length synthetin were linked to ephrin B via the C-terminal portion. In the end, the syntenin and ephrin Bl could be co-immunoprecipitated from transfected Cos-1 cells, indicating that the binding of the PDZ domain of ephrin B occurs in the cells. These results indicate that the C-terminal portion of ephrins B provides a binding site for specific proteins that contain the PDZ domain, which potentially localize transmembrane ligands to interactions with Eph receptors or participate in signaling within cells expressing ephrin B. Widely stated the present invention relates to a complex comprising an ephrin of class B and a protein containing the PDZ domain. The invention is also directed to a peptide derived from the PDZ binding domain of an ephrin of class B. The invention also contemplates antibodies specific for the complexes and peptides of the invention. The present invention also provides a method for modulating the interaction of an ephrin of class B and a protein containing the PDZ domain, comprising the administration of an effective amount of one or more of the following: (a) a complex comprising an ephrin of class B and a protein containing the PDZ domain; (b) a peptide derived from the PDZ binding domain of an ephrin of class B; or (c) the enhancers or inhibitors of the interaction of an ephrin of class B and a protein containing the PDZ domain. The invention further provides a method for identifying a substance that binds to a complex comprising an ephrin B of class B and a protein containing the PDZ domain comprising: (a) reacting the complex with at least one substance that can potentially link to the complex, under conditions that allow the binding of the substance and the complex; and (b) link detection, where the detection of the link indicates that the substance binds to the complex. The link can be detected by the evaluation of the substance-complex conjugates, or for the activation of ephrin B of class B or the protein containing the PDZ domain. The invention also contemplates methods for identifying substances that bind to other intracellular proteins that interact with the complexes of the invention. Still further, the invention provides a method for evaluating a compound for its ability to modulate the interaction of an ephrin of class B and a protein containing the PDZ domain. For example, a substance that inhibits or increases the interaction of the molecules in a complex of the invention, or a substance that binds to the molecules in a complex of the invention, can be evaluated. In one embodiment, the method comprises the provision of a complex of the invention, with a substance that binds to the complex, and a test complex under conditions that allow the formation of conjugates between the substance and the complex, and removal and / or detection of conjugates. In yet another embodiment, the method comprises the provision of an ephrin of class B and a protein that contains the PDZ domain, and a test compound under conditions that allow the binding of class B ephrin to the protein containing the PDZ domain; and (b) detection of the link, wherein detection of the linkage increased or decreased relative to the linkage in the absence of the test compound, indicates that the test compound modulates the interaction of a class B ephrin and a protein containing the PDZ domain. The present invention also contemplates a peptide of formula I that interferes with the interaction of an ephrin of class B and a protein containing the PDZ domain.

X-X1-X2-K-V wherein X represents 0 to 70, preferably 0 to 50, more preferably 2 to 20 amino acids, and X1 and X2 each represent tyrosine or phosphotyrosine. The invention also relates to analogs of the peptides of the invention. Furthermore, the invention relates to a method for modulating the interaction of an ephrin of class B and a protein containing the PDZ domain comprising the change of the terminal amino acid Val in an ephrin of class B.

The complexes, peptides and antibodies of the invention, and the substances and compounds identified using the methods of the invention, can be used to modulate the interaction of an ephrin of class B and a protein containing the PDZ domain, and these can be used to modulate cellular processes of cells associated with ephrines of class B and / or proteins containing the PDZ domain (such as proliferation, development and / or differentiation, in particular axonogenesis, nerve cell interactions and regeneration of the same) in which the compounds or substances are introduced. Accordingly, complexes, antibodies, peptides, substances and compounds can be formulated into compositions for administration to individuals suffering from disorders associated with a class B eryrin such as central nervous system disorders (e.g., neurodegenerative diseases and cases). of nerve damage). Therefore, the present invention also relates to a composition comprising one or more of a complex, peptide, or antibody of the invention, or a substance or compound identified using the methods of the invention, and a carrier, excipient or diluent pharmaceutically acceptable It is also provided a method to modulate proliferation, development and / or differentiation of cells associated with ephrines of class B and proteins that contain the PDZ domain, which comprises the introduction into cells, of a complex, peptide or antibody of the invention, a compound or substance identified using the methods of the invention or a composition containing the same. Methods for treating proliferative and / or differentiative disorders associated with ephrines of class B and / or proteins containing the PDZ domain using the compositions of the invention are also provided. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating the preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described with reference to the drawings in which: Figure 1 shows the amino acid sequence of the cytoplasmic domains of human ephrins B (SEQ ID Nos. 15, 16 and 17); Figure 2A shows a preferred linker sequence of FAP-1 PDZ5 (SEQ ID No. 18, 19 and 20) below a schematic representation of the protein FAP-1 tyrosine phosphatase, complete; Figure 2B are diagrammatic representations of the proteins containing the PDZ domain identified through an expression screen with a biotinylated peptide probe of the C-terminal sequence of ephrin B3; Figure 2C shows the alignment of the amino acid sequence of FAP-1 PDZ5 and the isolated PDZ domains in the expression screen (SEQ ID No. 21 to 27); Figure 2D shows the alignment of the amino acid sequence of PHIP (SEQ ID No. 1) and PAR-3 (SEQ ID No. 34); Figure 3A is a spot showing the binding of FAP-1 PDZ5 GST fusion proteins to ephrin Bl; Figure 3B is a spot showing the binding of FAP-1 PDZ5 fusion proteins to ephrin Bl; Figure 3C is a spot showing the binding of the GST proteins from synthetin to the effine Bl; Figure 3D is a spot showing the binding of the GST proteins from synthetin to the effine Bl; Figure 4A is a blot showing the blocking of FAP-1 PDZ5 binding to ephrin Bl by the addition of peptides corresponding to the C-terminal sequence of ephrin B; Figure 4B is a blot showing blockade of the syntenin to effine Bl binding by the addition of peptides corresponding to the C-terminal sequence of ephrins B; Figure 5A is a graph showing the fluorescence polarization analysis of GST-FAP-1 PDZ3 and GST-FAP-1 PDZ5 which binds fluorescein-labeled peptides, corresponding to the C-terminus of ephrin Bl; Figure 5B is a graph showing the fluorescence polarization analysis of the GST-syntheline linkage to the fluorescein-labeled peptides corresponding to the C-terminus of ephrin Bl; Figure 6 is a spot showing the co-immunoprecipitation of syn- tenin-FLAG with ephrin Bl; Figure 7 is a graph showing a fluorescence polarization analysis of GST-PHIP PDZ3 which binds to the fluorescein-labeled peptides corresponding to the C-terminus of ephrin Bl; and Figure 8 is an immunoblot showing that PHIP PDZ3 binds specifically to ephrin Bl phosphorylated with V-Src in mixtures of GST.

DETAILED DESCRIPTION OF THE INVENTION Definitions Unless indicated otherwise, all terms used herein have the same meaning as they would have for a person skilled in the art of the present invention. Practitioners are particularly directed to Current Protocols in Molecular Biology (Ansubel) for definitions and terms of technique. Abbreviations for amino acid residues are the standard 3-letter and / or single-letter codes used in the art to refer to one of the 20 common L-amino acids. Likewise, abbreviations for nucleic acids are the standard codes used in the art. "Antibody" refers to intact monoclonal or polyclonal molecules, and to immunologically active fragments (e.g., a Fab or (Fab) 2) a heavy chain of antibody and antibody light chain, a single chain Fv molecule engineered by genetic engineering (Ladner et al., U.S. Patent No. 4,946,778), or a chimeric antibody, for example, an antibody that contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies that include monoclonal and polyclonal antibodies, fragments and chimeras can be prepared using methods known to those skilled in the art. Antibodies that bind to a complex, or the peptide of the invention, can be prepared using peptides or intact fragments containing an immunization antigen of interest. The polypeptide or oligopeptide used to immunize an animal can be obtained from the translation of the RNA or chemically synthesized, and can be conjugated to a carrier protein, if desired. Suitable carriers that can be chemically coupled to the peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled peptide can then be used to immunize the animal (e.g., a mouse, a rat, or a rabbit).

"Effrin of class B" refers to a family of proteins that bind to Eph receptors and possess a transmembrane element, and a highly conserved cytoplasmic tail comprised of 82-88 C-terminal residues (17-22). Examples of ephrins of class B include ephrin Bl (also known as LERK-2, Elk-L, EFL-3, Cek-L, and STRA1), ephrin B2 (also known as Htk-L), ELF-2, LERK-5, and NLERK-1), and Ephrin B3 (also known as NLERK-2, Elk-L3, EFL-6, ELF-3, and LERK-8). The family also includes proteins with substantial sequential identity (eg homologs) and portions of the proteins (see for example SEQ ID No. 15, 16 or 17). Ephrines of class B used in the complexes and methods of the invention contain a binding domain that binds to a protein that contains the PDZ domain. The link domain contains the consensus sequence YYKV. The term "isolated", as used herein, refers to nucleic acid and amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and more preferably 90% free of other components with which these are naturally associated.

The term "modulated", as used herein, refers to a change or alteration in the biological activity of a protein. The modulation may be an increase or decrease in the activity of the protein, a change in the binding characteristics, or any other change in the biological, functional or immunological properties of a protein. The term "agonist" as used herein, refers to a molecule which when linked to a complex of the invention or a molecule in the complex, increases the amount of, or prolongs the duration of, the activity of a Ephrin of class B or the protein that contains the PDZ domain, or increases the formation of the complex. Agonists can include proteins, nucleic acids, carbohydrates, or any other molecules that bind to a complex or molecule of the complex. The agonists also include a peptide or peptide fragment derived from the PDZ binding domain of a class B ephrin but will not include the full length sequence of the wild-type molecule. Peptide mimetics, synthetic molecules with physical structures designed to mimic the structural characteristics of particular peptides, can serve as agonists. The stimulation can be direct, or indirect, or through a competitive or non-competitive mechanism.

