MXPA97001904A - Linking proteins of novedos pka and uses of mis - Google Patents

Abstract

The present invention provides novel PKA binding polypeptides, nucleic acids encoding polypeptides and antibodies specifically immunoreactive with polypeptides.

Description

NOVELTY PKA LINK PROTEINS AND USES OF THE SAME FIELD OF THE INVENTION The present invention relates, in general terms, to proteins that bind protein kinase A. More specifically, the present invention relates to novel proteins and nucleotide sequences that encode these proteins that localize protein kinase A inside cells. BACKGROUND OF THE INVENTION Extracellular proteins, such as hormones and cytokines, modulate many cellular processes by "activating adenylate cyclase, increasing the intracellular levels of cAMP and the subsequent activation of kinase-dependent cAMP (PKA)." PKA is a ubiquitous enzyme which functions in many intracellular pathways, for example, the regulation of glycogen metabolism by the reversible phosphorylation of glycophosphorylase (Alsh et al., J. Biol. Chem., 243: 3763-3765 (1969)), and MAP kinase regulation signaling by inhibition of Raf-1 activation by Ras (Vajtefc et al., Cell, 74: 205-214 (1993) and Hafner et al., Mol. Cell Biol., 14: 6696-6703 (1994).) The inactive PKA exists with tetramer where two identical catalytic subunits are linked to a dimer of two regulatory subunits.The activation of PKA by cAMP is effected by the binding of a molecule of cAMP on each of its regulatory conditions (R) which causes the release of the active catalytic subunit (O. While only one form of subunit C has been identified, there are two subunit classes R, Rl and RII, with apparently distinct subcellular distributions. The isoforms of Rl (RIalfa and RIbeta) are reported as predominantly cytoplasmic and are excluded from the nucleus, while up to 75% of the Rl isoforms (RII alpha or Rubeta) are particles and are related to any of the following: plasma membrane , cytoskeletal components, granules are re, galgi apparatus, centro or aβ or possibly nuclei (Scott, Pharmac .. Ther., 50: 123-145 (1991)). It is considered that differences < either physical or physiological) in the various subunits of R provide a means by which cells can restrict the activity of subunit C to a desired pathway. Recent evidence indicates that the cells can be targeted towards PKA activity by locating the inactive enzyme in the vicinity of potential substrates, thereby restricting the activity of the C subunit after release by the cAMP link on the R subunit. This "compartmentalization" segregates PKA with participants in a given signaling pathway and contributes to the specificity of PKA in response to different intracellular stimuli. The PKA comparison occurs, at least in part, due to the interaction or blockade of the R subunit with specific proteins that localize, or anchors the inactive haloenzyme at specific intracellular sites. Proteins that specifically secrete PKA have been designated kinase anchor proteins or AKAPs (Hirsch et al., J. Biol. Chem., 267: 2131-2134 (1992)). Due to the fact that some AKAPs bind and anchor other proteins besides PKA, the family of proteins is generally known as anchoring proteins. To date, numerous anchoring proteins have been identified (this is discussed below) that apparently link PKA by means of a secondary structural motif of common carbohydrate terminal that includes an amphipathic helical region (Scott and McCartney, Mol. Endo ., 8: 5-11 (1994)). The PKA linkage on most, if not all, of the identified anchor proteins can be blocked in the presence of a peptide (Ht31) that mimics this common helical secondary structure, while a Ht31 utapte peptide, which contains a unique amino acid substitution which disrupts the helical nature of the peptide, has no effect on the PKA / anchor protein binding (Carr et al., J. Biol. Chem., 266: 14188-14192 (1991)). Even when the PKA / anchoring pratein interaction is effected by means of a common secondary structure, anchor proteins (or homologous anchor proteins found in different species) generally have a unique primary structure in accordance with what is evidenced by the growing number of anchorage proteins that have been identified in several organisms. Supposedly, the unique structure of amino acids, most notable in the amino terminal regions of proteins, partly explains the anchorage proteins identified as co or unique for several specific cell types and for the various specific intracellular compartments where the location has been observed. of PKA. For example, anchor proteins predominantly expressed in mammalian brain have been identified in the rat (AKAP 150) and in the cow (AKAP 75) (Begman, et al., J. Biol. Chem. 266: 7207-7213 (1991 )), as well as in humans (AKAP 79) (Carr, et al., J. Bio, Chem. 267: 16816-16823 (1992)). The identity of the amino acids and the immunological cross-reactivity between these specific proteins for neurons suggest that they represent homologs between species. To mention another example, AKAP 100 appears to be specific for the skeletal and cardiac muscle of the human being and the rat, while it is expressed to a lesser degree in the brain cells of these mammals. As another example, AKAP Ht31 (Carr et al., J. Biol. Chem., 267: 13376-13382 (1992)) appears to be specific for thyroid cells. Conversely, AKAP 95 has been shown to be expressed in numerous cell types, and apparently does not have a specificity for cell type or tissue. Regarding localization in specific intracellular compartments, AKAP 75, a protein associated with micratubules (MAP-2) (Theurkauf and Val lee, J. Biol. Chem., 257: 3284-3290 (1982) and DeCa illi et al. , J. Cell Biol., 103: 189-203 (1986)), AKAP 79 (Glantz et al., J. Biol. Chem., 268: 12796-12804 (1993)) and AKAP 150 (Glantz et al., Mol. Biol. Cell, 3: 1215-1228 (1992)) are closely related to itoesquelet les structural proteins, and AKAP 75 is more specifically related "With postsynaptic densities (Carr et al., J. Biol. Chem., 267: 16816-16823 (1992)) It has been shown that other anchoring proteins localize less diffused cell structures, including association of AKAP 350 with centrosomes (Keryer et al., Exp. Cell Res., 204: 230-240 (1993 )), AKAP 100 with the sarcaplasmic reticulum in rat heart tissue (McCartney et al., J. Biol. Chem. 270: 9327-9333 (1995)), and an 85 kDa AKAP that binds PKA to the golgi apparatus ( Rios et al., EMBO J., 11: 1723-173 1 (1992)). AKAP 95, with an apparent zinc finger DNA binding region, appears to be found exclusively in the nucleus (Coghlan et al., J. Biol. Chem., 269: 7658-7665 (1994)) . The DNA binding domain of AKAP 95 provides a function for the direct involvement of PKA in genetic transcription, possible by positioning PKA for phosphorylation of transcription factors. Other diverse cellular activities which are influenced by PKA anchor / binding protein have been demonstrated by the disruption of the interaction, for example, it has been shown that the disruption of the PKA binding / T cell anchor protein reverses the suppression induced by cAMP of the expression of interleukin 2 (Lockerbie et al., J. Cell Biochem., Supplement 21A: 76, Abstract D2155 (1995)) and it has been shown that the disruption of the PKA / anchor protein binding in. The hippocampal pebbled attenuates whole cell currents through alpha-amino-3-hydroxy-5- and il-4-isoxazole propionic acid / kainate ratiamate receptors (Rosenmund et al., supra). The ability of anchor proteins to regulate the expression of IL-2 and to regulate glutamate receptor activity, in combination with a previous demonstration that anchor proteins can bind calcineurin, suggest multiple therapeutic applications for unbound protein and molecules that modulate the binding of anchor proteins on cellular components. Taking into consideration the diversity, both in terms of expression of cell types, subcellular location and physiological activities of the anchorage proteins identified to date, there is a need in the art to continue identifying novel anchor proteins and nucleic acids that encode them. The unique character of primary structures of anchor protein provides a target to specifically regulate the localization of PKA, and therefore its function in specific cellular processes. SUMMARY OF THE INVENTION The present invention provides purified and isolated nucleotide poly sequences encoding proteins that have the biological properties of binding PKA and subcellular compartmentation. UIF presently preferred polynucleotide is presented in SEQ ID NO: 5. The polynucleotides of the present invention also encompass polynucleotides that hybridize under stringent conditions of hybridization to the polynucleotide of SEQ ID NO: 5. The polynucleotides of the invention can be DNA or RNA and can hybridize in the sense strand or in the antisense strand of the DNA molecule. The DNA can be cDNA, genomic DNA or chemically synthesized DNA. The polynucleotides of the present invention can be identified by standard techniques such as, for example, complementation, looser hybridization, and chain reaction of pslimerase using primers generated based on knowledge of the polynucleotide sequences of the invention. In the present invention there are also provided recombinant expression constructs containing polynucleotides of the invention operably linked with transcriptional regulatory elements with, for example, transcription promoters and terminators. The elements of transcription regulation can be homologous or heterologous.

