GAMMA II ADAPTIN

Abstract: A mammalian protein involved in receptor-mediated endocytosis specifically binds to phosphatidylinositol 3-kinase. More specifically, it binds to the Bcr homology domain of the p85 subunit of phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase also binds to platelet derived growth factor receptor. Other receptors may also bind, including those for insulin, insulin-like growth factor-1, colony stimulating factor 1, nerve growth factor, hepatocyte growth factor, stem cell growth factor, and epidermal growth factor. Mitogenesis is the consequence of the binding and internalization of these growth factors. Inhibition of the process of receptor-mediated internalization inhibits mitogenesis.

Description: GAMMA π ADAFΠN This application is a continuation-in-part of U.S. Serial No. 60/019,462 filed June 13, 1996, and U.S. Serial No. 60/018,162 filed May 23, 1996, the disclosures of which are expressly incorporated herein. TUΓKGHOTTNTI OF TTTF. TNWIVTTON Receptor-mediated endocytosis is the mechanism by which a variety of nutrients, hormones, and growth factors are specifically and efficiently transported into the cell as described in Goldstein et al, Annu. Rev. Cell Biol. 7:1-39 (1985). During this process, receptors are selectively concentrated in clathrin-coated pits from which they are rapidly internalized and delivered to endosomes; some receptors like the low density lipoprotein (LDL) receptor are constitutively clustered and internalized in the absence of ligand, while others, such as the epidermal growth factor (EGF) receptor are concentrated in the coated pits and internalized only after binding ligand, as described in Trowbridge, Current Opin. in Cell Biol. 5:634-641 (1991). The protein complexes that link clathrin to transmembrane proteins are called adaptors. Plasma-membrane adaptors contain an α-adaptin and a β-adaptin subunit, while adaptors found in the Golgi apparatus contain a γ-adaptin and a β'- adaptin subunit, as described in Robinson, J. of Cell Biol. Ul: 2319-2326 (1990). Despite these general outlines of the process of receptor-mediated endocytosis, the identities of all of the molecules involved and the interactions among them are not known. Thus, there is a continuing need in the art for identification of components of this important process. It is an object of the invention to provide a newly identified adaptin protein involved in receptor-mediated endocytosis. _ . It is an object of the invention to provide a fusion protein comprising a portion of the new adaptin molecule It is another object of the invention to provide an isolated polypeptide which consists of a portion of the new adaptin molecule. It is still another object of the invention to provide a preparation of antibodies which specifically bind to the new adaptin molecule. It is another object of the invention to provide a polynucleotide which encodes the new adaptin molecule. It is yet another object of the invention to provide methods of screening for agents useful in inhibiting mitogenesis. These and other objects of the invention are provided by one or more of the embodiments described below. Li one embodiment of the invention an isolated and purified mammalian γll-adaptin protein is provided. The protein binds to the Bcr homology domain of phosphatidylinositol 3-kinase. In another embodiment of the invention a protein is provided which comprises the sequence shown in SEQ ID NO: 1. In another embodiment of the invention a fusion protein is provided. The fusion protein comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment consists of at least eight contiguous amino acids of a mammalian γll-adaptin protein. In yet another embodiment of the invention an isolated polypeptide is provided. The polypeptide consists of at least eight contiguous amino acids of a mammalian γll-adaptin protein which binds to the Bcr homology domain of phosphatidylinositol 3-kinase. In yet another embodiment of the invention an isolated preparation of antibodies is provided. The preparation specifically binds to a mammalian γLI- adaptin protein, wherein said protein binds to a Bcr homology domain as shown in amino acids 132-322 of