The term "antagonist", as used herein, refers to a molecule which, when linked to a complex of the invention or to a molecule in the complex, decreases the amount of or duration of activity of an ephrin. of class B or a protein that contains the PDZ domain, or decreases complex formation. Antagonists also include a peptide or peptide fragment derived from the PDZ binding domain of a class B ephrin but will not include the full length sequence of the wild-type molecule. Peptide mimetics, synthetic molecules with physical structures designed to mimic the structural characteristics of particular peptides, can serve as antagonists. The stimulation can be direct, or indirect, or through a competitive or non-competitive mechanism. "Protein containing the PDZ domain" refers to proteins or peptides, or portions thereof that comprise or consist of a characteristic structural portion known as the PDZ domain. (See the database of the Structural Protein Classification (SCOP) for the characteristics of the domain). Examples of the proteins include GRIP, synthetin, and FAP-1, and homologs and portions thereof. Other proteins that contain the PDZ domains can be selected using public databases such as GENEPEPT and ENTREZ. The present inventors isolated a novel protein containing the PDZ domain designated "PHIP" as more particularly described herein. Examples of proteins containing the PDZ domain include GRIP, GRIP PDZ6 and PDZ7 of SEQ. ID. No. 22 and 23, FAP-1 PDZ5 of SEQ. ID. No. 21, amino acid residues 1 to 299 of the synthetin, PDZ1 and PDZ2 synthenin of SEQ. ID. No. 26 and 27, PHIP PDZ2 of SEQ. ID. No. 24, and PHIP PDZ3 of SEQ. ID. No. 25. A "binding domain" is that portion of the molecule in a complex of the invention that interacts directly or indirectly with another molecule in a complex of the invention. The binding domain can be a sequential portion of the molecule, for example a contiguous amino acid sequence, or it can be conformational, for example a combination of non-contiguous amino acid sequences which, when the molecule is in its native state, form a structure which interacts with another molecule in a complex of the invention. By being "derived from" a binding domain, is meant any molecular entity that is identical or substantially equivalent to the native binding domain of a molecule in a complex of the invention. A peptide derived from a specific binding domain may encompass the amino acid sequence of a naturally occurring binding site, any portion of that binding site, or another molecular entity that functions to bind to an associated molecule. A peptide derived from such a binding domain will interact directly or indirectly with an associated molecule in a manner such as to mimic the native binding domain. Such peptides may include competitive inhibitors, peptide mimetics, and the like. The term "interaction" refers to a stable association between two molecules due, for example, to electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions under physiological conditions. Certain interacting molecules interact only after one or more of them has been stimulated. For example, a protein containing the PDZ domain can only bind to a substrate, if the substrate is phosphorylated (for example phosphorylated). "Peptide mimetics" are structures that serve as substitutes for peptides in the interactions between molecules (See Morgan et al. (1989), Ann. Reports Med. Chem. 24: 243-252 for a review). Peptide mimetics include synthetic structures which may or may not contain amino acids and / or peptide bonds but which retain the structural and functional characteristics of a peptide, or agonist or antagonist of the invention. Peptide mimetics also include peptoids, oligopeptoides (Simón et al. 1972) Proc Nati. Acad. Sci USA 89: 9367); and peptide libraries containing peptides of a desired length representing all possible amino acid sequences corresponding to a peptide, or agonist or antagonist of the invention. The following terms are used to describe the sequential relationships between two or more nucleic acid molecules or proteins: "reference sequence", and "substantial sequence identity". A "reference sequence" is a defined sequence used as a basis for a sequential comparison; a reference sequence may be a subgroup of a larger sequence, eg, a segment of a full-length cDNA or a gene sequence given in a sequence listing, may comprise a complete cDNA or a gene sequence. The optimal alignment of the sequences for the alignment of a comparison window can be driven by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Match 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for the similarity method of Pearson and Lipman (1988) Proc. Nati Acad. Sci. (USA) 85: 2444, or through computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Résease 7.0, Genetics Computer Group, 575 Science Dr. Madison, Wis .; ClustalW program (55) and the Genestream Alignment Program). As applied to peptides, the term "substantial sequence identity" means that two peptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs using the default empty space, share at least 90% sequential identity, preferably at least 95 percent sequential identity, more preferably at least 99 percent sequential identity or more. Preferably, residual positions that are not identical differ by conservative amino acid substitutions. For example, substitution of amino acids that have similar chemical properties such as charge or polarity are not likely to affect the properties of a protein. Examples include glutamine by asparagine or glutamic acid by aspartic acid.

Complexes of the Invention The complexes of the invention comprise an ephrin protein of class B and a protein containing the PDZ domain. It will be appreciated that the complexes may comprise only the binding domains of the interaction molecules and other flanking sequences of this type are necessary to maintain the activity of the complexes. In an embodiment of the invention, the protein that contains the PDZ domain in the complex is GRIP, GRIP PDZ6 and PDZ7 of the SEQ. ID. No. 22 and 23, FAP-1 PDZ of the SEQ. ID. No. 21, the amino acid residues 1 to 299 of synthetin, PDZ1 and PDZ2 synthenin of SEQ. ID. No. 26 and 27, PHIP PDZ2 of SEQ. ID. No. 24, and PHIP PDZ3 of SEQ. ID. No. 25. Examples of complexes of the invention include ephrin B3 / GRIP; Ephrin B3 / GRIP PDZ6 and PDZ7 of SEQ. ID. No. 22 and 23; efrina Bl / FAP-1 PDZ of the SEQ. ID. No. 21; efrina Bl B3 / residues of amino acids 1 to 299 of synthetin; Ephrin Bl or B3 / Syntenyne PDZ1 and PDZ2 of SEQ. ID. No. 26 and 27; efrina Bl or B3 / PHIP PDZ2 of SEQ. ID. No. 24 and efrina Bl or B3 / PHIP PDZ3 of SEQ. ID. No. 25. The complexes may comprise a portion of class B ephrin, or a peptide of the invention. For example, the complex may comprise YYKV (SEQ ID No. 5), GPPQSPPNIpYYKV (SEQ ID.

No. 6), NipYpYKV (SEQ ID No. 7), NIpYYKV (SEQ ID No. 8), NIYpYKV (SEQ ID No. 9), NIYYKV (SEQ ID No. 10), GNYYYKV (SEQ ID No. 28), GNIpYpYKV (SEQ ID No. 29), GNIpYYKV (SEQ ID No. 30), and GNIYpYKV (SEQ ID No. 31). Examples of such complexes include FAP-1 PDZ / NIYYKV, synthetin / NIYYKV, synthetin PDZ1 and PDZ2 / NIYYKV, PHIP PDZ3 / GNYYYKV, and PHIP PDZ2 / GNYYYKV. As illustrated herein, ephrin of class B or a portion thereof, or peptide of the invention, in a complex of the invention, can be phosphorylated. Therefore, a complex of the invention comprising a protein containing the PDZ domain as a component, may comprise an ephrin of the phosphorylated class B or a portion thereof, or a phosphorylated peptide of the invention as another component. For example, the complex may comprise FAP-1 PDZ / NIpYYKV, FAP-1 PDZNIpYpYKV, syntinin / NIYYKV, syntinin / NIpYYKV, PDZ1 and PDZ2 / NIYYKV synthenin, PDZ1 and PDZ2 / NIpYYKV synthenin, PHIP PDZ3 / GNIpYpYKV and PHIP PDZ3 / GNIpYYKV . The invention also contemplates antibodies specific for the complexes of the invention. The antibodies can be intact monoclonal or polyclonal antibodies, and immunologically active fragments (eg, a Fab or (Fab) 2 fragment), an antibody heavy chain, and a light chain of antibody, a single chain Fv molecule engineered by genetic engineering (Lander et al., U.S. Patent No. 4,946,778), or a chimeric antibody, e.g., an antibody that contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies that include monoclonal and polyclonal antibodies, fragments and chimeras can be prepared using methods known to those skilled in the art. Antibodies specific for the complexes of the invention can be used to detect complexes in tissues, and to determine their tissue distribution. The methods of detection in vi tro and in si tu that use the antibodies of the invention, can be used to help in the prognostic and / or diagnostic evaluation of proliferative and / or differentiative disorders associated with an ephrin of the class B, for example disorders of the nervous system. Some genetic diseases may include mutations in the binding domain regions of the interaction molecules in the complexes of the invention. Therefore, if a complex of the invention is involved in a genetic disorder, it may be possible to use PCR to amplify the DNA from the binding domains for Quickly verify if a mutation is contained within one of the domains. The primers can be elaborated corresponding to the flanking regions of the domains and the standard sequencing methods can be used to determine if a mutation is present. This method does not require prior chromosomal mapping of the affected gene, and can save time by omitting sequencing of the entire gene encoding a defective protein.

PHIP protein Broadly stated, the present invention contemplates an isolated protein comprising the amino acid sequence shown in Figure 2D and SEQ. ID. No. 1. The invention contemplates a truncation (for example portion) of a protein of the invention, an analogue, an allelic or species variant thereof, or a protein having substantial sequence identity with a protein of the invention (eg. homologous example), or a truncation thereof. (Truncations, analogs, or allelic or species variations, and homologs are collectively referred to herein as "PHIP-related proteins").

The truncated proteins may comprise peptides of between 3 and 70 amino acid residues, in the size range of a tripeptide to a 70 mer polypeptide, preferably 12 to 20 amino acids. In an aspect of the invention, fragments of the PHIP protein are provided having an amino acid sequence of at least 5 consecutive amino acids in Figure 2D and in SEQ. ID. No. 1, wherein no amino acid sequence of five or more, six or more, seven or more, or eight or more consecutive amino acids present in the fragment is present in a protein other than a PHIP protein. In one embodiment of the invention, the fragment is a stretch of amino acid residues of at least 12 to 20 contiguous amino acids from a particular sequence such as a sequence underlined in Figure 2D. The fragments may be immunogenic and preferably are not immunoreactive with antibodies that are immunoreactive to proteins other than a PHIP protein. In one aspect of the invention, isolated nucleic acids (eg, SEQ ID No. 33, fragments thereof, complementary and homologous sequences), are provided comprising sequences encoding the PHIP protein or PHIP-related proteins.