Another aspect of the present invention is transformed or trans-transfected host cells with polynucleotides of the invention. The host cells can be prokaryotic or eukaryotic. Host cells transformed or transfected in this manner are especially useful for the expression of PKA binding polypeptides of the present invention which can be isolated from the cells or from the culture medium. Another aspect of the present invention are PKA binding polypeptides encoded by the polynucleotides of the present invention. A preferred PKA binding polypeptide is encoded by the polynucleotide presented in SEO. ID NO: 5. The polypeptides of the present invention can be purified from natural sources or produced by recombinant methods using the host cells of the present invention. Palpeptide variants that maintain the biological activity of a wild-type polypeptide are also contemplated, including analogs where additions, pre-motions or substitutions of conservative amino acids are incorporated which modulates the functional or immunological characteristics of the traced polypeptide to PKA. Other variant polypeptides include fusion proteins where additional sequences of polypeptides that facilitate purification or immobilization in assay supports are incorporated. Additional polypeptides of the present invention can be identified by immunological cross-reactivity with the polypeptide encoded by the polynucleotide of SEQ ID NO: 5. The present invention also provides "" polypeptides and non-polypeptide molecules that specifically bind to the PKA-binding polypeptides described above Preferred binding molecules include antibiotics (eg, polyclonal, monoclonal, recombinant antibodies or binding fragments thereof) The binding molecules are useful for purification of PKA-binding polypeptide-β. , Identification of cells expressing PKA binding proteins and modulation of the in vivo interaction between PKA and PKA binding polypeptides. Hybridoma cell lines producing antibodies specifically immunoreactive with PKA binding polypeptides are also provided. prior invention. Ridomas can be produced and identified by techniques well known in the art. Assays are also provided that identify molecules that disrupt the interaction between PKA and PKA binding proteins of the present invention (eg, immobilized binding assays, solution binding assays, scintillation proximity assays, inhibited screening assays, and Similar). In some cases, it may be desirable to modulate the link between PKA and the polypeptides of the present invention. In other cases, it may be desirable to specifically modulate the link between a polypeptide of PKA linkage and a cellular component (another PKA) to which it is linked. In any case, the polypeptides of the present invention provide a sieve target useful for the assays of the present invention. Assays of the present invention can be carried out in varioß farmatoß, including cell-based assays, such as for example complementation assays or disinfected screening in accordance with that described in US Patent No. 5,283,173 and in the Published Document of the Cooperation Treaty. on Patents (PCT) No. WO 91/16457, respectively. Assays of this type are especially useful for evaluating the intracellular efficacy of the compounds. Non-cell-based assays of the present invention include scintillation proximity assays, cAMP competition assays, ELISA assays, radioimmunoassays, chemolysis assays, and the like. Such assay procedures are well known in the art and generally described, for example, in Boudet et al., J. Immunol. Meth., 142: 73-82 (1991)? Ngai et al., J. Immunol. Meth., 158: 267-276 (1993); Prußlin et al., J. Immunol. Me.th., 137: 27-35 (1991)? Udenfriend et al., Proc. Nati Acad. Sci. USA, 82: 8672-8676 (1985)? Udenfriend et al., Anal. Biochem., 161: 494-500 (1987); Bosworth and Towers, Nature, 341: 167-168 (1989); ßilman, Proc. Nati Acad. Sci. USA, 67: 305-312 (1970);; and in U.S. Patent No. 4,568,649. The utility of the compounds that model the binding of anchor proteins is evident. For example, it may be found that small molecules inhibit either the PKA / anchor protein binding or the interaction of anchor protein with specific cellular β components. Modulators of this type of delocal would raise specific PKA sets and would affect a target signaling guide. The identification of anchor protein binding modulators on other cellular components can be equally beneficial. For example, factors that affect the activity of calcineurin in a manner similar to previously identified immunosuppressants, but that have fewer side effects, may be useful in the treatment of conditions treated now with more toxic immunosuppressants. In addition, the identification of factors that modulate the participation of anchor proteins in cellular proteins may also be useful to replace the currently accepted therapeutic interaction. For example, factors regulating the regulation of IL-2 expression anchor protein may be useful to replace the administration of recombinant, hexogenous IL-2. DETAILED DESCRIPTION OF THE INVENTION The following examples are offered to illustrate the present invention but not to limit it. Example 1 focuses on the identification of a specific anchor protein for T cells from a human cDNA library. Example 2 describes the binding specificity of RII of the identified anchor protein. Example 3 presents the determination of the nucleotide sequence of the anchor protein. Example 4 presents the expression of the clone of the anchor protein. Example 5 refers to the cellular and tissue distribution of the anchor protein. Example 6 describes the potential therapeutic applications of the anchor protein and molecules that modulate the binding of the anchor protein. EXAMPLE 1 IDENTIFICATION OF ANCHOR PROTEINS EXPRESSED IN CELLS T In an attempt to identify novel T cell anchoring proteins, a human Jurkat T cell cDNA library subcloned in ZAPII Express (Stratagene, La Tolla, CA) was screened by Rllalfa coating techniques in accordance with that described in Carr. et al., J. Biol. Chem., 267: 16816-16823 (1992). Briefly, one μl of the library phage (5? 10,000 pfu) was added to 600 μl of the E. coli strain XL-1 Blue MRF '(Stratagene) in 10 mM of MgSO4 cultured at 0D600 = 0.5. Bacteria and phage were incubated at 37 ° C for 15 minutes, after which 7.5 ml of upper agar (NZY medium (VA weight / volume) of NZA ina type A, 0.5% (pe? volume) of yeast extract, 86 mM NaCl, 8 mM MgSO4.7H20, 1.5% (pe / volume) of Bacto agar, pH 7.5), 0.7 * /. of agarsßa). The resulting mixture was immediately placed in NZY dishes preheated to 374 > C. The plates were allowed to cool to room temperature and allowed to incubate at 42 ° C for 4 hours. Ne trocelulssa fibers, previously wetted in 10 M isopropyl 1-1-t io-beta-D-galactopyranoside (IPTG), were placed in the plates and the plates were further incubated for 4 hours at 37 ° C. The filters were removed and washed 3 times in TBS (50 M Tris, pH 7.5, 150 mM NaCl), and blocked overnight at 4 β C in the block (TBS, 5% nonfat milk, 0.1 * 4 BSA ). A second set of nitrocellulose fibers prepared in the same manner was placed on the plates and incubated at 4'C overnight. The filters were washed (in accordance with that described above) and blocked (also as previously described) for 1 hour at room temperature. Approximately 4 μg (6 μl) of recombinant mouse Rllalfa was mixed with 2.35 μg (0.5 μl) of bovine catalytic subunit replacing PKA in a reaction containing 2.5 μl (32 P) ATP (25 μCi, 3000 Ci / mol), and 1 μl of buffer (containing 0.5 M of M0PS, pH 7.0, 0.5 M NaCl, 20 mM-MgC12, and 10 mM DTT). The reaction proceeded for 30 minutes at 30 ° C, after which "the unincorporated label was removed using a column of Execeluloßa.