SEQ ID NO: 4. According to another embodiment of the invention a subgenomic polynucleotide is provided. The polynucleotide encodes a mammalian γll-adaptin protein, wherein said protein binds to a Bcr homology domain of a p85 subunit of phosphatidylinositol 3-kinase. According to still another embodiment of the invention a method of screening for agents useful in inhibiting mitogenesis is provided. The method comprises the steps of: contacting a test compound with a mammalian γll-adaptin and a protein comprising &Bcr homology domain, wherein the protein binds to the γll-adaptin in the absence of the test compound; determining the amount of at least one of γU-adaptin and the protein which is bound or unbound in the presence of the test compound, wherein a test compound which decreases the amount bound or increases the amount unbound, of at least one of γπ-adaptin and the protein is an agent useful for inhibiting mitogenesis. According to still another aspect of the invention, a method of screening test compounds to identify agents useful for inhibiting mitogenesis is provided. The method comprises the steps of: contacting a cell with a test compound, wherein the cell comprises: a first fusion protein comprising (1) a DNA binding domain and (2) all or a portion of a mammalian γJJ-adaptin, wherein the portion is capable of binding to p85 subunit of phosphatidylinositol 3-kinase; a second fusion protein comprising (1) a transcriptional activating domain and (2) all or a portion of a p85 subunit of phosphatidylinositol 3-kinase, said portion comprising a Bcr homology domain of p85 subunit of phosphatidylinositol 3-kinase consisting of amino acids 134-322 as shown in SEQ ID NO: 4, wherein the interaction of the first and second fusion proteins reconstitutes a sequence- specific transcriptional activating factor; and a reporter gene comprising a DNA sequence to which the DNA binding domain of the first fusion protein specifically binds; and measuring the expression of the reporter gene, wherein a test compound that decreases the expression of the reporter gene is a potential inhibitor of mitogenesis. According to still another aspect of the invention another method is provided for screening test compounds to identify agents useful for inhibiting mitogenesis. The method comprises the steps of: contacting a cell with a test compound, wherein the cell comprises: _ _ a first fusion protein comprising (1) a DNA binding domain and (2) all or a portion of a p85 subunit of phosphatidylinositol 3-kinase, said portion comprising a Bcr homology domain of p85 subunit of phosphatidylinositol 3-kinase consisting of amino acids 134-322 as shown in SEQ ID NO: 4; a second fusion protein comprising (1) a transcriptional activating domain and (2) all or a portion of a mammalian γU-adaptin, wherein the portion is capable of binding to p85 subunit of phosphatidylinositol 3-kinase; wherein the interaction of the first and second fusion proteins reconstitutes a sequence-specific transcriptional activating factor; and a reporter gene comprising a DNA sequence to which the DNA binding domain of the first fusion protein specifically binds; and measuring the expression of the reporter gene, wherein a test compound that decreases the expression of the reporter gene is a potential inhibitor of mitogenesis. The present invention thus provides the art with methods and reagents for drug discovery. BKTEF imSCRTPTTON OF THE SEQUENCES SEQ ID NO: 1 provides the amino acid sequence of mouse γll adaptin. SEQ ID NO: 2 provides the polynucleotide sequence encoding mouse γll adaptin. SEQ ID NO: 3 provides a peptide sequence used for immunization of sheep. SEQ ID NO: 4 provides the Bcr homology domain of phosphatidylinositol 3-kinase. nFTATT,FJ> nESCRTPTTON OF THE PREFERRED EMBODIMENTS The inventors have discovered a new member of the family of mammalian adaptin polypeptides. This polypeptide has been named γU adaptin. The polypeptide binds to the Bcr homology domain of phosphatidylinositol 3-kinase (PI 3-kinase), shown in SEQ ID NO: 4. PI 3-kinase is described in Carpenter et al, Mol. Cell. Biol. 13: 1657-1665 (1993). While not wishing to be bound by any theory or mechanism