The nucleic acids of the invention can be inserted into an appropriate vector, and the vector can contain the necessary elements for the transcription and translation of an inserted coding sequence. Accordingly, the vectors can be constructed, which comprise a nucleic acid of the invention, and where appropriate, one or more transcription or translation elements linked to the nucleic acid molecule. A vector of the invention can be used to prepare transformed host cells expressing a PHIP protein or a PHIP-related protein. Therefore, the invention further provides host cells that contain a vector of the invention. The invention also contemplates non-human, transgenic mammals whose germ cells and somatic cells contain a recombinant molecule comprising a nucleic acid molecule of the invention, in particular one that codes for an analog of a protein PHIP, or a truncation of a PHIP protein. A PHIP protein or PHIP-related protein can be obtained as an isolate from natural cell sources, but are preferably produced by recombination procedures. In one aspect of the invention, this provides a method for preparing a PHIP protein or a PHIP-related protein, using an isolated nucleic acid molecule of the invention. In one embodiment, there is provided a method for preparing a PHIP protein or a PHIP-related protein, comprising: (a) transferring a vector of the invention having a nucleotide sequence encoding a PHIP protein or protein related to PHIP, within a host cell; (b) selection of transformed host cells from non-transformed host cells; (c) the cultivation of a transformed host cell, selected, under conditions that allow the expression of the PHIP protein or the PHIP-related protein, and (d) the isolation of the PHIP protein or the PHIP-related protein. The invention further contemplates broadly a recombinant PHIP protein or PHIP-related protein obtained using a method of the invention. A PHIP protein or PHIP-related protein of the invention can be conjugated with other molecules, such as proteins, to prepare fusion proteins or chimeric proteins. This can be achieved, for example, by synthesis of N-terminal or C-terminal fusion proteins. The invention further contemplates antibodies that have specificity against an epitope of a PHIP protein or PHIP-related protein of the invention. The antibodies can be labeled with a detectable substance and used to detect proteins of the invention in tissues and cells. The invention also allows the construction of nucleotide probes that are unique to the nucleic acid molecules of the invention and consequently to the proteins of the invention. Therefore, the invention also relates to a probe comprising a nucleic acid sequence encoding a protein of the invention, or a portion thereof. The probe can be labeled, for example, with a detectable substance and this can be used to select from a mixture of nucleotide sequences, a nucleic acid molecule of the invention that includes the nucleic acid molecules that code for a protein showing one or more of the properties of a protein of the invention. The invention further provides a method for identifying a substance that binds to a protein of the invention, which comprises reacting the protein with at least one substance which can be linked potentially with the protein, under conditions that allow the binding of the substance and the protein; and link detection, where the detection of the link indicates that the substance binds to the protein. The link can be detected by evaluating the protein-substance complexes, or by the activation of the protein. The invention also contemplates methods for identifying substances that bind to other intracellular proteins that interact with a PHIP protein or a PHIP-related protein. Methods that identify compounds that bind to the gene regulatory sequences (eg, promoter sequences) can be used. Additionally, the invention provides a method for evaluating a compound for its ability to modulate the biological activity of a PHIP protein or a PHIP-related protein of the invention. For example, the compound may be a substance that binds to proteins or a substance that inhibits or enhances the interaction of the protein, and a substance that binds to the protein (e.g., an ephrin of class B). In one embodiment, the method comprises the provision of a PHIP protein or a PHIP-related protein, a substance that binds to the protein, and a test compound under conditions that allow the binding of the substance and protein, and linkage detection, wherein detection of the linkage increased or decreased relative to the linkage detected in the absence of the test compound, indicates that the test compound modulates the activity of a PHIP protein or a protein related to PHIP The link can be detected by evaluating the substance-protein complexes, the free substance, and / or the free protein, or the activation of the protein. The activation of PHIP or a PHIP-related Protein can be evaluated by measuring the phosphorylation of the protein, or the binding of the protein to cellular proteins, or by evaluating a biological effect on the cell, such as inhibition. or stimulation of proliferation, differentiation or migration. Compounds that modulate the biological activity of a protein of the invention can also be identified using the methods of the invention by comparing the pattern and level of expression of a PHIP protein or a PHIP-related Protein of the invention, in tissues and in cells, in the presence, and in the absence of the compounds. Substances and compounds identified using the methods of the invention can be used to modulate the biological activity of a PHIP protein or a PHIP-related Protein of the invention, and these can be used in the treatment of conditions that require the modulation of proteins or other molecules that bind to a PHIP protein or a PHIP-related protein (e.g. ephrin of class B).

Peptides The invention provides the peptide molecules that bind to and inhibit the interactions of the molecules in the complexes of the invention. The molecules are derived from the binding domain of an ephrin of class B that binds to a protein that contains the PDZ domain. For example, the peptides of the invention include the amino acids YYKV of ephrin Bl, B2 or B3 that bind to a protein containing the PDZ domain. Other proteins that contain these binding domain sequences can be identified with a protein homology search, for example by searching the available databases such as GenBank or SwissProt - and various search algorithms and / or programs can be used , including FASTA, BLAST (available as part of the sequence analysis package GCG, University of Wisconsin, Madison, Wis.), or ENTREZ (Center National for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD). According to one embodiment of the invention, specific peptides that mediate the binding of an ephrin of class B and a protein containing the PDZ domain are contemplated. In particular, a peptide of the formula I is provided, which interferes with the interaction of an ephrin of class B and a protein containing the PDZ domain: X-X1-X2-K-V wherein X represents 0 to 70, preferably 0 to 50 amino acids, more preferably 2 to 20 amino acids, and X1 and Z2 each represents tyrosine or phosphotyrosine. In specific embodiments, X1 is tyrosine and X2 is phosphotyrosine, X1 is phosphotyrosine and X2 is tyrosine, or X1 and X2 are phosphotyrosine. In one embodiment of the present invention, there is provided a peptide of formula I, wherein X represents NI, GNI, CPHYEKVSGDYGHPVYIVQ (E, D) (M, G) PPQSP (A, P) A (SEQ ID NO .: 2) , GDYGHPVYIVQ (ECD) (M, G) PPQSP (A, P) A (SEQ ID NO .: 3), PPQSP (A, P) A (SEQ ID NO .: 4), GPPQSPPNI (SEQ ID NO .: 32 ).

Preferred peptides of the invention include the following: YYKV (SEQ ID NO .: 5), GPPQSPPNIpYYKV (SEQ ID NO .: 6), NIpYpYKV (SEQ ID NO .: 7), NIpYYKV (SEQ ID NO .: 8), NIYpYKV (SEQ ID NO .: 9), NIYYKV (SEQ ID NO .: 10), GNYYYKV (SEQ ID NO .: 28, GNIpYpYKV (SEQ ID NO .: 29), GNIpYYKV (SEQ ID NO .: 30), and GNIYpYKV (SEQ ID NO: 31) All the peptides of the invention, as well as substantially homologous molecules, complementary or otherwise functionally or structurally equivalent to these peptides, may be used for purposes of the present invention. In addition to the full-length peptides of the invention, truncations of the peptides in the present invention are contemplated. The truncated peptides may comprise the peptides of about 7 to 10 amino acid residues. The truncated peptides may have an amino group (-NH2), a hydrophobic group (eg, carbobenzoxyl, dansyl or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxycarbonyl group (PMOC) or a macromolecule including but not it is limited to conjugates of lipid-fatty acid, polyethylene glycol, or carbohydrates at the amino-terminal end. The truncated peptides may have a carboxyl group, an amido group, a T-butyloxycarbonyl group, or a macromolecule including but not limited to lipid-acid conjugates fatty acid, polyethylene glycol, or carbohydrates at the carboxyl-terminal end. The peptides of the invention may also include analogs of a peptide of the invention and / or truncations of the peptide, which may include, but are not limited to the peptide of the invention containing one or more amino acid insertions, additions or deletions, or both of them. Analogs of the peptide of the invention show the characteristic activity of the peptide, for example, interference with the interaction of an ephrin of class B and a protein containing the PDZ domain, and may also possess additional advantageous characteristics such as increased bioavailability , increased stability or immune recognition of the host, reduced. One or more amino acid insertions can be introduced into a peptide of the invention. The amino acid insertions may consist of a single amino acid residue or sequential amino acids. One or more amino acids, preferably one to five amino acids, can be added to the left or right ends of a peptide of the invention. Deletions may consist of the removal of one or more amino acids, or discrete portions of the peptide sequence. The suppressed amino acids may or may not be contiguous. The length of the lower limit of the resulting analogue with a deletion mutation is about 7 amino acids. It is anticipated that if the amino acids are inserted or deleted in sequences outside of a NIX1X1KV sequence, that the resulting analog of the peptide will show the activity of a peptide of the invention. The invention also includes a peptide conjugated to a selected protein, or a selectable marker (see below) to produce fusion proteins. The peptides of the invention can be prepared by using recombinant DNA methods. Accordingly, the nucleic acid molecules encoding a peptide of the invention can be incorporated in a known manner into an appropriate expression vector, which ensures good expression of the peptide. Possible expression vectors include, but are not limited to, cosmids, plasmids or modified viruses, as long as the vector is compatible with the host cell used. The expression vectors contain a nucleic acid molecule that codes for a peptide of the invention and the regulatory sequences necessary for the transcription and translation of the inserted protein sequence. The appropriate regulatory sequences can be obtained from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes. (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA. The selection of appropriate regulatory sequences is dependent on the chosen host cell, and may be easily achieved by a person of ordinary skill in the art Other sequences, such as an origin of replication, additional DNA restriction sites, enhancers and sequences that confer inducibility of transcription, may also be incorporated within the expression vector. Recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells Suitable selectable marker genes are genes encoding proteins such as G418 or hygromycin, which confer resistance to other drugs, β-galactosidase, chloramphenicol-acetyltransferase , firefly luciferase, or an immunoglobulin or portion thereof such as the fc portion of an immunoglobulin, preferably IgG. Selectable markers can be introduced on a separate vector from the nucleic acid of interest.

Recombinant expression vectors may also contain genes encoding a fusion protein which provides increased expression of the recombinant peptide; increased solubility of the recombinant peptide; and / or assist in the purification of the recombinant peptide by acting as a ligand in affinity purification. For example, a proteolytic cleavage site can be inserted into the recombinant peptide to allow separation of the recombinant peptide from the fusion portion, after purification of the fusion protein. Examples of fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione-S-transferase (GST), maltose binding protein E, or protein A, respectively, to the recombinant protein. Recombinant expression vectors can be introduced into host cells to produce a transforming host cell. Transforming host cells include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention. The terms "transformed with", "transfected with", "transformation" and "transfection" are intended to include the introduction of nucleic acid (eg, a vector) into a cell by one of many techniques known in the art. For example, prokaryotic cells can be transformed with the nucleic acid by electroporation or calcium chloride mediated transformation. The nucleic acid can be introduced into mammalian cells using conventional techniques such as calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transformation and transfection of host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)), and other laboratory manuals. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the peptides of the invention can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods m Enzymology 185, Academic Press, San Diego, CA (1991).

The peptides of the invention can be phosphorylated tyrosine using the method described in Reedijk et al. (The EMBO Journal 11 (4): 1365, 1992). For example, tyrosine phosphorylation can be induced by infection of bacteria harboring a plasmid containing a nucleotide sequence encoding a peptide of the invention, with a bacteriophage? Gtll coding for the cytoplasmic tyrosine kinase domain Elk as a lacZ-Elk merger. The bacteria that contain the plasmid and the bacteriophage as a lysogen, are isolated. After induction of the lysogen, the expressed peptide is phosphorylated by the Elk tyrosine kinase. The peptides of the invention can be synthesized by conventional techniques. For example, peptides can be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid phase or solution phase syntheses (see for example, JM Stewart, and JD Young, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co., Rockford III (198r and G. Barany and RB Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solid-phase synthesis techniques, and M Bodansky, Principies of Peptide Synthesis , Springer-Verlag, Berlin 1984, and E. Gros and J.

Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biologu, supra, Vol. 1, for the classical synthesis in solution). By way of example, the peptides can be synthesized using the solid phase chemistry of 9-fluorenylmethoxycarbonyl (Fmoc) with direct phosphotyrosine incorporation as the N-fluorenylmethoxy-carbonyl-O-dimethylphosphono-L-tyrosine derivative. N-terminal or C-terminal fusion proteins comprising a peptide of the invention conjugated with other molecules, can be prepared by fusion, through recombinant techniques, of the N-terminus or the C-terminus of the peptide, and the sequence of a selected protein or selectable marker with a desired biological function. The resulting fusion proteins contain the peptide fused to the selected protein or marker protein, as described herein. Examples of proteins that can be used to prepare the fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc. The cyclic derivatives of the peptides of the invention are also part of the present invention. Cyclization may allow the peptide to assume a more favorable conformation for association with molecules in the complexes of the invention. The cyclization can be achieved using techniques known in the art. For example, disulfide bonds can be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond can be formed between an amino group of one component and a carboxyl group of another component. Cyclization can also be accomplished using an amino acid containing azobenzene, as described by Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The side chains of Tyr and Asn can be ligated to form cyclic peptides. The components that form the bonds can be amino acid side chains, non-amino acid components or a combination of the two. In one embodiment of the invention, cyclic peptides having a beta turn in the right position are contemplated. Beta turns can be introduced into the peptides of the invention by the addition of Pro-Gly amino acids in the right position. It may be desirable to produce a cyclic peptide that is more flexible than cyclic peptides containing peptide bonds, as described above. A more flexible peptide can be prepared by introducing cysteines in the right and left position of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are arranged for Do not deform the beta sheet and turn over. The peptide is more flexible as a result of the length of the disulfide bond and the smaller number of hydrogen bonds in the beta sheet portion. The relative flexibility of a cyclic peptide can be determined by molecular dynamic simulations. Peptide mimetics can be designed based on the information obtained by the systematic replacement of the L-amino acids by the D-amino acids, the replacement of the side chains with groups having different electronic properties, or by systematic replacement of the peptide bonds with Amide bond replacements. Local conformational constraints may also be introduced to determine the conformational requirements for the activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote the reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogs can be used to constrain amino acid residues to particular conformational states. The mimetics may also include mimetics of inhibitory peptide secondary structures. These structures can model the three-dimensional orientation of the amino acid residues in the known secondary conformation of the proteins.

The peptoids can also be used, which are oligomers of N-substituted amino acids and can be used as portions for the generation of chemically diverse libraries of novel molecules. Peptides that interact with the molecules in a complex of the invention can be developed using a biological expression system. The use of these systems allows the production of large libraries of random peptide sequences and the selection of these libraries for peptide sequences that bind to particular proteins. Libraries can be produced by cloning synthetic DNA encoding the random peptide sequences within the appropriate expression vectors. (See Christian et al., 1992, J. Mol. Biol. 227: 711; Devlin et al., 1990 Science 249: 404; Cwirla et al., 1990, Proc. Nati, Acad. Sci. USA, 87: 6378). Libraries can also be constructed by concurrent synthesis of overlapping peptides (see U.S. Patent No. 4,708,871). The peptides of the invention can be used to identify the guide compounds for drug development. The structure of the peptides described herein can be easily determined by a number of methods such as Nuclear Magnetic Resonance (NMR) and X-ray crystallography. A comparison of peptide structures similar in sequence, but different in the biological activities that they promote in target molecules, can provide information regarding the structure-activity of the target. The information obtained from the examination of structure-activity relationships can be used to design either modified peptides, or other small molecules or guide compounds that can be tested for the properties predicted in relation to the target molecule. The activity of the guide compounds can be evaluated using assays similar to those described herein. Information regarding structure-activity relationships can also be obtained from co-crystallization studies. In these studies, a peptide with a desired activity is crystallized in association with a target molecule, and the x-ray structure of the complex is determined. The structure can then be compared to the structure of the target molecule in its native state, and information from such a comparison can be used to design compounds that are expected to possess the desired activity. The peptides of the invention can be converted to pharmaceutical salts by reaction with inorganic acids such as hydrochloric acid, acid sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, acid benzoic acid, salicylic acid, benzenesulfonic acid, and toluenesulfonic acids. The peptides of the invention can be used to prepare antibodies. Conventional methods can be used to prepare the antibodies. Peptides and antibodies specific for the peptides of the invention can be labeled using conventional methods with various enzymes, fluorescent materials, luminescent materials and radioactive materials. Suitable enzymes, fluorescent materials, luminescent materials, and radioactive material are well known to a person skilled in the art. Antibodies and labeled antibodies specific for the peptides of the invention can be used to select the proteins that contain the PDZ domain binding sites. Computer modeling techniques known in the art can also be used to observe the interaction of a peptide of the invention, and truncations and analogs thereof with a molecule in a complex of the invention, for example a protein containing the PDZ domain (for example, Homology Insight II and Discovery available from BioSym / Molecular Simulations, San Diego, California, USA) . If computer modeling indicates a strong interaction, the peptide can be synthesized and tested for its ability to interfere with the binding of the molecules of a complex discussed in the present. , Methods to Identify or Evaluate Substances / Compounds The methods described herein are designed to identify substances and compounds that modulate the activity of a complex of the invention which thus potentially affects cellular processes associated with ephrines of class B and / or proteins containing the PDZ domain. . They are therefore contemplated the "novel substances that bind to molecules in complexes, or bind to other proteins that interact with molecules, to compounds that interfere with, or improve the interaction of molecules in a complex, or other proteins that interact with molecules.

Substances and compounds identified using the methods of the invention include but are not limited to peptides such as soluble peptides that include Ig-tail fusion peptides, members of random peptide libraries and molecular libraries derived from combinatorial chemistry., made of amino acids of the d- and / or L- configuration, phosphopeptides (including members of libraries of 'directed, random or partially degenerated phosphopeptides'), antibodies [eg, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single strand, fragments, (eg, Fab, F (ab) 2, and Fab expression library fragments, and epitope-binding fragments thereof)], and small organic and inorganic molecules. The substance or compound can be an endogenous physiological compound or it can be a natural or synthetic compound. Substances that modulate the activity of a complex of the invention can be identified based on their ability to bind to a molecule in the complex. Therefore, the invention also provides methods to identify the novel substances that bind to the molecules in the complex. The substances identified using the methods of the invention can be isolated, cloned or sequenced using conventional techniques. The novel substances that can be linked to a molecule in a complex of the invention can be identified by reacting one of the molecules with a test substance which potentially binds to the molecule, under conditions that allow the molecule to be linked and the test substance, and the detection of the link. The link can be detected by the assay for the substance-molecule conjugates, for the free substance, or for the non-complexed molecules, or the activation of the molecule. The conditions that allow the formation of the substance-molecule conjugates can be selected with respect to factors such as the nature and the amounts of the substance and the molecule. The substance-molecule conjugate, the substance-free and non-complexed molecules can be isolated by conventional isolation techniques, for example, by saline displacement, chromatography, electroporesis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination or combinations thereof. To facilitate the testing or evaluation of the components, the antibody against the molecule or the substance, or the labeled molecule, or a marked substance. The antibodies, proteins or substances can be labeled with a detectable substance as described above. Activation can be assessed by measuring the phosphorylation of a molecule, the binding of cellular receptors or proteins to a molecule, or in a cellular assay, by evaluating a biological effect on the cell, such as inhibition or stimulation of proliferation, differentiation or migration. A molecule or complex of the invention, or the substance used in the method of the invention can be insolubilized. For example, a molecule or substance can be linked to a suitable carrier such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethylcellulose, polystyrene, filter paper, ion exchange resin, plastic film, plastic tube, glass spheres, copolymer of polyamine-methylvinyl ether-maleic acid, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier can be in the form of, for example, a tube, test plate, spheres, disc, etc. The protein or insolubilized substance can be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, coupling with cyanogen bromide. The invention also contemplates a method for evaluating a compound for its ability to modulate the biological activity of a complex of the invention, by assaying for an agonist or antagonist of the binding of the molecules in the complex. The basic method for the evaluation of whether a compound is an agonist or antagonist of the binding of the molecules in a complex of the invention, is to prepare a reaction mixture containing the molecules and the test compound, under conditions that allow the molecules they bind and form a complex. The test compound can be initially added to the mixture, or it can be added subsequent to the addition of the molecules. Control reaction mixtures without the test compound or as a placebo are also prepared. The formation of the compounds is detected and the formation of the complexes in the control reaction but not in the reaction mixture indicates that the test compound interferes with the interaction of the molecules. The increased formation of the complex relative to a control reaction indicates that the test compound increases the interaction of the molecules. The reactions can be carried out in the liquid phase or the molecules, or the test compound can be immobilized as described herein. It will be understood that agonists and antagonists that can be evaluated using the methods of the invention can act on one or more of the binding sites on the interaction molecules in the complex including the agonist binding sites., binding sites to the competitive antagonist, non-competitive antagonist binding sites or allosteric sites. The invention makes it possible to select antagonists that inhibit the effects of an agonist from the interaction of molecules in a complex of the invention. Thus, the invention can be used to evaluate a compound competing for the same binding site of a molecule in a complex of the invention. The invention also contemplates methods for identifying novel compounds that bind to proteins that interact with a molecule of a complex of the invention. Protein-protein interactions can be identified using conventional methods such as co-immunoprecipitation, cross-linking and co-purification, through gradients or chromatographic columns. Methods that result in the simultaneous identification of the genes encoding the proteins that can be used can also be employed. interact with a molecule. These methods include probing the expression libraries with labeled molecules. Additionally, studies of X-ray crystallography can be used as a means to evaluate interactions with substances or molecules. For example, recombinant molecules purified in a complex of the invention when crystallized in a suitable form, are available for the detection of intramolecular interactions by X-ray crystallography. Spectroscopy can also be used to detect interactions and in particular can be used to detect interactions. A hybrid quadrupole / time of flight instrument (QqTOF) will be used. Double hybrid systems can also be used to detect protein interactions in vivo. In general, the plasmids encoding two hybrid proteins are constructed. A first hybrid protein consists of a DNA binding domain of a transcription activating protein, fused to a molecule in a complex of the invention, and the second hybrid protein consists of the activating domain of the transcription activating protein, fused to an unknown protein, encoded by a cDNA that has been recombined within the plasmid as part of a cDNA library. Plasmids are transformed into a yeast strain (e.g., S. cerevisiae) that contains a reporter gene (for example, lacZ, luciferase, alkaline phosphatase, horseradish peroxidase) whose regulatory region contains the binding site to the transcription activator. Hybrid proteins alone can not activate transcription of the reporter gene. however, the interaction of the two hybrid proteins reconstitutes the functional activator protein and results in the expression of the reporter gene, which is detected by an assay for the reporter gene product. It will be appreciated that fusion proteins and recombinant fusion proteins can be used in the methods described above. It will also be appreciated that the complexes of the invention can be reconstituted in vi tro using recombinant molecules and the effect of a test substance can be evaluated in the reconstituted system. Suitable reagents for the application of the methods of the invention for evaluating substances and compounds can be packaged in convenient equipment by providing the necessary materials packaged within suitable containers. The equipment may also include suitable supports useful in carrying out the methods of the invention.