GF-5 (Pierce). The filters were tested with (32P) RIIalfa (100,000 cpm / ml in block) for 6 hours at room temperature. After incubation, the filters were washed three times in PBS containing VA of Tween-20 and exposed to X-ray films for 16 hours. Among approximately one million screened plates, a positive plate was identified, plate number 11 as a labeled RIalpha link. A secondary sieving was performed on plate number 11, by techniques described in the initial screening, which indicated that the pragency of plate number 11 could also bind radiolabelled Rllalfa. EXAMPLE 2 SPECIFICITY OF THE RALPHAL LINK ON PLATE NUMBER 11 Based on previous reports according to which peptide Ht31 (SEQ ID NO: 1) can generically block the PKA binding on anchor proteins and that a proline mutant of Ht31 ( see SEQ ID NO: 2 below, where the proline substitution is indicated in bold and underlined), also described above can not block it, the binding specificity of Rllalfa on plate number 11 was determined in parallel experiments where they led to coatings with Rllalfa in the presence of any of the peptide β Ht31. Asp-Leu-Ile-Giu-Glu-Ala-Ala-Ser-Arg-Ile-Val- - ^,. Asp-A) a-Vjü-Ilc-Glu-Gln-Val-Lys-Ala-Ala-GIy-Al * (SEQIDN0: 1) Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg- £ tff-Val-Asp-Ala-Vil-ne.Glu-Gte-VaJ.Lyf.Ata-Ala-Gly-All < SEQIDNO2) Briefly, nitrocellulose filter risers were prepared according to that described in example 1, except that the resulting plate elevators were preincubated for 15 minutes at room temperature in block containing 1 μM of Ht31 peptide or mutant Ht31 peptide of proline. After preincubation, the filters were tested with < 32P) RIIalfa according to that described in example 1, and the filters were subsequently subjected to autoradiography. The autoradiograms revealed that the preincubation of plate number 11 with the peptide Ht31 blocked the binding of (32P) RIIalfa, while preincubation with the peptide Ht31 with proline mutation had no effect. They are refuted. indicate that the binding of Rllalfa ß on the polypeptide encoded by plate number 11 ß effected by means of a secondary structure of plate number 11 similar to that employed by anchor protein previously identified ß. EXAMPLE 3 CLONING OF PLATE cDNA. NUMßHÓ 11 In an attempt to determine the nucleotide sequence of the bulb in the phage of plate number 11 and to de the amino acid sequence of the encoded protein, the cDNA area of plate number 11 was removed in vivo using an ExAssißt system / XLOLR (Stratagene) in accordance with the manufacturers' instructions. Briefly, plate number 11 was removed from the NZY plate and mixed with 500 μl of SM buffer < 100 mM NaCl, 8 M MgSO4.7H20, 50 M Tri-HCl, pH 7.5, 0.01% (w / v) gelatin) and 20 μl chloroforin. The mixture was ßometida to vortex and stored at 4ßC (phage storage). The XL-1 Blue MRF 'and XL0LR cells (both from Stratagene) were cultured overnight at 30 ° C in an LBM medium supplemented with 10 mM MgSO4.7H20 containing 0.2% maltose (volume / volume).

A 1/100 dilution of the XL-1 Blue MRF 'cells was prepared with 0.5 ml of the culture medium overnight and 50 ml of LBM medium and the dilution was cultured at 37 ° C for 2-3 hours until the semi-logarithmic phase ( 0D600 * 0.2-0.5 for XL-1 Blue MRF "or 0D600 = 0.5-1.0 cells for the XL0LR cells.) The culture was centrifuged at 1500 μg and the resulting pellet was resuspended in 10 mM MgSO4.7H20 to a density of 0D600 = 1.0., 200 μl of the XL-1 cells, 250 μl of the phage stock suspension as described above and 1 μl of the ExAssist phage (Stratagene) were combined and incubated for 15 minutes at 37 ° C. 3 ml of LBM medium were added and the mixture was further incubated for 2.5 hours at a temperature of 37 ° C with shaking. After incubation, the mixture was centrifuged for 15 minutes at 200 x g. The supernatant was removed, incubated at 70 ° C for 15 minutes, and centrifuged at 4000 ° C. g for 15 minutes. The resulting supernatant contained entose-like phages that wrapped the DNA of plate number 11 in phagemid pBK-CMV. The phagemid were recovered by mixing 200 μl of XL0LR cells (prepared according to the above) with 10 μl of the mother phagemid and by incubation for 15 minutes at 37 ° C. After the incubation, 300 μl of LBM medium was added. and the mixture was further incubated for 45 minutes at 37 ° C. The resulting cell suspension was placed in a proportion of 200 μl / p ato in LMB containing 50 μg / ml kanamycin. Plasmid preparation was performed by means of standards and included the use of a Wizard Miniprep Kits (Promega) The DNA of the plasmid isolated from plate number 11 was called clone number 11. The cDNA insert was removed from the vector by means of digestion with E.coRI and Ba HI and the resulting fragments were separated using agarose gel electrophoresis, and the insert of clone number 11 was determined to be 2850 bp with an Erase-a-Base system (Promega, Madison, Wl). nested deletions of clone number 11 and clone number 11 was sequenced using Universal primers T3 (ATTAACCCTCACTAAAG (SEQ ID N0? 3 > and T7 (SATATCACTCAGCATAA (SEQ ID N0: 4) and a Pris Ready Reactisn DyeDea ™ and Terminator Cycle Kit (Perkin Elmer) on an ABI373 DNA sequencer (Perkin Elmer, Foßter City, CA) The DNA sequence of clone number i 1 is presented in SEQ ID NO: 5. Because no appropriate initiation codon could be detected in the ß nucleic acid sequences, it was not possible to determine a ded amino acid sequence or a molecular weight for clone number 11. A Blast Nucleotide-level search (June 16, 1995, 14:01? 37 EDT) of the sequence obtained from the T3 primer showed homology with a clone named "3 'end of homosapiens cDNA similar to none" (access number T32770), while the sequence data obtained from the T7 primer showed 98% homology over a 343 base-length of 1905-2248 of clone number 11 with a clone called the '5' end of the partial cDNA of Ho orphans. similar to none "; (access number T31099). In addition, clone number 11 presented a homology of 98% over a length of 332 baseß of nucleotides 2308-2640 with a -ion called "partial cDNA sequence of Homosapiens, clone 66D04 (accession number Z24883). ^ ^ * EXAMPLE 4 EXPRESSION OF CLON NUMBER 11 To determine an appropriate molecular weight for the genetic product of clone number 11, an overnight culture of clone number 11 was performed in XLOLR cells (prepared according to that described in example 3) in LBM medium. / tetracycline (12.5 μg / ml) and subsequently used to inoculate 250 ml of the same medium The incubation was allowed to take place at 37 * C to 0D600 = 1.2, after