of action proposed, it is believed that γLI adaptin, like other members of the adaptin family, is involved in receptor-mediated endocytosis. Phosphatidylinositol 3-kinase binds to the receptor for PDGF. Upon PDGF (platelet derived growth factor) stimulation, cells are induced to undergo mitosu. In addition, the cellular localization of γU adaptin has been found to change upon PGDF stimulation. Upon stimulation of resting cells with PGDF, the amount of γU adaptin at the membrane is decreased, and the concentration of γll adaptin in the perinuclear region increases. This change in localization is consistent with a role in endocytosis. Mammalian γU adaptin protein as provided herein can be isolated and purified according to conventional techniques. Provided with the sequence of the protein, antibodies can be routinely generated against all or portions of it. Antibodies can be affinity purified, using the γll adaptin protein or polypeptide portions thereof. Such antibodies can, in turn, be used to purify γll adaptin from cell lysates, for example, or to quantitate the amount of γU adaptin in cells. Techniques for immunizing host animals to raise antiserum are well known in the art. Similarly, techniques for using such immunized host animals to raise monoclonal antibodies are also well known. Both types of antibodies are contemplated within the scope of the invention. Mammalian γLI adaptin proteins and polypeptides can also be made using recombinant host cells. The nucleotide sequence of one gene which encodes a mammalian γll adaptin protein is provided in SEQ ID NO: 2. This gene, or other genes which are obtained from other mammals by hybridization under stringent conditions, or modifications of the gene which retain the coding capacity for a protein which binds to the Bcr homology domain of phosphatidylinositol 3-kinase, can all be used in recombinant cells to produce a γU adaptin protein. Preferably such proteins will be at least 85 % , and more preferably at least 90 % identical to the sequence provided in SEQ ID NO: 1. Techniques for producing recombinant proteins given the nucleotide sequence encoding the protein are well known in the art. Selection of an appropriate technique is well within the purview of the skilled artisan. Portions of the gene can also be used to express portions of the γll adaptin protein. These may be used, even if they do not retain the ability to bind to phosphatidylinositol 3-kinase, to raise antibodies, as discussed above. Typically a minimum number of contiguous amino acids to encode an epitope is 6, 8, or 10. However, more may be used, for example, at least 15, 25, 25, or 50, especially to form epitopes which involve non-contiguous residues. Fusion proteins are also provided here, which comprise all or a portion of γU adaptin covalently linked to a second protein or protein domain. Typically, at least 8 contiguous amino acids of γll adaptin will be used, in order to constitute an epitope. The second protein can be an unrelated protein such as β-galactosidase or glutathione-S-transferase, which are useful for processing and purification with standard reagents. Alternatively, the second protein can harbor a transcription activation domain or a specific DNA binding domain. Such fusion proteins are useful for methods employing the two-hybrid technique, for example, in yeast cells. Techniques for making fusion proteins, either genetically or by covalently linking two proteins, are well known in the art. For some uses, such as in the two hybrid assays, it is desirable that the fusion protein retain the ability to bind to phosphatidylinositol 3-kinase. Polynucleotide molecules which encode γll adaptin are also provide herein. Such molecules are typically subgenomic, i.e., they are fragmented with respect to the state in which they exist in the genome. Thus they may comprise PI or YAC genomic clones. They may also be cDNA clones, and thus intron-free. They may contain their own native expression control sequences, or they may be fused to expression control sequence from other genes or even other organisms, depending on the expression system in which they will be used. The polynucleotide molecules may be