Compositions and Treatments The complexes, peptides and antibodies of the invention, and the substances and compounds identified using the methods of the invention can be used to modulate cellular processes such as proliferation, development and / or differentiation of cells associated with ephrines of class B and / or proteins that contain the PDZ domain (in particular axonogenesis, nerve cell interactions and nervous system regeneration). Therefore, these can be used to treat conditions in a subject in which compounds or substances are introduced. In this way, the substances can be used for the treatment of disorders associated with a class B eryrin such as nervous system disorders including neurodegenerative diseases and cases of nerve damage. Accordingly, the complexes, peptides, substances, antibodies and compounds can be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible formula, suitable for in vivo administration. By "biologically compatible form suitable for administration in vivo" is meant a form of the substance to be administered, in which any toxic effects are overcome by the therapeutic effects. The substances can be administered to living organisms including humans and animals. The administration of a "therapeutically effective amount" of the pharmaceutical compositions of the present invention is defined as an effective amount, at doses and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a substance can vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the antibody to promote a desired response in the individual. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active substance can be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance can be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions that can inactivate the compound.

The compositions described herein can be prepared by methods known per se for the preparation of pharmaceutically acceptable compositions that can be administered to the subject, such that an effective amount of the active substance is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the compositions include, but are not limited to, solutions in the substances or compounds in association with one or more pharmaceutically acceptable carriers or diluents, and contained in buffered solutions, with a suitable pH and iso-osmotic with the physiological fluids. The activity of the complexes. Substances, compounds, antibodies and compositions of the invention can be confirmed in experimental model systems, with animals. The invention also provides methods for studying the function of a complex of the invention. Non-human cells, tissues and animals that lack the complexes or that partially lack the molecules in the complexes, can be developing recombinant expression vectors of the invention that have deletion or insertion mutations, specific, in the molecules. A recombinant expression vector can be used to activate or alter the endogenous gene by homologous recombination, and thereby create cells, tissues or animals deficient in the complex. Null alleles can be generated in cells and can then be used to generate non-human transgenic animals. The following non-limiting example is illustrative of the invention: Example EXPERIMENTAL PROCEDURES Synthesis of peptides A C-terminal ephrin B peptide probe of the biotin-Aca sequence-GPPQSPPNIpYYKV (SEQ ID NO: 6), the related peptides NIpYpYKV (SEQ ID NO .: 7), NIpYYKV (SEQ ID NO .: 8), NIYpYKV (SEQ ID NO .: 9), NIYYKV (SEQ ID NO .: 10), and DHQpYND (SEQ ID NO .: 11), were synthesized as previously described (29).

Isolation of the cDNA clones that code for the PDZ domain A 10.5 day mouse embryo expression library,? EXlox (Novagen) was plated at an initial density of 10,000 15 cm petri dish / plate forming units. The selection of the library was performed using a biotinylated peptide probe conjugated to streptavidin-alkaline phosphatase following a procedure similar to that described by Sparks et al. (30) To isolate more coding sequence for PHIP, an EcoR1 / Pst1 fragment of PHIP cDNA (coding for amino acid residues 462-602) was radiolabelled with [a32P] dCTP and used to select the 10.5 mouse embryo library. days? Exlox. DNA sequencing of the positive clones was carried out using the automatic ALF DNA sequencer (Amersham Pharmacia Biotech).

Antibodies, constructions and mutagenesis Anti-ligand antibodies (Santa Cruz) were produced against residues 329-346 of effine Bl.

Anti-FLAG M2 monoclonal antibodies were purchased from Eastman Kodak Company. The expression construction of The cDNA of the Bl in the pJE14 vector has been described (18). The full length synthetin cDNA was intrastructurally subcloned into the mammalian expression vector pFLAG M2 (Eastman Kodak) using standard cloning procedures. For the GST ususe constructs, the synthenin cDNA sequences (full length: residues 1-299; PDZ 1 + 2: residues 101-299; PDZ1: residues 101-211; PDZ2: residues 172-299) were cloned into pGEX4T2 (Amersham Pharmacia Biotech). FAP-1 (the atase associated with Fas) PDZ3 and FAP-1 PDZ5 have also been described (1). The mutation by suppression of the kidney of the kidney was constructed by eliminating the nucleotides coding for the C-terminal V346 using a protocol mediated by PCR. The PpuMI / EcoRI PCR fragment possessing the mutated region was subcloned into the cDNA of the full-length Bl in pJFE14. This mutation and all the constructions of use were signed by sequencing both strands of the region to ectada.

Immunoprecipitation and Western blot analysis Cos-1 cells were maintained in DMEM supplemented with 10% bovine serum serum (FBS). Transient transitions were made using the lipofectin reagent and Opti-MEM medium (Life Technologies Inc.) as described by the manufacturer. To reduce phosphorylation of ephrin Bl by binding to endogenously expressed EphB receptors, or by stimulation with serum growth factors, transfected cells were transferred from 10 cm plates to 15 cm 24 hours after transfection and deprived of serum in 0.5% FBS with DMEM, 12 hours before cell lysis. The transfected cells were rinsed once in PBSA and used in the PLC lysis buffer (5) with 10 μg / ml of aprotinin, 10 μg / ml of leupeptin, 1 mM sodium vanadate and 1 mM phenylmethylsulfonyl fluoride added. The immunoprecipitations were performed for one hour at 4 ° C using 1 μg of anti-ephrin antibody Bl or 1 μg of anti-IL-3 receptor antibody with protein A-sepharose. The GST mixing experiments were carried out by incubation of 1 hour at 4 ° C of the lysate with 5-10 μg of the immobilized fusion protein on glutathione-sepharose. For the peptide competition experiments, the peptides were included in the incubation with the GST fusion proteins at a final concentration of 100 μM. The spheres for the immunoprecipitations and the GST mixing experiments were washed 2 to 3 times in HNTG buffer (5). The proteins were separated by SDS-PAGE to %, transferred to the Immobilon-P membrane (Millipore) and subjected to immunoblotting with the appropriate antibody. The spots were revealed by Augmented Chemiluminescence (Pierce).

Fluorescence Polarization Analysis The determination of the binding constant and peptide proficiency studies were carried out using fluorescence polarization on a Beacon 2000 Fluorescence Polarization System (Pan Vera, Wl) equipped with a 100 μl sample chamber. The fluorescein-labeled probes were prepared through the reaction of the C-terminal peptides of ephrin B with 5- (y-6) -carboxyfluorescein, succinimidyl ester (Molecular Probes, OR) and purified by high performance liquid chromatography. resolution (HPLC) of reverse phase. The authenticity of the fluorescein-labeled peptides was confirmed by mass spectroscopy. In the binding studies, the fluorescein-labeled peptide probe was dissolved in 20 mM phosphate pH 7.0, 100 mM sodium chloride, and 2 mM DTT at a concentration of 25 nM and a known amount of the fusion protein was added to GST The reaction mixtures were allowed to stand for 10 minutes at room temperature before of each measurement. All fluorescence polarization measurements were conducted at 22 ° C.

RESULTS Identification of potential link partners for the PDZ link site, putative of the Ephrin B As a procedure towards the identification of proteins that interact with cytoplasmic tails of type B ephrines, the C-terminal regions of transmembrane ephrines were initially examined for conserved peptide portions that can bind to the modular domains of signaling proteins intracellular The carboxyl terminus of the three known Ephrins B has a conserved sequence reminiscent of known or predicted binding sites for the PDZ domains (Figure 1). Two strategies were used to identify proteins containing the PDZ domain with the potential to recognize ephrines B. Firstly, the comparison of the known binding specificities of PDZ domains, predicted through the use of a peptide library technique oriented, revealed the fifth PDZ domain of cytoplasmic tyrosine phosphatase FAP-1 (Fas-associated phosphatase) as a possible link partner to ephrin B (Figure 2A). FAP-1 (also known as PTP-bas and PTP-L1) has at least six PDZ domains, an element related to the cytoskeletal polypeptide Band 4.1, and a C-terminal tyrosine-phosphatase domain (31-33). The fifth PDZ domain binds in vi tro to the peptides with the consensus E- (I / Y / V) -Y (Y / K) - (V / L / I), which closely matches the conserved C-terminus of ephrines type B (YYKV) (1). A more direct procedure to isolate ephrin B binding proteins was undertaken by selecting a cDNA expression library from a 10.5 day mouse embryo with a peptide probe based on the putative PDZ domain binding site of Ephrin B3. The probe was a biotinylated peptide, biotin-Aca-GPPQSPPNIpYYKV (SEQ ID NO: 6), conjugated to streptavidin-alkaline phosphatase. Although this peptide contained a phosphotyrosine residue in the -3 position relative to the C-terminal valine, it was anticipated that the alkaline phosphatase used in the selection could at least partially dephosphorylate the probe, allowing the detection of the link dependent and independent of phosphorylation. of tyrosine. The selection of approximately 500,000 cDNA clones produced four distinct cDNA products that bound to the C-terminal peptide of ephrin B3, of which three were subsequently found as possessors of the PDZ domains after the sequential analysis (Figures 2b and c) One of these cDNAs codes for a portion of the GRIP adapter protein, from the sixth PDZ domain to the carboxyl terminus (amino acid residue 642-1112 ) GRIP is a protein of approximately 180 kDa composed of seven PDZ domains, originally identified by its ability to bind to the C-terminus of AMPA receptors through domains 4 and 5 of PDZ (34). procedure contained the complete coding sequence for the syn- tenin protein containing the PDZ domain.Syn- tenin was first reported as a sub-transcribed transcript during melanoma differentiation (termed MDA-9), and it was subsequently shown to interact via its two domains PDZ with the C-terminus of transmembrane syndecan proteins (35,36) A third clone identified in this selection was a partial cDNA coding for the carboxyl-terminal fragment of a novel protein containing the PDZ domain (referred to as PHIP for abbreviating protein for interaction with ephrin). Sequence analysis of the PHIP cDNA fragment revealed the presence of two adjacent PDZ domains followed by a C-terminal stretch of 50 amino acids. The PHIP cDNA fragment was subsequently used as a probe to isolate a transcribed from a mouse embryo library of 10.5 days. The predicted PHIP sequence indicates that it codes for a total of three PDZ domains and is closely related to PAR-3, a protein of C. elegans involved in the regulation of early embryo polarity (Figure 2D) (37). Of these candidates, FAP-1 PDZ5 and synthetin were subsequently investigated for binding to ephrins B.