which the bacteria were pelleted at 6000 xg for 15 days. The pellet was weighed and resuspended in 10 volumes (bp / volume) of FP regulator (1% Triton X-100, 130 mM NaCl, 1 M EGTA, 1 mM EDTA, 10 M Tris, pH 7.4 , 1% aprotinin, 0.2% NaN3.) The cells were broken in u na Frenen Prese and the lysate was clarified by centrifugation at 40,000 x g for 30 minutes. The lysate was then concentrated using a Centricon-10 (Amicon). An aliquot of the concentrated lysate was loaded on a 10% tris-glycine gel (Novex), subjected to electrophoresis and transferred to Immobilon (Millipore). The spot was tested with (32P) RIIalfa. A single band of approximately 120 kD was detected that was partially removed by the Ht31 peptide. These results indicate that the clone number '11 encodes a PKA binding protein that can be used in the assay to identify inhibition of the link between the PKA and PKA binding polypeptides. EXAMPLE 5 CLON TISSUE CELLULAR DISTRIBUTION No. 11 To determine the cellular and tissue distribution of the expression of clone no. 11, a reverse transcriptase poly erase chain reaction (RT-PCR) was used to evaluate the no. Of RNA from clone no. 11. In summary, the initiatereß were initially designed to span 300 bp of the sequence of clone no. 11, based on the nucleic acid sequence determined in example 3. In the sequence for clone no. 11 (SEQ ID NO: 5), the 2T3 initiator corresponds to nucleotides 266-283, the M2T3 initiator corresponds to nucleotides 434-453, the R2T3 initiator corresponds to nucleotides 601-622, the R2T7 initiator corresponds to nucleotides 2229 -2250, the M2T7 primer corresponds to nucleotides 2337-2400, and the 2bT7 primer corresponds to nucleotides 2256-2592. The RNA was prepared from various cell and tissue types (described below in the discussion of the results) using an RNa isolation types (Stratagene). RT-PCR was performed in accordance with the following. Initially approximately 1 μg RNA was denatured in 10 μl of water) by incubation at 80 ° C. for 3 minutes, after which the RNA was incubated further on ice until reverse transcriptase reactions were carried out in the following manner. The denatured RNA was mixed with 8 μl of 5X buffer MMuLV-RT (Boehringer), 8 μl of a mixture of 2.5 mM dNTP, 1 μl of water containing 0.5 μg of each of the 2T3 and R2T3 primers or of the primers 2bT7 and R2T7, 1 μl of RNase inhibitor (Boehringer), 1 μl of MMuLV-TY (Boehringer) and 11 μl of water and incubated for 1 hour at 42 * C. The polymerase chain reactions were carried out in the following manner: 2 μl of the preferred reverse transcriptase reaction were mixed with 3 μl of a mixture of 2.5 mM dNTP, 3 μl of 10X Taq polymerase buffer (Boehringer), 3 μl (0.3 μg) of the 2T3 primer with 3 μl (0.3 μg) of the R2T3 primer, to 3 μl (0.3 μg) of the initiator of the 1 2bT7 primer with 3 μl (0.3 μg) of the R2T7 primer, 0.5 μl of Taq polymerase and 14.5 μl of water. The mixture was heated at 94 ° C for 4 minutes, after which 30 reaction cycles were carried out (94 ° C for 1 minute, 60. β C for 1 minute and 72 β C for 1 minute). The products of the amplification from the polymerase chain reactions were separated by electrophoresis in 1% agarose gel, and subsequently transferred to a Nytran Plus membrane (S + S) by standard procedure. The PCR products were crosslinked on the membrane with UV irradiation and the membrane was subsequently prehybridized for 3 hours at 42 ° C in 5X SSPE, 0.5% SDS, 0.1 mM Tris, pH 7.5, and 2X Denhardt. Hybridization probes were prepared by end-labeling in the following manner. 2 μl <200 ng) of M2T3 primer were mixed with 2 μl of M2T7 primer (200 ng), 2 μl of regulator 10X polynucleotide kinase (Boehringer), 10 μl 32P-ATP (100 μCi, 1000 Ci / mmol), 2 μl (20 μg) units) of T4 polynucleotide kinase (Boehringer), and 2 μl of water. The reaction proceeded at 37 ° C for 30 minutes after which the reaction was stopped by the addition of 2 μl of 0.5 MEDTA and the unincorporated label was removed by centrifugation with a Centristep column. { Princeton Separation, Inc.). The membrane was then probed overnight at 42 ° C in the same prehybridization buffer but also containing 400 ng (200 ng each) of 32 P labeled M2T3 and M2T7 regulators. After hybridization, the β membrane β was washed down. temperature - ambient 3 times for 10 minutes each in 0.5X SSC, with 0.2% SDS, and subsequently autoradiographed. The cell-washed results indicated that the clone did not. 11 was expressed in Ramos cells (cell B), Jurkat cell (T cell), U973 cells (monocyte), T84 cells (colon carcinoma), cells-. , .HL60 (pro ielocítica leukemia), A549 cell (pulmonary epithelium), and HeLa (epithelial carcinoma). The results of the tissue analyzes indicated that the clone no. 11 ß expressed in testicles, liver and polite or? Ipital of the human brain. EXAMPLE 6 POTENTIAL THERAPEUTIC APPLICATIONS The above demonstration that AKAP 79 binds to calcineurin is relevant by taking into consideration the fact that calcineurin is the target of 2 potent immunosucesoreß and clinically útileß, ciclosporin and FK506, both inhibitors of calcineurin activity . As described below, both cyclosporine and FK506 are useful in the treatment of various diseases, but have important side-effect collaterals. Probably, the factors that modulate the pratein binding of anc 1 age / ca lc i neuri na can ultimately modulate the activity of calcineupna in a similar way to the activities of the ciclopopna or of F 506. The idenificación of such modulator, Especially with fewer side effects than those observed with other immunosuppressants, it would have a broad therapeutic use in the treatment of numerous diseases currently treated with ci > : 1ospor i na a bien Fl-506. Numerous clinical indications of 3rd cycle cyclospine and FK506 have been reinforced. For example, immunosuppression has defined the standard for immunosuppression after transplantation, making liver, lung, bowel and pancreas transplants possible, even though it is generally considered that Fl 5 '"is a more potent immunosuppressant. Patients who do not tolerate or who do not react to ciclospopna or FK506 are sometimes successfully switched to the other drug.As another example, inflammatory bowel disease (IBD) is a common term for two diseases that have clinical appearances Differently, Crohn's disease and ulcerative colitis (UC). Cyclopspna has been used successfully to treat Crohn's disease and statistically significant results of treatment have been demonstrated at at least one index of the activity of the disease (Bryneolov, Dan, ed.Bul 1. 41: 332-344, 1994)).