propagated in vectors, either prokaryotic or eukaryotic, and these may be maintained and expressed in host cells. Suitable vectors and host cells are available for many different purposes, and the particular choices are well within the skill of those in the art. The polynucleotide molecules of the invention may encode portions of a γπ adaptin protein, especially those portions which retain binding ability to the p85 subunit of phosphatidylinositol 3-kinase. In addition, they may encode fusion proteins and polypeptide portions of γll adaptin which are useful for raising antibodies. Typically these molecules will encode at least about 8 contiguous amino acids of γll adaptin, and thus will comprise at least 24 contiguous γπ adaptin- encoding nucleotides. Such polynucleotides which encode less than a full length γπ adaptin are also useful as nucleotide probes for obtaining γπ adaptin variants and γll adaptin genes from other species. Techniques for hybridizing polynucleotide probes and primers to obtain related family member genes are well known in the art. Methods are provided for performing screening assays on agents which may be useful in inhibiting mitogenesis. The basis for all of these assays is the discovery of the binding interaction between γπ adaptin and phosphatidylinositol 3-kinase, and the functional consequence of the binding in mediation of the intracellular signaling of PGDF. Since PGDF is a known mitogen, agents which are found to inhibit the interaction of γH adaptin and phosphatidylinositol 3-kinase will be useful in inhibiting mitogenesis. According to one method a γπ adaptin and a protein comprising a Bcr homology domain are incubated together in the presence of a test compound. In the absence of the test compound the protein binds to γπ adaptin. The amount of bound and/or unbound proteins is determined according to any technique known in the art, including any immunological technique. In order to facilitate the assay, one of the proteins may be bound to a solid support, or may be labeled with a radiolabel, or other detectable label. A useful agent is identified which decreases the amount of protein bound or increases the amount of protein unbound. The proteins can be prebound prior to the introduction of the test compound, or the test compound can be contacted with one of the two binding partners prior to incubation. The protein which has a Bcr homology domain may be, for example, phosphatidylinositol 3-kinase, rho-GAP, n-chimerin, pl90, Bem2, Bem3, 3BP1, Bcr, or phosphatidylinositol 3-kinase. Alternatively, a protein containing the Bcr homology domain of one of the proteins can be used. A two-hybrid assay can also be used to assay for useful agents for inhibiting the interaction between γπ adaptin and phosphatidylinositol 3-kinase. According to such an assay, fusion proteins of each of the binding partners are used which each contain at least the domains necessary for the binding interaction. One of the binding partners is fused to a DNA binding domain and the other is fused to a transcriptional activating domain. The two fusion proteins interact to reconstitute a sequence-specific transcriptional activating factor. The two fusion proteins are produced in a cell which also contains a reporter gene which is sensitive to the activation of the reconstituted sequence-specific transcriptional activating factor. In the absence of test compound the cell expresses the reporter gene. Test compounds are added to the cell and the effect on the reporter gene's expression is monitored. A test compound which disrupts the binding of the γπ adaptin and the phosphatidylinositol 3-kinase domains will have a negative effect on the transcriptional activation ability of the reconstituted sequence-specific transcriptional activating factor. Thus the expression of the reporter gene will be reduced. The assay may also be conducted in the presence of agents which may bind directly or indirectly to phosphatidylinositol 3-kinase, including PGDF, insulin, IGF-1, CSF1, NGF, hepatocyte growth factor (HGF), stem cell growth factor, EGF. Example 1 Cloning the γ II Adaptin gene by Yeast 2-Hybrid System using Bcr homology domain of P85 as