Sintenin and FAP-1 PDZ bind to effine Bl in vitro To determine whether the syntenin or FAP-1 could interact with Bl in vi tro ephrin, the GST fusions containing the fifth PDZ domain of FAP-1 or the full-length synthetin were incubated with the lysates of the Cos-1 cells transfected with the efrina Bl. Recovery of these immobilized GST fusion proteins and immunoblotting of the proteins associated with anti-ephrin antibody Bl revealed that FAP-1 PDZ5 and full-length synthetin were available to bind to intact efrin BL (Figures 3A and 3C) . The region of the synthetin required for the binding to the effine Bl was mapped using GST fusions containing the defined fragments of the protein synthetin. The minimum sequence necessary for a Strong interaction included both PDZ domains of the synthetin, but not the amino-terminal third of the protein (Figure 3D). Interestingly, both PDZ domains of the synthetin are also required for the link to the C-terminal sequence of the syndecans, suggesting that the involvement of two PDZ domains at the link of a single target site may be a common feature of the interactions. of synthetin (36). While the PDZl domain of synthetin alone was unable to associate with an efrin Bl, the second PDZ of the synthetin alone, showed a very weak interaction. In these experiments, neither GST alone nor a fusion of GST with the third PDZ domain of FAP-1 showed detectable link to the efrin Bl. The identity of the approximately 40 kD band recognized by GST-FAP-1 PDZ3 is not known but its apparent size does not co-relate to any of the three known ephines b. Consistent with this finding, the binding specificity of FAP-1 PDZ3, as previously described using an oriented peptide library, is significantly different from that of FAP-1 PDZ5, with a preference toward target sequences such as the QSLV-COOH moiety. in the Fas antigen (1.33). The inability of the FAP-1 PDZ3 domain to bind to Bl ephrin indicates a degree of specificity in Bl recognition by PDZ domains. A notable mark of many PDZ domain binding sites is a requirement for a C-terminal hydrophobic residue that makes contact with the PDZ domain through its side chain and the C-terminal carboxylate group (1,38,39). The involvement of the Val C-terminal efrin Bl in the specific link to the synthetin and FAP-1 PDZ5 was initially evaluated by the expression of a deletion mutant of the efrin Bl that lacks the Val terminal residue in the Cos-cells. 1. Removal of C-terminal Val from full-length efrin Bl abrogated its binding to the synthetin and FAP-1 PDZ5 GST fusion proteins (Figures 3B and 3C). As an alternative procedure towards investigating the specificity of ephrin Bl interactions with PDZ domain proteins, a specific peptide modeled on the C-terminus of ephrin type B was used in the competition experiments. For this purpose, lysates from cells transfected with ephrin Bl were incubated with either GST-synthetin or GST-FAP-1 PDZ5 in the presence or absence of a corresponding peptide in sequence at the six C-terminal residues of ephrins B. The peptide successfully blocked the syntenin and the binding of FAP-1-PDZ5 to a peptide concentration of 100 μM (Figures 4A and 4B). The addition of the unrelated peptide, DHQpYpYND (SEQ ID NO: 11), did not decrease the binding, indicating the specificity of the peptide competence (Figure 4A).

FAP-1 PDZ5 and synthetin show differential binding to phosphopeptides The binding of ephrins B to their cognate Eph B receptors, the expression of an activated Src tyrosine kinase or the treatment of cells expressing the ligand with PDGF results in the tyrosine phosphorylation of residues in the cytoplasmic domain of Ephrin (26.27). Preliminary evidence based on specific substitutions of the Tyr residues in the effine Bl tail indicates that the two tyrosines at positions -2 and -3 within the PDZ domain binding site are between the phosphorylation sites. To investigate whether the tyrosine phosphorylation of these residues can affect the binding to the PDZ domain, the C-terminal peptide used for the competence of the peptide described above was also synthesized such that one or both of the tyrosine residues -2 and -3 They were phosphorylated. The phosphorylated and non-phosphorylated peptides were labeled with fluorescein and used in the polarization experiments of fluorescence to obtain quantitative measurements of their affinities for the PDZ domains of FAP-1 and of synthetin. GST-FAP-1 PDZ5 linked to a NIYYKV peptide labeled with fluorescein (SEQ ID NO: 10) with an affinity of 9.9 ± 1.0 μM, whereas the binding of GST-FAP-1 PDZ3 was much weaker (65.0 ± 9.6 μM) (Figure 5a). This is consistent with the GST mixing experiments that indicated that FAP-1 PDZ3 does not interact stably with the Bl ephrin. Similar results were obtained when the linkage to the three different phosphorylated peptides was investigated, indicating that the alternative tyrosine phosphorylation states of the C-terminal sequence of ephrin B had little effect on the binding of GST-FAP-1 PDZ5. Similar binding affinity values of 6.8 ± 0.8 μM, 15.4 ± 3.4 μM and 8.412.5 μM were obtained for the peptides NIpYYKV, NIYpYKV and NIpYpYKV (SEQ ID Nos .: 8, 9 and 7 respectively), respectively. Fluorescence polarization experiments measuring the GST-synthetin fusion protein that binds to NIYYKV peptides labeled with fluorescein (SEQ ID NO: 10) and NIpYYKV (SEQ ID NO .: 8) produced almost identical binding curves (Figure 5B). Affinity values of 17.7 ± 1.2 μM and 15.4 ± 0.5 μM were obtained, indicating that tyrosine phosphorylation at position -3 did not significantly affect the PDZ-domain interaction.

However, the GST-synthetin fusion protein was linked to the peptide pYpYKV (SEQ ID NO .: 12) with a much lower affinity of 151.0 ± 20.9 μM, indicating that the phosphorylation in Tyr -2 can have a damaging effect on the binding to the synthetin. A similar low affinity interaction was observed for the YpYKV peptide.

Ephrin Bl and synthetin can associate in cells The possibility that type B ephrins could interact with PDZ domain proteins was achieved by testing whether Ephrin Bl and synthetin are associated when co-expressed in Cos-1 cells. In cells co-transfected with ephrin Bl and synthetin (labeled at the N-terminus with a FLAG epitope) the immunoprecipitation of ephrin Bl co-specifically precipitated the synthetin (Figure 6). Precipitation with protein A sepharose alone or with an arbitrarily chosen antibody did not produce detectable synthetin, indicating that the interaction is specific. In addition, co-immunoprecipitation experiments with the Val suppression mutant of Bl ephrin Bl, which fails to interact with the PDZ domains in vi tro, showed that Bl ephrin lacking Val C-terminal was not detectably associated with synthetin (Figure 6). While the truncated protein could be successfully immunoprecipitated by antibodies against ephrin Bl, the synthetin can not be co-immunoprecipitated with the mutant protein. These results demonstrate that Bl ephrin and synthetin can associate in cells, and show that an intact PDZ domain binding site to Bl ephrin Bl is necessary for its interaction with in vivo synthetin.

DISCUSSION In an effort to identify components of the cytoplasmic domain that may contribute to the function of ephrin B, it was shown that the C-terminal residues of ephrin B constitute a binding site for the PDZ domains, a class of protein module that is knows is a mediator of specific protein-protein interactions. Several lines of evidence indicate that the C-terminal YYKV sequence, conserved among the three known Ephrins B, represents a binding site to the PDZ domain. First, a biotinylated peptide probe with a sequence corresponding to the C-terminal residue of ephrin B3 identified the cDNAs encoding the known proteins synthetin and GRIP containing the PDZ domain, as well as a cDNA for PHIP, a novel protein containing the domain of PDZ. In addition, a Fourth protein containing PDZ, FAP-1, was identified as a binding candidate based initially on the predicted binding specificity of its fifth PDZ domain. Second, in vitro studies with syntenin and FAP-1 have demonstrated the specific interactions of the PDZ domains of these proteins with the C-terminus of the efrin Bl. The finding that the Val C-terminal effine Bl residue is absolutely required for these interactions, indicates that the binding occurs in a manner characteristic of other interactions of the PDZ domain with the C-terminal target sequences. Similar results were also obtained from in vitro binding experiments with ephrin B2, suggesting that PDZ domain interactions may be common to all Ephrin B. In vi tro experiments were also performed with GST fusions separated from GRIP PDZ6 and GRP PDZ7. Interactions with ephrin Bl or with the fluorescent GNYYYKV peptide (SEQ ID NO: 13) were not detected in fluorescence polarization and GST mixture experiments. The finding to effine Bl may require PDZ6 and PDZ7 of GRIP in a manner reminiscent of the requirement of the PDZ domains of syntenin for the link. In the end, it was shown that PDZ domain interactions with ephrin B can occur in vi ve, since the synthetin can be successfully co-immunoprecipitated with the full-length Bl ephrin but not with the effine BL truncated at its PDZ domain target site. The effect of the phosphorylation status of two adjacent tyrosines at positions -2 and -3 relative to the C-terminal Val of the target site of the PDZ domain, was examined using a fluorescence polarization assay. Structural studies of PDZ domains have suggested that interactions between PDZ domains and residues at positions -2 and -3 of the C-terminal target site confer binding specificity (38-40). In one case, the modification of the residues in these positions by the phosphorylation of serine, has been reported to regulate the PDZ domain binding. The specific association between the second PDZ domain of PSD-95 and the internal rectifying potassium channel (K +) Kir2.3 is disorganized or disintegrated by protein kinase A-mediated phosphorylation of a key serine residue at position -2 from the C extreme of Kir2.3 (41). The results with ephrines of class B and PDZ FAP-1 and synthetin domain proteins suggest that the phosphorylation of the residues with the PDZ domain binding site has different effects on different PDZ domains. The results with FAP-1 PDZ5 suggest that residues of the PDZ domain that make contact with the tyrosines in the binding site of ephrin B are able to accommodate the addition of two phosphate groups. This is consistent with the observations that the simple PDZ domain of AF-66 binds to a non-phosphorylated peptide with the consensus target sequence AYYV (SEQ ID NO: 14) and a corresponding peptide phosphorylated at the residue Tyr-2 with approximately equal affinity. In contrast, GST-synthenin showed significantly decreased binding to the phosphorylated peptides at residue -2 of the PDZ domain binding site. These data indicate a mechanism through which tyrosine phosphorylation of Bl ephrin can regulate interactions with modular cytoplasmic proteins. Possible roles for the PDZ-Ephrin B domain associations can be proposed based on the known functions of the PDZ domains. Several examples have shown the importance of PDZ domain interactions in the appropriate location and accumulation of transmembrane proteins (42,43). For example, the placement of NMDA receptors and K + channels at the post-synaptic ends is probably dependent on the specific interactions of these receptors with the proteins contained in the PDZ domain (34,44-47). In Drosophila larvae, null mutations of the gene encoding the PDZ protein result in poor placement of the K + channel of Shaker (48). The clustering of the Shaker K + channels via the PDZ domain interactions has also been demonstrated in the C0S7 cells that co-express the channel with either its link partners, PSD-95 or chapsin 110 (49). A requirement for correct localization and correct grouping figure prominently in the proposed functions of ephrines of class b. Since the ephrins B-EphB interactions involve direct cell-cell contact, ephrins must be present at the contact sites with the cells expressing the receptor. This location can be mediated by associations of the PDZ domain with the C-terminus of Ephrin B. In this regard, it is of interest that PHIP is a close relative of PAR-3, a protein of C. elegans that regulates symmetry and polarity in the early embryo. It is possible that PHIP has a similar function in mammalian cells in controlling the symmetric distribution of the proteins with the binding portions to the PDZ domain. Studies involving the soluble forms of the extracellular domain of ephrins have revealed a requirement for the pooling of the ligand in receptor activation. While the treatment of cells expressing the receptor, with soluble versions of the ligands does not result in activation of the receptor and subsequent autophosphorylation, the artificial aggregation of soluble ephrins by clustering antibodies allows activation of the receptor (18). Since the co-culture of cells expressing ephrin with cells expressing the Eph receptors leads to receptor activation, the ligands bound to the membrane must also come to be grouped in some way. In addition, recent studies in a system of renal endothelial cells have indicated that the oligomerization state of ephrin B is important in the determination of alternative receptor signaling complexes, as well as coupling and assembly responses in the cell that has receiver (50). Although the binding of both higher order ligands and oligomers causes autophosphorylation of the receptor, only the tetrameric forms of the ligand were able to induce the coupling response and stimulate the recruitment of low molecular weight phosphotyrosine phosphatase to the activated receptor. Given the known role of PDZ domains in the clustering of transmembrane proteins, interactions of the PDZ domain with ephrin Bl may play a role in the presentation of the ligand in the correct oligomeric form to promote specific responses in the cell expressing the receptor .