Other index- », although they correlate better with the resolution of exacerbations showed insignificant tendencies toward improvement. Cyclospopna has also exhibited severe acute asteroid-resistant UC activity (data are not significant since the trial was stopped for ethical reasons). Another trial of patients with sclerosing cholangitis and UC demonstrated a signi fi cant limit to a milder UC course. Recurrence was frequent after withdrawal and treatment was limited by reoccurrence concerns with toxicity (Choi and Targan, Dig.Dis and Sci. 39: 1885-1892 (1994)). In addition, other immunosuppressants have been used successfully in IBD, such as methotrexate, a.atioppm, and 6-MP. As another example, it has been shown that ciclosporin is effective for the treatment of rheumatoid arthritis in several trials when it was used as a second or third line therapy for the disease, ie in patients who had not responded to other established therapies and They had serious illness. In these trials, it was found that ciclospopna is generally as effective and toxic as other second-line agents, such as gold, antimalarial agents, azathiopana, D-penicillium, and methotrexate (Wells and Tugwell, Br.J. .Rheum., 32 (suppl l): 51-56 (1993)); Forre et al., Arth.Rheu., 30: 88-92 Í997)). The trials report only the treatment of "refractory active RA, very severe" due to the "potentially irreversible toxicity" of the body (Dougados and Tarley, Br.J.Rheum, 3 (suppl 1): 57-59). (1993)). It is thought that renal toxicity has been mediated primarily through renal vasoconstriction that exacerbates the nephritis of NS ID and the renal disease inherent in rheumatoid arthritis' Lealer and Cairni, Br. J. Hosp. Me. , 52: 520-534 (1994); Sturroc et al 1., Nephrol .Dial. Transplant, 9: 1149-0 11 ^ 6 (1994); Ludwin and Ale? Opolulou, Br. J-.Rhe? M. , 32 (ß ppl 1): 60-64 (1993)). Approximately 10% of the renal biopsies of patients with PA treated with those who showed morphological characteristics of rotslope toxicity (International and Idney Biopsy Pegistry of 5 Cyclospopne, Autoimmune Diseases, Br. J. Rheu., 32 (supp 1 1): 65-71 (1993)). As an additional example, the porosine has been reinforced because it is effective for the treatment of steroid-dependent asthma. In one trial, some patients were < ") to read results between cyclespopna or placebo, and the group of patients receiving the drugs presented an increased flux and FVC, as well as a smaller number of rescue courses with prednisolone.As another example, it has been shown that ciclopopna is effective in the treatment of necrotic syndrome of minimal change disease dependent on steroid This study showed that patients had lower steroid requirements at a low dose of cyclosporin, but had recurrences when discontinuing the drug. The forms resistant to the steroids of the cytoplasm and necrotic have only a response of 20 to 35% to the cyclespopna (Meyper, Nephral Dial Transplant, 9: 596 ~ 5C? 8) 1994) Huí ton et al. , Pedi r.Nephrol., 8: 401-403 (1994).) Regarding the treatment of systemic lupus erythematosus (SLE), one study indicated a significant decrease in SLE activity indices in an uncontrolled study. , not alea tari a do, prospective (Tokuda et al., Arthr.Pheumat. , 37: 551-558 (1994)). Other studies, however, have shown efficacy in SLE. As another example, it has been shown that cislospopna induces the reduction in patients suffering from diabetes mellitus and its insulin-dependent loss when it occurs at an early hour after the initial presentation. The reviews had an average duration of one year, although some lasted up to 850 days (Jenner et al., Diabetol ogía, 35: 884-888 (1992), Bougneres et al., Diabetes, 39: 1264-1272 ( 1990)). A non-lasting effect of ciclospopna was observed in long-term follow-up in one study (Martin et al., Diabetologia, 34: 429-434 (1991)). In another study, without .8 However, renal function deteriorated over a period of 12 to 18 months and did not fully return to the placebo level indicating that chronic kidney damage may have occurred (Feldt-B-ismussep et al., Diabetes Medicine, 7: 429-433 (1990)). An earlier intervention may be necessary to increase the effect of immunosuppressive therapy on the course of diabetes mellitus and inodepend lens. Some researchers are screening first-degree relatives and treating prophylactically with it; i or relatives with diabetic markers (Elliott and Chase, Di abetolo ía, 34: 362-365 (1991)). As another example, psoriasis has been effectively treated with cislaspopna (Cuellar et al., Balliere's Clin.Rheum., 8: 483-498 (1994); Ellis et al., TAMA 256: 3110-3116 (1986)) . A high dose therapy was effective for the treatment of psoriatic arthritis, a particularly severe form of destructive arthritis, and the suspension of therapy was generally followed by an exacerbation of skin and joint disease. Due to the potential side effects of the need for contiguous long-term treatment, ciclospopna is indicated only in the case of refractory psapatic arthritis not adequately treated by other meios. In addition, it has been shown that cholesterol is effective for the treatment of severe atopic dermatitis in placebo controlled studies (Van Joost et al., Br.J. Derm., 130: 634-640 (1994); Coaper, J. Invest. Der., 102: 128-137 (1994)). Side effects such as nausea, abdominal discomfort, paresthesias, cholestadis, and renal insufficiency of the drug were preferred by patients to the disease without treatment. After a double-blind, randomized, placebo-controlled study, it was found that treatment with the poprin cycle significantly increases the quality of patients with severe atopic dermatitis (Salek e * al., Br.J. Derm., 129: 422-430 (1993)). The cutaneous lesions presented recurrences quickly after the suspension of the treatment with cipoles, but the quality of life remained improved. As another example, ciclopopna has been used in the treatment of chronic dermatitis of the hands, a disease with a reinforced prevalence of 4-22%, and typically treated with topical steroids to which, however, many patients do not respond. It has been found that a low dose of c i c 1 ospopna effec- tively treated 6-7 patients in an open-label study (Reita o and Granlund, Br.J. Derm., 130: 75-78 (1994)). Approximately the ital of the patients refrained after the suspension of the treatment with c ulospop na. ;or Co or another example, cyclaspopna has been used in the treatment of urticaria and angioedema, skin diseases that present urticaria and subcutaneous mellitus. The pathology is related to the mast cells, and the treatment is again ineffective. In one trial, three patients with refractory urticaria and angiaede were treated with ciclosporin and all symptoms disappeared within a week (Fradin et al., JM Acad. De., 25: 1065-1067 (1991)). All the patients had to discontinue the therapy due to side effects, * the symptoms reappeared after 1 to discontinuation of the therapy. As for other rheumatic diseases, the studies indicate an effective treatment by the ciclospopna of other less common autoimmune diseases, including Behcet's disease (PQ COG et a.1., Clin.Rheu., 13: 224-227 (1994)), and Wegner's null and void (Alien et al., Cyr.l ospop n A Therapy for Wegner 6ranu] ornaments in ANCA-Associa ed Vascul les: Immunologic l