Bait The yeast 2-hybrid system as described in U.S. Patent No. 5,283,173 and Chien et al, Proc. Nat'lAcad Sci USA 38:9578-9582 (1991) was used to screen a mouse thymus cDNA library, using the Bcr homology domain of the p85 subunit of PI 3 kinase as bait. See Cell 6 * 5:91-104 (1991), which teaches the characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp66 <>4rc complexes, and phosphatidylinositol 3-kinase. Yeast strain Y190 is a Y153 derivative that is resistant to cyclohexamide (2.5 ug/ml) due to a mutation in the CYH2 gene (genotype MATa gal4 gal80 his3 trp-1- 901 ade2-101 ura3-52 leu2-3,-112 +URA3::GA1> lacZ, LYS2::GAL(UAS)- HIS3 cyh " ). Y190 was supplied with 2-hybrid kits by Dr. Stephen J. Elledge of Baylor College of Medicine at Houston, TX. A bait plasmid and a cDNA library cloned into vector PACT were transformed into strain Y190. The bait plasmid consisted of the Bcr homology domain of the p85 subunit of PI 3 kdnase cloned into pASl- CYH, a GAL4 DNA-binding domain fusion vector. Genes and Development 7:555- 569. The prey plasmid PACT encodes the GAL 4 transactivation domain. Upon reconstitution of the GAL4 transcription activating factor, lacz and HIS3 are induced. Transformants were selected for tryptophan prototrophy. Expression of the fusion protein was verified by western blotting with antibodies against the Bcr homology domain of the p85 subunit of PI 3 kinase. The resulting strain was confirmed by checking for its growth properties on SC-His plates containing 3-AT (3-aminotriazole, A8056, SIGMA, St. Louis, MO) and for its ability to activate the LacZ reporter. These tests were carried out relative to control strains carrying SNF1 in the pASl vector. 3-AT concentrations of 25 mM to 50 mM are sufficient to select against pASl clones that fail to activate transcription on their own. A strain that failed to activate transcription was selected for transformation by the mouse thymus cDNA library. A colony was used to inoculate 50 ml of SC-Trp and grown overnight at 30°C. An optical density reading (OD) of the culture at absorbance of 600 nM was taken and the culture was subsequently diluted to 0.1 (OD wo) in 250 ml medium YEPD. [YEPD medium is made (for a one liter batch) with 10 g bacto-yeast extract, 20g bacto-peptone, 20 g glucose, and 40 mg adenine sulfate.] Synthetic complete minus Tip media is used to select for pASl but YEPD gave the best transformation efficiencies. The cells were harvested at 5,000 rpm for 7 to 8 minutes in a small laptop centrifuge. The cells were washed once with TE (approximately 20 mis) and resuspended in 20 mis LiSORB and incubated at 30°C for 15 to 30 minutes. The cells were spun down as above and resuspended in 2 ml of LiSORB (LiSORB consists of 100 mM LiOAc, 10 mM Tris pH 8, 1 mM EDTA, and 1M Sorbitol), and an aliquot of 100 ul was placed into each of 20 tubes, and held on ice. The DNA carrier mix was then prepared by boiling 200 ul of 20 mg/ml sheared salmon sperm DNA for 7 to 10 minutes. 800 ul LiSORB at room temperature was added and mixed by pipetting up and down. The mixture was cooled to room temperature, making sure, however, that the temperature did not go below room temperature so that the mixture did not gel. Forty ug of mouse thymus library cDNA was added. One hundred ul of the DNA mix was mixed with 100 ul of yeast Y190 cells in one of the 20 tubes of aliquoted cells. One hundred ul of the cells with DNA, 900 ul of 40% PEG-3350 in 100 mM LiAc/TE was added then the mixture was incubated at 30°C for 30 minutes and the mixture was heat shocked at 42°C for 5 minutes. The cells were harvested and resuspended in 300 ul mis TE and plated at about 300 ul per 150 mm plate at 50 mM 3-AT. 30 ul of Sc-Trp Leu was plated to test transformation efficiency. Colonies that grow after 3 to 5 days were tested for β-galactosidase activity using a standard X-Gal colony filter assay. Blue colonies were taken for further study. The efficiency of transformation was about 5 x 10 4 to 10 s colonies per ug of cDNA library. A positive control was pSEllll (an SNF4 yeast transcription factor fused to the activation domain in pSE1107) and pSE1112 (an SNF1 yeast transcription factor fused to the