Another role ascribed to the proteins that contain the PDZ domain is to act as a scaffold to organize the signaling complexes. This is well illustrated by the function of the InaD protein in the photo-transduction pathway of the compound eye of the Drosophi la. Key components of this cascade, which include the potential calcium channel of the transient receptor (TRP), the eye form of protein kinase C and the phospholipase C-β are linked by the PDAD domains of Inad to form a compartmentalized signaling complex. (51.52). Mutations in the specific PDA domains of InaD that suppress the linkage result in defects in the kinetics of the phototransduction cascade. In the case of ephrin B, genetic evidence together with biochemical studies indicating that tyrosine residues in the intracellular domain become phosphorylated after binding to the receptor or treatment with PDGF, has led to the hypothesis that the The cytoplasmic tail of ephrins may have an intrinsic signaling function (2,6,26,27). . The phosphorylated tyrosine residues represent potential platform sites for proteins with phosphotyrosine recognition modules such as the SH2 or PTB domains. The downstream components of this possible phosphotyrosine-dependent signaling pathway can be assembled around a protein that contains the PDZ domain, in a manner similar to the InaD complex. In addition, the PSD-95 protein that contains the PDZ domain associates with the glutamate receptors and the K + channels, also interacts through its PDZ domains with the neuronal nitric oxide synthase and a GTPase activation protein of Ras (pl35 SynGAP) (53, 54). The proteins containing the PDZ domain can thereby serve as adapters to directly activate the signaling pathways. In this context, it is of interest that the phosphorylation of the Tyr residues in the portion of the Bl C-terminal ephrin can regulate the interactions with the PDZ domains, as is suggested by the results with the synthetin. Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated that those skilled in the art can modify the invention in accordance and detail without departing from such principles. All modifications that fall within the scope of the following claims are claimed. All publications, patent applications and patents referred to herein are hereby incorporated in their entirety to the same degree as if each individual publication, patent or patent application. was specifically and individually indicated as incorporated by reference in its entirety.

Detailed Description of the Drawings Figure 1. Amino acid sequence of the cytoplasmic domains of human ephrins B. The residues conserved between the three ephines B are underlined. The asterisks mark the conserved tyrosines that are potential phosphorylation sites. The potential PDZ domain link site is underlined. Figure 2A-D. Identification of the candidates that contain the PDZ domain for the link to ephrin B. Figure 2A. The preferred binding sequence of FAP-1 PDZ5 is shown below a schematic representation of the complete tyrosine phosphatase protein FAP-1. The PDZ domain specificity of FAP-1 was deduced from an oriented peptide library technique (1). The residues within the optimal link sequence that are coupled to the C-terminal sequence of ephrin B are indicated in bold. The organization of PDZ domains of FAP-1 shown in this figure follows the numbering described by Sato et al. (33). Figure 2B. Diagrammatic representations of the proteins that contain the PDZ domain identified through an expression screen with a biotinylated peptide probe of the C-terminal sequence of ephrin B3. The brackets mark the portions of the protein encoded by the cDNAs isolated from the sieve. PDZ domains are represented by gray boxes. Figure 2C. Alignment of the amino acid sequence of FAP-1 PDZ5 and PDZ domains isolated on the expression screen. The numbering of the PDZ domains is as shown in Figure 2B. The conserved waste is placed with bold. The alignment was made with the ClustalW program (55). Figure 2D, alignment of the amino acid sequence of PHIP and PAR-3. The conserved residues are placed in bold and the PDZ domains are underlined. The alignment was made with the Genestream Alignment program. Figure 3A-C. FAP-1 PD5 and the synthetin are specifically bound to the efrin Bl in the GST mixtures. Cos-1 cells were transiently transfected with either the wild type Bl ephrin (W.T.) or the ephrin Bl (Val?) Val deletion or were not transfected. The cell lysates were incubated with the GST fusion proteins as indicated and analyzed by immunoblotting with anti-Ephrin antibody Bl. Immunoprecipitated effine Bl or ephrin Bl Val? They were included as a positive control. Figure 3A and Figure 3B show the mixtures of GST with the fusion proteins of FAP-1. C and D, the mixtures of GST with the syntein fusion proteins. Figures 4A and 4B. FAP-1 PD5 and the syntenin that bind to Bl ephrin Bl can be blocked by the addition of the corresponding peptides to the C-terminal sequence of ephrins B. The peptides of the indicated sequence were included at a concentration of 100 μM in incubations of the GST fusion proteins with the lysates of Cos-1 cells transfected with the efrine Bl. The associated proteins were separated on a 10% polyacrylamide gel / SDS and analyzed by immunostaining with the antibodies against ephrin Bl. Figure 4A, Competition of FAP-1 PDZ5 binding to Bl Ephrin using the indicated peptides. A peptide from the DHQpYpYND sequence was added at a concentration of 100 μM as a negative control. Immunoprecipitation of efrin Bl was included as a positive control. Figure 4B, peptide competence of the full-length synthelin binding to effine Bl. Figures 5A and 5B. Fluorescence polarization analysis of GST-FAP-1 PDZ3, GST-FAP-1 PDZ5 and GST-synthenin that binds to the peptides labeled with Fluorescein corresponding to the C-terminus of ephrin Bl. Figure 5A, solutions containing the indicated final concentration of GST-FAP-1 PDZ3 (O) or GST-FAP-1 PDZ5 (•) which are fusion proteins, in mixtures containing the NIYYKV peptide probe labeled with fluorescein 25 nM , 20 mM phosphatase pH 7.0, 100 mM sodium chloride, and 2 mM dithiothreitol (DTT) were checked periodically for fluorescence polarization at 22 ° C. The GST-FAP-1 PDZ5 fusion protein was also measured to the phosphorylated peptides, NIpYYKV (T), NiYpYKV (A) and NIpYpYKV (I). The fluorescence polarization values obtained for the peptide in the absence of the aggregated GST fusion protein have been subtracted from the polarization values shown. Figure 5B, a link of a GST fusion of full-length synthetin to the peptides NIYYKV (•), NIpYYKV (T), and NIpYpYKV (B) as measured by fluorescence polarization. Figure 6. Co-immunoprecipitation of syntenin-FLAG with ephrin Bl. The Cos-1 cells were co-transfected with either Ephrin Bl and Synthene FLAG or with Val suppression of Ephrin Bl and Sintenin-FALG as indicated. Cell lysates were immunoprecipitated with antibodies against ephrin Bl or the a-receptor of IL-3, or were treated with protein A sepharose only. The immunocomplexes were subjected to SDS-PAGE (10%) and stained with anti-FLAG antibodies. Figure 7. Fluorescence polarization analysis of GST-PHIP PDZ3 that binds to the fluorescein-labeled peptides corresponding to the C-terminus of ephrin Bl. Solutions containing the indicated final concentration of the GST-PHIP PDZ3 fusion protein in mixtures containing the 25 nM fluorescein-labeled peptide probe, 20 mM phosphate pH 7.0, 100 mM sodium chloride, and 2 mM DTT were verified periodically for fluorescence polarization at 22 ° C. The GST-PHIP PDZ fusion protein was measured for binding to the phosphorylated peptides NIpYYKV (Y), NiYpYKV (A) and NIpYpYKV M and the unphosphorylated peptide NIYYKV (•). The fluorescence polarization values obtained for the peptide in the absence of the aggregated GST fusion protein have been subtracted from the polarization values shown. Figure 8. PHIP PDZ3 binds specifically to ephrin Bl phosphorylated with V-Src, in mixtures of GST. Cos-1 cells were transiently co-transfected with V-Src and wild-type ephrin Bl or ephrin Bl with suppression Val (V?) Or transfected with either wild type Bl ephrin Bl or ephrin Bl with Val suppression alone. The lysates Cells were incubated with the GST fusion proteins as indicated and as analyzed by immunostaining with the anti-phosphotyrosine antibody. Immunoprecipitated efrin Bl was included as a positive control.