and Clincal Aspects, Gross ed. Plenum Press (1993)), and tro boc i immune mediated topenia (Srhultz et al., Blood 85: 1406-108 (1995)). In many of the trials described above, the use of ciclospopna or F1'506 was associated with many undesired effects. In general terms, the increased risk of infection, malnutrition is related to general immunosuppression, and it is unlikely that immunosuppressant related to proteins of anl3 e does not present similar risks. Other side effects can be avoided or reduced, however, by the tissue specificity of the anchor protein. The most severe collateral effect of both the c.c. osporina and Ff'506 is the nef rotox? > : idad, which, at least to some extent, is related to the dose and occurs in most patients, usually in the form of a - 1 decrease in the glomerular filtration rate during the treatment. This collateral effect, however, is when efíos is partially reversible when the administration of the drug is suspended (Leal r and Cairns, supra). Typically, progressive renal failure does not develop when more follow-up is necessary to obtain a definitive evaluation. Chronic damage was also observed in patients who received low doses of ciclospapna (3-4 mg / lg / d), approximately 4% of the biopsies of these patients showed changes in interstitial fibrosis, tubular atrophy, and artholopathy ( Varstad et al., Nephrol .Dial. Transplant, 9: 1462-1467 (1994); Young et al.), Fidney International, 46; 1216-1222 (1994)). Changes in the celiac cells were also apparent in isthological sections' ahan, N.Engl. J. ed. , 321: 1725-1748 (1989)). The possibility was posed that nephrotic disease was the primary result of artepolar vasoconstruction and chronic-grade ischemia (Lealer and Capns, supra), even though it was also shown that the drugs had a direct toxicity on the cells tubular and vascular »interstitial cells (Platz et al., Transplantat ion, 5 58: 170-178 (1994)). Some reports indicate that the incidence and severity of nephotaxia may be slightly higher with Fl-506 (Platz et al., Supra). Another significant toxicity reported from both ciclospopna and FK506 was neurotoxicity, with 0 clinical manifestations including "attacks, confusion, blindness, coma, headache, ataxia, Parkinson's syndrome, parestesis, psychosis, focal deficits, asymmetric mutism, tremors, neuropathies, and sleep disorders (Shimi zu et al, Pediatr Nephrol., 8: 4983-385 (1994); W? 3son et al., 5 Muse le and Nerve, 17: 528-532 (1994); Peece et al., Bone Marrom Transpl., 8: 393-401 (1991), Eidelman et al., Transpl.Proc., 23: 3175-3178 (1991), de Groen et al., N.Engl. Med., 317: 861-566 (1987).) After a liver transplant, moderate to severe neurotoxicity was observed in 10-20% of patients treated with Fl 506 and 3-12% of patients treated. with cyclosporine The neurotoxity was also associated with serum fluid abnormalities and hepatic dysfunction Other collateral effects of ciclospopna and / or F 506 * - "include hepatotox d, glucose intolerance, hypertension, hirsutism, gastrointestinal symptoms, thrombosis, pancreatitis, and ginyival hyperplasia (Morris, J. Heart Lung Transplant, 12: S286 (1993); Fung et al., Transpl. Proc., 23: 3105-3108 (1991); Mason, Phannacol. Fev., 42: 423-434 (1989); ahan, N.Engl. T.Med. , 321: 1725-738 (1989); Thomason et al., Renal Fail? Re, 16: 731-745 (1994)). Therefore, taking into account the widespread use of cyclasapopne and FK506 and the collateral effects inherent in its use, it could be extremely beneficial to develop end-tidal immunosupersors. For example, it is possible that the deslocal ionization of calcineupan from a putative T-cell anchoring protein can inhibit the activity of calcineupna in the activation of T cells, and consequently provide a specific immunosuppressant for T cells. that has the utility of ciclopopna or of FK506, but with less side effects. The previous observation that the deocalization of PKA ion from a T-cell anchor prstin increased the expression of IL-2 in stimulated cells indicated that PKA localized by anchor protein somehow mutates to a regulatopa function in the expression of IL-2 during the activation of T cells. The deslocal ion-specific T-cell of PKA can therefore provide a means to increase the secretion of IL-2 in vivo, thus mimicking the administration of recin b inants of JL-2 and possibly reducing the previously reinforced toxicity of treatment can IL-2 coma = will be described below. IL-2 has been approved for the treatment of metastatic renal carcinoma and approximately 15-20% of patients with metastatic renal cell carcinoma or malignant melanoma respond to 1L-2 therapy. Some of these responses are durable, with a duration greater than 66 months (Dill an, C ncer Biotherapy, 9: 183-209 (1994); Whittington and Faulds, Drugs 46: 446-514 (1993)). While high-dose bolus therapy has been associated with seborrheal side effects (as described below), continuous infusion or subcutaneous therapy with low doses produced a limited response rate (12%) while reducing toxicity ( Vogelzang et al., J .Cl in. Incol., 11: 1809-1816 (1993)). IL-2 therapy (with and without interferon-alpha and other agents) has been investigated for the treatment of other diseases. For example, were sustained clinical responses, but not cures, obtained in direct application of JL-2 on tumor beds after? Glia as resection (Merchant et al., J.Neuro., 8: 173-188 (1990)). In other trials, limited efficacy was reported in the treatment of lymphoma (Di liman, supra), colorectal carcinoma (Whittington and Faulds, supra), limited AML 'Bruton v oeller, Pharmacotherap, 14: 635-656 (1994)), cancer of the ovaries as well as early cancer of the bladder (Whittington and Faulds, supra). The number of participants in each of these studies was too small to allow significant confusions in terms of effectiveness. IL-2 has also been used in combination with adoptive innotherapy and has been shown to be effective for the treatment of metastatic renal carcinoma (Pierce et al., Se .Oncol., 22: 74-80 (1995); Belldegrun et al. al., J.Urol., 150: 1384-1390 (1993)). In addition, I-2 can also be effective for the treatment of certain infectious diseases, decreasing the bacterial load of the skin and antigen levels in patients with leprosy after intradermal injection (Kaplan, J. Infect. Dis., 167 (suppl. l): S18-22 (1993)). It has also been observed that, compared to healthy controls positive for PPD, the lmfocitos of patients with tubeculosia produce levels lower than IL-2 (Sánchez et al., Inf.I un., 62: 5673-5678 (1994)) , suggesting that 1? Therapy with TI-2 may be valuable for the treatment of mycobacterial infections. Despite the potential therapeutic value of TL-2, the cytokine is also associated with significant toxicity unless stated otherwise, the sources being Whittington and Faulds, Di liman and Bruton and Koeller, supra). The side effects that limit the treatment to a greater extent are the capillary leak syndrome. The administration of IT-2 increases vascular permeability, causing intimal and pulmonary edema, and patients develop hypotension and a substantial number of them require prescriptions. A vigorous resuscitation of fluid can cause life-threatening pulmonary edema. Up to 20% of patients may require incubation and mechanical ventilation. The ad ti nst ra