DNA-binding domain of GAL4 in pASl). These are both inserted as BamHI fragments and can serve as a positive control for X-gal staining and 3-AT resistance. A negative control was pSF1112. The weak activation of pASl appears to decrease when genes are cloned into it. Plasmids were harvested from the confirmed positive yeast colonies, which were then transformed into bacteria. Ten to 20 colonies of bacteria containing a library plasmid which is derived from a yeast colony were analyzed for the presence of library plasmid. The library plasmids were then reintroduced into yeast Y190 with the original bait (pAS-Bcr) or a PAS-lamin construct, to confirm the specifidty of the interaction. One plasmid which encodes the 3' portion of γ π adaptin (amino adds 518-791) was found to specifically interact with the Bcr homology domain of p85. A third test method for confirming positive clones was employed and involved switching the bait and prey in their respective plasmids because the majority of false positives will not interact in this test. Some true positives may not activate either in this switch, so only a positive result is reliable. The pASl polylinker has been placed into pACT to facilitate this transfer, creating pACT2. All general yeast protocols in this example are described in Methods in Enzymology Vol. 194 "Guide to Yeast Genetics and Molecular Biology" by Guthrie and Fink. Also Ausubd et al (1994) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, (Greene Publishing Associates and John Wiley & Sons, New York, NY), and Sambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, New York), are appropriate general references. Fynmplp 2 Generation of Polyclonal Antibodies In Sheep Against a Hydrophilic Peptide of γ π Adaptin Protein A 5.1 mg purified hydrophilic peptide of γ π adaptin protein was coupled through the terminal cysteine to diphtheria toxoid (DT) with the heterobifunctional cross-linking agent 6-maleimido-caproic acid N- hydroxysuccinimide ester (MCS) in the ration of 5.1 parts of peptide to 24.2 parts of DT (w/w). The host selected for the immunization was a sheep Leicester/Merino cross, 3 to 5 years old. The immunization was conducted with a peptide-DT conjugate that was suspended in purified water at a concentration of 34.8 mg/ml, and emulsified by mixing with two volumes of Complete Freund's Adjuvant and injected intramuscularly, for a total volume of 1.0 ml (3.48 mg of conjugate, and 0.6 mg of peptide) per immunization. A second similar immunization followed 2 weeks later, this time using Incomplete Freund's Adjuvant. A third immunization, a boost, was performed on week 12 using Incomplete Freund's Adjuvant. The animals were bled from the jugular vein using a cannula. The blood was heated at 37°C for 30 minutes, chilled at 4°C for 15 hours and centrifuged. The serum was stored at -20°C. On day 1 a pre-immune bleed was conducted, followed by the initial immunization. On day 14 the second immunization was conducted. On day 35 the first bleed was performed; on day 98 the second bleed; and on day 112 the third bleed. All of the sera were tested with an enzyme linked immunosorbent assay (ELISA) with the carrier protein (DT) as coat. For all sera, the anti-peptide antibody titer was determined with an ELISA with a saturated level of the biotinylated form of the peptide (Ac-VERGDPHVKEGGKEKQTEAQP-GSGS- Biocytin-OH) immobilized on an avidin coated microtiter plate (500 ng/well). Also as a negative control, all sera were titrated on avidin only coated microtiter plates (without peptide coating). The assay was conducted by a standard biotin ELISA protocol. Either streptavidin or avidin can be used to coat the microtiter plates for the purpose of detecting anti-peptide antibodies because they produce similar activities. The results were expressed as the reciprocal of the serum dilution that results in an absorbance of 1.0 above the background at 405 nm wavelength setting, and the background was established with HRP-anti Sheep IgG conjugate and peroxidase dye. Two sheep were used to produce the polyclonal antibodies, sheep # 2238 and # 2239 and the results of the experiment indicated high antibody titer in crude sera with