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It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. 451 ATCGCTTGGA GCATGGAGAT GGAGGGATTC TAGACCTGGA TGACATCCTC 501 TGTGACGTTG CTGATGACAA AGACAGACTG GTAGCAGTAT TTGATCAACA 551 GGATCCCCAC CA7GGAGGAG ATGGTACCAG CGCCAGCTCC ACGGGAACCC 601 AGAGTCCAGA GATATTCGGC ACTCACCTGG CCACCAACAA TC? TTCTGCT 651 TTTCAGCCTT ATCAAGCCAC? AGTGAAATT GAGGTCACGC CTTCAGTTCT 701 TCGGGCAAAT ATGCCTCTTC ATGTCCGCCG GAGCAGCGAC CCAGCTTTAA 751 CTGGCCTTTC CACTTCTGTC AGTGATAACA ACTTTTCCTC AGAGGAGCCC 801 TCCAGCAAAA ACCCCACCCG CTGCTCCACG ACAGCTGGCT TTCTCAACC? B51 GAACACCGCT GGAACTCCCA AAACCTGCGA CACGAACAAA GATGAAAACT 901 ACAGAAGCCT TCCACGGGAT CCCAGTAGCT GGTCCAACCA GTTCCAGCGA 951 GACAACGCCC GCTCCTCCCT GAGCGCCAGC CACCCAATGC TAGACCCCTC 001 GCTGGAGAAG CAAGAACAGG ATGAGGAAGG CACAGAAGAA GACAGCAGCC 1051 CAGTGGAGCC GGTTGGACAT GCTGATACCG GATTGGAGAA CATGCCCAAC 1101 TTTTCCCTCG ATGATATGGT AAAGCTCGTA CAAGTCCCCA ACGATGGAGG 1151 GCCCCTGGGA ATCCATGTAG TGCCTTTCAG TGCTCGAGGC GGCAGAACAT 1201 TGCCCTTCTT ACTCAACCGG TTGGAGAAAG GCGGTAAGGC TGAGCAAGAA 1251 AACCTTTTCC ATGAGAATGA CTGCATTGTG AGGATTAACG ATGGAGATCT 1301 TCGAAACAGA AGATTTGAGC AAGCACAACA TATGTTCCGC CAAGCTATGC 1351 GTGCGCGTGT CATTTGGTTC CATGTGGTCC CTGCAGCAAA CAAGGAGCAA 1401 TATGAACAAC GTCCCAACG CGAGAAGAAC AACTACTCCC CAGGCCGCTT 1451 CAGCCCTGAC AGCCACTCTC TCGCCAACAG GAGTGTGGCC AACAATGCCC 1501 CTCAAGCATT GCCCAGAGCA CCC? GACTG? GTCAGCCACC CCACCACCTG 1551 GATGCTCACC CCCGACTACC TCATAGTGCT CACGCCTCAA CCAAACCACC 1601 CGCAGCCCCG GCCTTGGCTC CACCCAGTGT GCTTAGTACC AACGTAGGCA 1651 GTGTGT? CA? CACGA? GAAA GTAGGCAACA GGCTCAACAT CCAGCTTAAG 1701 AAAGGTACAG AAGGACTGGG ATTCAGCATC ACCTCCCGGG ACCTCACCAT 1751 AGGTGGCTCA GCTCCCATTT ATGTCAAGAA TATCCTTCCT CGAGGGGCTG 1801 CCATTCAGGA TGGCAGACTC AACGCAGGAG ACCGGCTAAT AGAGGTCAAT 18S1 GGAGTAGATT TAGCAGGCAA ATCCCAGGAG GAAGTTGTTT CCCTGTTGAG 1901 AACC? CC ?? G ATGGAGGGGA CTGTGAGCCT TCTCCTCTTT CGTCAGGAAG 1951 AGGCTTTCCA CCCAAGGGAA ATGAATGCTG AACCAACCCA GATGC? GACT

Claims (31)

92 CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An isolated complex, characterized in that it comprises ephrin of class B and a protein containing the PDZ domain.

2. An isolated complex according to claim 1, characterized in that the ephrin of class B is ephrin Bl or ephrin B

3. 3. An isolated complex according to claim 1 or 2, characterized in that the protein containing the PDZ domain is GRIP, GRIP PDZ6 and PDZ7 of SEQ. ID. No. 22 and 23, FAP-1 PDZ of the SEQ. ID. No. 21, the amino acid residues 1 to 299 of the sintenin, PDZI and PDZ2 synthenin of SEQ. ID. No. 26 and 27, PHIP PDZ2 of SEQ. ID. No. 24; and PHIP PDZ3 of the SEQ '. ID. No. 25.

4. An isolated complex according to claim 3, characterized in that it is ephrin B3 / GRIP; Ephrin B3 / GRIP PDZ6 and PDZ7 of SEQ. ID. No. 22 and 23; efrina Bl / FAP-1 PDZ of the SEQ. ID. No. 21; Ephrin Bl or B3 / synthetin PDZl and PDZ2 of SEQ. ID. No. 26 and 27; Ephrin Bl or B3 / residues 1-299 of synthetin; efrina Bl or B3 / PHIP 93 PDZ2 of the SEQ. ID. No. 24, Ephrin Bl or B3 / PHIP PDZ3 of SEQ. ID. No. 2

5. 5. A peptide derived from the PDZ binding domain of an ephrin of class B.

6. A synthetic peptide of the formula I, characterized in that it interferes with the interaction of a class B ephrin and a protein that contains the PDZ domain. X-X1-X2-K-V I wherein X represents 0 to 70 amino acids, and each of X1 and X2 represents tyrosine or phosphotyrosine.

7. A peptide according to claim 6, characterized in that X represents 2 to 20 amino acids.

8. A peptide according to claim 7, characterized in that X represents NI, GNI, CPHYEKVSGDYGHPVYIVQ (E, D) (M, G) PPQSP (A, P) A (SEQ ID No. 2), GDYGHPVYIVQ (E , D) (M, G) PPQSP (A, P) (SEQ ID No. 3), PPQSP (A, P) A (SEQ ID No. 4), GPPQSPPNI (SEQ ID No. 32) ).

9. A peptide according to claim 7, characterized in that it is YYKV (SEQ ID No. 5), GPPQSPPNIpYYKV (SEQ ID No. 6), NIpYpYKV (SEQ ID No. 7), NIpYYKV (SEQ. ID No. 8), NIYpYKV (SEQ ID No. 9), 94 NIYYKV (SEQ ID No. 10), GNYYYKV (SEQ ID No. 28), GNIpYpYKV (SEQ ID No. 29), GNIpYYKV (SEQ ID No. 30), or GNIYpYKV (SEQ ID. No. 31).

10. A complex comprising a peptide according to claims 6, 7, 8, or 9 and a protein containing the PDZ domain.

11. A complex according to claim 10, characterized in that the protein containing the PDZ domain is GRIP, GRIP PDZ6 and PDZ7 of SEQ. ID. No. 22 and 23, FAP-1 PDZ of the SEQ. ID. No. 21, residues of amino acids 1 to 299 of syntenin, PDNI synthetin and PDZ2 of SEQ. ID. No. 26 and 27, PHIP PDZ2 of SEQ. ID. No. 24; and PHIP PDZ3 of the SEQ. ID. No. 25.

12. A complex according to claim 10, characterized in that it is FAP-1 PDZ of the I KNOW THAT. ID. No. 21 / NIpYYKV, FAP-1 PDZ of the SEQ. ID. No. 21 / NIpYpYKV, sinten to / NIYYKV, synthenin / NIpYYKV, PDZl and PDZ2 synthenin of SEQ. ID. No. 26 and 27 / NIYYKV, PDZl and PDZ2 synthetin of SEQ. ID. No. 26 and 27 / NIpYYKV, PHIP PDZ3 of SEQ. ID. No. 25 / GNIpYpYKV, or PHIP PDZ3 of SEQ. ID. No. 25 / GNIpYYKV.

13. A method for modulating the interaction of an ephrin of class B and a protein containing the PDZ domain, characterized in that the method comprises the 95 administration of an effective amount of a complex according to claim 1.

14. A method for modulating the interaction of an ephrin of class B and a protein containing the PDZ domain, characterized in that it comprises the administration of an effective amount of a peptide according to claim 6.

15. A method for identifying a substance that binds to a complex according to claim 1, characterized in that the method comprises: (a) the reaction of the complex with at least one substance that can potentially link to the complex, under conditions that allow the link of the substance and the complex; and (b) link detection, where the detection of the link indicates that the substance binds to the complex.

16. A method according to claim 15, characterized in that the binding is detected by the evaluation of the substance-complex conjugates, or by the activation of ephrin of class B or the protein containing the PDZ domain.

17. A method for evaluating a compound for its ability to modulate the interaction of an ephrin of class B and a protein containing the PDZ domain, characterized the method because it comprises the provision of a 96 complex according to claim 1, 2 or 3, with a substance that binds to the complex, and a test compound, under conditions that allow the formation of conjugates between the substance and the complex, and the removal and / or detection of the conjugates.

18. A method for evaluating a compound for its ability to modulate the interaction of a class B ephrin and a protein containing the PDZ domain, characterized in that it comprises (a) the provision of an ephrin of class B and a protein that contains the PDZ domain, and a test compound, under conditions that allow the binding of ephrin of class B and the protein containing the PDZ domain; and (b) detection of the link, wherein detection of the linkage increased or decreased relative to the linkage in the absence of the test compound, indicates that the test compound modulates the interaction of a class B ephrin and a protein containing the PDZ domain.

19. A method for modulating the interaction of an ephrin of class B and a protein containing the domain PDZ, characterized in that it comprises the change of the Val-terminal amino acid in an ephrin of class B.

20. The use of a complex according to claim 1 or a peptide according to claim 6, in the preparation of a medicament for 97 modulate the interaction of an ephrin of class B and a protein that contains the PDZ domain.

21. The use of a complex according to claim 1 or a peptide according to claim 6, in the preparation of a medicament for modulating cellular processes of cells associated with ephrines of class B or proteins containing the PDZ domain.

22. The use according to claim 21, wherein the cellular processes are axonogenesis, nerve cell interactions, and nerve cell regeneration.

23. A composition, characterized in that it comprises a complex according to claim 1 or a peptide according to claim 6, and a pharmaceutically acceptable carrier, excipient or diluent, effective for administration to individuals suffering from disorders associated with an effine of class B.

24. A method for modulating the proliferation, growth or differentiation of cells associated with ephrines of class B or proteins containing the PDZ domain, characterized in that the method comprises the introduction into cells of a complex of according to claim 1 or a peptide according to claim 6.

25. A method for treating proliferative or differentiating disorders associated with ephrines of class B or proteins containing the PDZ domain, characterized in that it is used in the compositions according to with claim 23.

26. An isolated protein, characterized in that it comprises the amino acid sequence of SEQ. ID. No. 1.

27. A variation of truncation, an analog, an allelic or species variation of a protein according to claim 26, or a protein characterized in that it has substantial sequence identity with the protein according to claim 26.

28. A fusion protein, characterized in that it comprises an isolated protein according to claim 26, conjugated to a protein.

29. Antibodies, characterized in that they have specificity against an epitope of a protein according to claim 26.

30. A method for identifying a substance that binds to a protein according to claim 26, characterized in that it comprises reacting the protein with at least one substance that can potentially link to the protein, under conditions that allow the binding of the substance and the protein, and the detection of the link, where the detection of the link indicates that the substance binds to the protein.

31. A method for evaluating a compound for its ability to modulate the biological activity of a protein according to claim 26, characterized in that the method comprises the provision of the protein, a substance that binds to the protein, and a test compound under conditions that allow the binding of the substance and the protein, and the detection of the link, wherein the detection of the Increased or decreased linkage relative to the linkage detected in the absence of the test compound indicates that the test compound modulates the activity of the protein.