tion of t > High doses cause more severe leaks than low doses or slow continuous infusions, and in some groups, 100% of patients require TCT support during treatment with IL-2. Myocarditis, cardio-oopathy as well as cardiac skills have also been observed. Acute renal failure can also occur as a result of hypotension induced by capillary leak syndrome. IL-2 can also cause severe diarrhea with electrolyte imbalances, cholestasis, thyroid abnormalities, and acute pancreatitis. In 15-20% of treated patients it is seen half-heartedly requiring transfusions (MacFarlane et al., Cancer 75: 1030-1037 (1995)). the top and top with hemorrhage may occur and defects of the clotting pathway are common. More than 70% of patients experience changes in mental status, including paranoid delusions, lucinations, loss of interest, sleep disturbances, and sobriety. S also reported coma, visual defects, ischemic attacks, and parestesis. These drawbacks associated with exogenous IL-2 suggest that alternatives such as the endogenous TL-2 solution that can be modulated and therefore eliminate the requirement for exogenous IL-2 treatment as potential therapeutic solutions should be explored. In addition to providing possible means to identify drugs and immunosuppressants and modulators of the IL-2 solution, the identification of the anchor proteins makes it possible to regulate other cellular activities due to the diverse metabolic pathways in which it has been shown that the proteins of Anchoring involved for example, AKAP 79 is important for the regulation of ion channels regulated by glutamate receptor in the post-smáptica density of neurons, probably by means of PIA, PC, and ca ine link. PKA regulates the activity of channels regulated by AMPA receivers, and the deslacal i zation or inhibition of PKA attenuates the activity of AMPA ion channel. PCK regulates the activity of regulation channels by NMDA receptors, and it has been shown that lupine decreases the NMDA receptor to stimuli. These observations indicate that localized kinases (P A and PKC) can regulate l a. activity of glutamate receptors in neurons. The dephosphorylation by cal i neur is the counterregulatory mechanism of NMDA receptors. This model corresponds physiologically to the evidence of attacks induced by the papna or Ft506. In addition, glutamate receptors have been associated with many neurological diseases. Glutamate and other excithate amino acids can produce an excitotoxicity in neurons, and excessive stimulation of the po- tassyptidic receptors of gliatamate has been shown to be toxic to neurons, causing acute neuronal degeneration. Hypaxia (such as after stroke or cardiac arrest) and traumas of the central nervous system have been shown to cause a marked effusion of glutamate in the tracellar space, which then interacts with glutamate receptors and triggers the excotoxin cascade. ic. It has been shown that anti-exc? They protect against brain damage in animal models (Olney, Neurobiology of Agmg, 15: 259-260 (1994)). It is interesting to note that NMDA antagonists are toxic to certain types of neurons indicating that glutamate can inhibit other excitatory pathways in these cells. It has been shown that macrolysis antibodies, such as FK506, protect against NMDA, but not camata, exc totaxity in cultured neuron * (Manev, et al., Brain Res., 624: 331-335 (1993 )). 59 Glutamate has also been implicated in Part-inson's disease. NMDA antagonists protect neuronal dopamine neurons in the a. black substance in monkeys exposed to MPTP, a chemical that induces Parí inson syndrome in humans and other primates. Amantanine and memantine are NMDA antagonists and have been used: in Europe to treat the disease of Par 1 mspn, however it has been shown that both cause psychosis in some patients. There is also some evidence that gluttaglidergic neurons may become overactive in Parson's disease and their inhibition may be reduced in the motor symptoms of the disease (Lange and Riedr r, Life Sciences, 55: 2067-2075 (1994)). Glutamate also plays a role in apoplectic disorders, participating in the initiation, development and • nanning of the attack activity. NMDA and non-NMDA antagonists are an iconvul if powerful (Meldrum, Neurology, 44: ísuppl 8): S14-S23 (1994)). AMPA receptors have also been implicated in ALS and one assay of a receptor antagonist is currently in progress' 49). Based on all these observations, it is not surprising that numerous other immunosuppressants are in clinical trials. The following information regarding such trials was obtained from Haydon and Hay, Balliere's Clín.Gastroentero. , 8: 455-464 (1994); Thomason and Starzi, I munol.Rev. 1993, 71-98 (1993); and Morris J. Heart Lung Transplant., 12: S275-S286 (1993). For example, azaßpyrene is a compound of SK'R that suppresses cellular infiltrates in intakes and the induction of IL-2R, and also abolishes the production of IL-2 and IFN-gamma. Appetically the azaspirano induces some type of suppressor cell and there is evidence of synergistic effects with the form cycles. As another example, mirof nolate is a Syntex compound that inhibits the synthesis of pupils and has a selective anti-prophylactic effect for T and B cells. It decreases the level of antibodies. Mycophenolate mofetial can also decrease the level of adhesion molecules on cell surfaces. While the drug appears to have low toxicity, it can cause leukopenia, and has been used in the treatment of psoriasis for 20 years. As another example, mizopbipa is a Sumi tome compound that inhibits DNA synthesis. The action maceni is identical to the icofenola to. As another example, brequinar is a compound of DuPont-Mercl that inhibits the synthesis of l a. pipmidine by the block of the hydrogenase dihydrorat. Complete reports of clinical trials are expected. It has been reported that the drug acts if it interacts with the claspop na, but it can p evokes rt omboc i topen i, der to 1111 s and mucos i 11 s, As another example, 15-deso ispergu 1 ino is a compound of Nippon-Kayat u that predominantly affects the function of the anus / macr phages, including the inhibition of oxidative metabolism, lysosomal enzyme synthesis, I Ll production, and similar surface expression of MHC claes II antigens. It is effective in 70-90% in refractory kidney rejection, but it can also be observed that bone marrow toxicity is higher in doses. _ * "As another example, lefluno ida is a compound of Hoeschst that inhibits the action of cytokines, blocks the activation of T cells and the synthesis of antibodies.It is a non-toxic product for the kidneys and bone marrow. As another example, raparm ina is a compound of Wyeth-Ayerst related to FK506. It is a prodrug that must bind to an immunophilin to be activated and inhibits calcineupna or blocks the production of cytokine, T cells. By an unknown mechanism, rapamycin blocks the transi tion from Gl to S. those skilled in the art will be able to design numerous modifications and variations to the present invention in accordance with what is presented in the illustrative examples. Accordingly, the present invention is limited only on the basis of the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Lockerbie, Robert O. I ashishian, Ada di) TITLE OF THE INVENTION: Novel PKA binding proteins and uses thereof, (ni) SEQUENCE NUMBER: 5 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Marshall, O'Toole, Gerstein, Murray Borun (B) STREET: 233 South Wacker Drive, 6300 Sears Tower (OR CITY: Chicago (D) STATE: Illinois (E) COUNTRY : US. (F) POSTAL CODE: 60606-6402 (v) COMPUTER LEGIBLE FORMAT: (A) MEDIA TYPE: Soft disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS- DOS ÍD) PROGRAM: Patentln Rel ase No. 1.0, Version Na. 1.25 (vi) CURRENT REQUEST DATA: (A) SOCIAL NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (vi) ATTORNEY / AGENT INFORMATION: (A) NOMBPF: Williams Jr., Jaseph A. ( B) REGISTRATION NUMBER: 38.659 (C) REFERENCE NUMBER / CEDULA: 27866 <? X) INFORMATION FOR TELECOMMUNICATION: (A) TELEPHONE: 312-474-6300 (B) TELEFAX: 312-474-0448 (C) TELEX : 25-3856 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (O No. OF HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (< i) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Asp Leu He Glu Glu Wing Wing Ser Arg He Val Asp Wing V l He 1 5 10 15 Glu Gln Val Lys Ala Wing Gly Wing 20 (2) INFORMATION OF SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (O No. OF HEBRAS: unique (D) ) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Asp Leu He Glu 81u Ala Ala Ser Arg Pro Val Asp Ala Val He 1 5 10 15 Blu Gln Val Lys Ala Ala Gly Wing 20 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nuclico acid ^ - "•" (C) No. OF HEBRAS : unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NOs 3s ATTAACCCTC ACTAAAG (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) No. OF HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4s GATATCACTC AGCATAA (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2850 base pairs (B) TYPE: nucl ical gone (C) No. OF HEBRAS: unique (D) TOPOLOGY: linear (ii) T MOLECULE IPO: cDNA ( xi) DESCR I SECUENC IA PC: SEQ ID NO s 5: GOC? Cß? Oß? GC? QC? OOTO C? OßCT ?OTO CTOTOC? OCT G? OOOCTOAC CCTOCCATC? 60? CG ?? CCTCT CCCCCTCO ?? G? CCTCTOTC CC ??? OT? OT OTCC? C? CCC CCC? ßTOTC? 120 C? ß? GCCTCC? ß ???? ßG ?? CTQTCC? CCO TO? OC? OCT OCCTOC? O? O CCC? C? CC? T lßO T? CTCC? G? C? C? CCC? CCT T? CCT ?? ß? T C? O? OTCCTC OOOC? TTCT? -CCT ?? C? CCX 240 C? G? C? Tß ?? ? TTOCß? CC? Oß ?? C? COC? C? ß? CO? C? OT? C ??? šCTO O? CCT? OCCC 300 T? C? G? T? S? T? CC? TCO? TTCCTC T? O? OTQCCC CCTTTC? TCC CC ??? OOOTO 360 T? CT? TTCTC C? GC ??? TC? OCTG? OQTGT CT ?? ßC ?? O? TTCCOCCTTC? CC? OCOTOC 420 C? ß? ?? OOT CC? OCC? OOC T? CCCCOT? O TCCCCßC? O? O? OCOT? ßC TCTCOCO? C? 480 GGCC ?? O? G? ß? C? ßOTOOß OCCO? OOO? CTOOTO? TOC COTOTTOOÃSO? C ???? OOTOC 540 5 TTO ?? G ?? GC TCTOTTOTCT COGO? ßC? TO TCTT? Β ?? TT OO? O ?? C? OC ?? OOOCCCC? 600 OCCTGGCCTC TTT? ß? ßOGO ß ?? G ?? G? T? ? GGCG ?? ß? O C? OCTC? TCC C? OGTTOOTO 660 CGGCC? ßTOC? OO? ßß ?? ß? OT? TOT? ßC? G? G? OTTOC C? CT? OOTT C? TC? GTCO 720 GCTC? C? C? ß? GCTßßC ??? GG? CC? TGCG GCGCC? ßC? C CCCC? OTCGC? Ú? CGCC ??? 760 CCCC? Cß? C? O? ßßTOTCO? GGO? ß ?? CTO GGC ?? TO? OO? OR? OCTTGß? T? G ??? Tß? O 840 G? GGGCTTGG? T? ß ??? Tß? Oß? GGGCTTG C? T? ß ??? TO? ßO? G? CCTT CC? TAG ??? T 900 GAGGAGGGCT TGC? T? G ??? TCAGCAGATT? AGCCOßCTß CCTTCC? G? T ?? TCTCCC ?? 960 ? 0 GTGATCTCAO? AGC ?? CCG? ACAGGTGCTG CCC? CCACßß TTGCC? OCT TGC? OOTOOT 1020 CTGTCTCACG CCACTCAGCT CCAAGGGCAG AAGGAAGAGA ßCTOTCTCCC? GTTCACC? ß 1060 AAAACTGTCT TGGGCCC? G? CACTCCCOAC CCTOCC? C? C? G? ßOCACC TCTTOCCCCG 1140 CCGGATGCTG CCCTCCCCTT GCCAGCCCTA CCAGCAGACO CCTC? CC? CC? CCA? C? CC \ 1200 TACOTCAGCT GCCTG ?? G? ß CCTTCTQTCC AGCCCCACCA AGGAC? ßT ?? OCCAAAT? TC 1260 TCTGCACACC? C? TCTCCCT GGCCTCCTGC CTG? CACT? CC? CCCC? O TG? G? Ofl? 1320 5 CCGGACCGG? CAGCCATCCT GGTGG ?? G? T OCC? CCT? T? TCACCTG? T CTC? C? C?? C 1380 AGCC ??? CTO TCCCTTT? ΒT ßßCTTCTCC? OC? C? CTGCT C? O? TTCTTT C? ßC? CTTC? 1440 OßßCTTG ?? ß? CTCTTTC? C? ß? ß? CC? OC TCO? ßCCCC? OOO? C ?? ßGC C? TC? CCCCO 1500 CC? CTGCC? O ??? OT? CTOT GCCCTTC? ßC ?? TOGOOTTC TO ?? OOOOO? OTTOTC? ß? C 1560 TTGGGGGCTO? CG? TCO? T? G? CC? TGG? T GCGG ?? GC? G? TC? TTC? ß? ßCTCTTC? C 1620? ß? ?? C? ßC? Tßß? TTCCOT ßß? T? ßCTßT TCCAOTCTCA? ß ?? ß? CTß? O? ßCTTCC ?? 1680 ?? TßCCC? ßß C ?? ßCTCC ?? CCCT ?? ß ?? ß OTCO? CCTC? TCATCTGOGA G? TCO? ßßTß 1740 CC ??? ßC? CT T? OTCßOTCO CCT ?? TTOßC ?? ßC? CGOGC ßCT? TGTC? C TTTTCTß ?? ß 1800 C ??? CATCT? CTOCC? ß? T CT? C? TTTC? ? CCCTßCCTT? C? CCC? ß? ß COTCCAG? TC 1860 TßCC? C? T? O ?? ßßCTCTC? AC? TC? TTT? ß? C ??? ßCOC TO ?? CTTß? T Tßßß ?? O ?? ß 1920 TTC ??? ß? OC TO ?? CCTC? C C ?? T ?? CT? C šCTCCCCCAT TOCCTTC? CT GOCACTOCCT 1980 TCTCTGCCGA Tß? C? TCCTG CCrCATßCT? CCT?? TGCC? TC? CCßTßß? OGTC? TTGTC 2040 CTCAACCAGC TC ?? TCCCO? ßC? CCTOTTC OTGC? ßC? CC? C? C? C? CCC TACCTTCCAC 2100 GCGCTGCCC? OCCTCß? CC? OC? O? TOT? C CTCTOTT? CT CTCAGCCÍOO K? TCCCC? CC 2160 TTGCCCACCC C? ßTßß ??? T ?? COOTC? TC TOTOCCGCCC CTßßTOCOO? COß? GCCTOO 2220 TOß8? OCCC ?? OTßßTTGC CTCCT? Cß? ß ß? ß? CC? C? A? CTCGAO? T TC?? T? COTO 2280 O? CT? COOCG O? T? T ?? ß? O ßßTß ?? OT? O? CßTOCTCC OOC ??? TC? ß OTCTO? CTTT 2340 OTC? CCCTOC COTTTC? OOO AßC? ß ?? ßTC CTTCTßß? C? GTOTO? TOCC CCTGTC? O? C 2400 O? TC? CC? ßT TTTC? CC? O? ? ßC? ß? TOCC ßCCATG? ßCO Aß? Tß? COOO O? T? C? ßC? 2460 CTOCTTOCTC AOßTß? C ?? G TT? C? ßTCC? ? CTC? CTCTO? TTC? ßCTGTOO? OT 2520 ßTßCTTOß? ß ATOAAGTGCT OTTGATAAAC CGßTCCCTGO TOß? ßCß? ßß CCTTßCCC? ß 2560 TGGGT? ß? C? OCT? CT? C? C ?? ßCCTTTO? CCCCC? TGCT OCTTCCTC? ß? GTCTTTTTT 2640 GC? CTOTTC? AATTßßOCTT GßC? CTC ?? ß TC ??? ß? Tß? ? C? TCGO ?? T ?? C ??? C? TT 2700 OTCCTCTCC? O ??? OTCCTT TCTTT? TCC? T? CTOT? ßTC CT? TTß? ß ?? ß? C? TTTCGT 2760 CTCTGAG ?? A ??? Gß? TOG? ? CT? TGGGTT CTCTTCGC ?? AßCC ??? CC? T? GTGTTT ?? 2820 C? GCC? ßCT G? TCT? TCCT OGCTC? T? CC 2850

Claims (15)

1. CLAIMS 1. A purified and purified purified polynucleotide comprising the protein coding sequence presented in SEQ ID NO: 5.
2. 2. The polynucleotide of claim 1 which is a DNA molecule.
3. 3. The DNA molecule of claim 2 which is a cDNA molecule.
4. 4. The DNA molecule of claim 2 that a genomic DNA molecule.
5. 5. The DNA molecule of claim 2 is a total or partially chemically synthesized DNA molecule.
6. 6. A purified and isolated polynucleotide selected from the group consisting of: a) the human DNA sequence presented in SEQ ID NO: 5, and b) a DNA molecule that hybridizes under the eßtrictaß condition with the non-coding DNA strand of to ) .
7. 7. A DNA expression cassette comprising a DNA molecule according to claim 2.
8. 8. A host cell transformed with a DNA molecule in accordance with the rei indicates ion 2.
9. 9. A method for the production of a human polypeptide encoded by the polynucleotide presented in SEQ ID NO: 5, which comprises culturing a huéeped cell according to claim 8 in a suitable medium and isolating the polypeptide from said host cell or from the host medium. culture.
10. 10. A purified and isolated polypeptide encoded by the polynucleotide sequence presented in SEQ ID NO: 5.
11. 11. A polypeptide capable of specifically binding to a polypeptide of claim 10.
12. 12. A polypeptide according to claim 11 which It is an antibody.
13. 13. An antibody according to claim 12, which is a monoclonal antibody.
14. 14. An anti-idotypic antibody specific for the monoclonal antibody of claim 13.
15. 15. A hybridoma cell line that produces the monoclonal antibody according to claim 13.