activity against DT, as compared to the controls as shown below. artivity against pgptifte Sheep prehleeri 2nd hleed 4th hlepri 2238 5 ml/112 titer 190 ml/882 titer 340 ml/1567 titer 2239 5 ml/68 titer 290 ml/459 titer 320 ml/ 1202 titer activity against DT Sheeri prehleeri 2nd hleeri 4th hleeri 2238 5 ml/128 titer 190 ml/3232 titer 340 ml/16416 titer 2239 5 ml/ND titer 290 πύ/5888 titer 320 ml/22976 titer activity against Avidin Sheep prehleeri 9nH hlftftH 4th hleeri 2238 5 ml/99 titer 190 ml/98 titer 340 ml/280 titer 2239 5 ml/86 titer 290 ml/50 titer 320 ml/ 70 titer Example.3 Co-Immunoprecipitation of γ H Adaptin with P85 Subunit Indicates the Function of γ II Adaptin The C-terminal one third of γ π adaptin cDNA encodes an ear portion of γ π adaptin molecule (amino acids 518-791), which is thought to interact with phosphatidylinositol 3-kinase, as does full-length γπ adaptin. Therefore, myc- tagged ear γ π was transfected into Cos cells. Cos cells transfected with myc- tagged ear portion of γH adaptin were cultured and lysed. (The myc tag is MEQKLISEEDL.) The cell lysate was immunoprecipitated with anti-p85 subunit of PI 3 kinase antibody. The precipitate was run on a reducing SDS- PAGE protein gel and transferred to a filter as described in Ausubel et al (1994) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, (Greene Publishing Associates and John Wiley & Sons, New York, NY), and Sambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, New York), for a Western hybridization. The filter was then blotted first with anti-myc monoclonal antibody to detect myc-taggged γ π adaptin and then blotted with alkaline phosphatase-conjugated anti-mouse antibodies, and then the filter was developed with an NBT and BCIP (Promega, Madison, WI) system. The detection of a myc-tagged ear portion of γπ adaptin confirmed the interaction between p85 and γ π adaptin in the cells. The reverse experiment using anti-myc antibody for immunoprecipitation and p85 for western blot also showed the co-immunoprecipitation between ear γ π and p85. Moreover, ear γ π was not found to complex with a p85 derivative that lacks the Bcr homology domain. Example 4 γ JJ adaptin-GFP Fusion Protein Is Internalized Upon PDGF Stimulation A recombinant γ π adaptin-green fluorescent protein (GFP) fusion protein was constructed to investigate the distribution of γ π adaptin in the cell. The fusion protein was transfected into Cos cells. Transfected Cos cells were starved overnight with 0.5% calf serum in DMEM. Both membrane and cytoplasmic distribution of γ H-GFP (green fluorescent protein) was observed in the starved cells. Upon 15 minutes PDGF (2nM) stimulation at 37°C, many γ π-GFP transfected cells displayed partial perinuclear distribution, while the membrane and cytoplasmic distribution was greatly diminished. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Cen, Hui Williams, Lewis T. (ii) TITLE OF INVENTION: Gamma II Adaptin (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Chiron Corporation (B) STREET: 4560 Horton Street (C) CITY: Emeryville (D) STATE: California (E) COUNTRY: U.S.A. (F) ZIP: 94608 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Releaβe #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Guth, Joseph H. (B) REGISTRATION NUMBER: 31,261 (C) REFERENCE/DOCKET NUMBER: 1189-003 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (510)923-3888 (B) TELEFAX: (510) 655-3542 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 791 amino acids - - (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NOtl: Met Val Val His Ser Leu Arg Leu Gin Asp Leu lie Glu Glu lie Arg 1 5 10 15 Gly Ala Lys Thr Gin Ala Gin Glu Arg Glu Val lie Gin Lys Glu Cys 20 25 30 Ala Gin He Arg Ala Ser Phe Arg Asp Gly Aβp Pro Leu Gin Arg His 35 40 45 Arg Gin Leu Ala Lys Leu Leu Tyr Val His Met Leu Gly Tyr Pro Ala 50 55 60 Hie Phe Gly Gin Met Glu Cyβ Leu Lys Leu lie Ala Ser Pro Arg Phe 65 70 75 80 Thr Aβp Lys Arg Val Gly Tyr Leu Gly Ala Met Leu Leu Leu Asp Glu 85 90 95 Arg His Aβp Ser His Leu Leu He Thr Aβn Ser He Lye Asn Aβp Leu 100 105 110 Ser Gin Gly Aβn Gin Pro Val Gin Gly Leu Ala Leu Cys Thr Leu Ser 115 120 125 Thr Met Gly Ser Ala Glu Met Cys Arg Aβp Leu Ala Pro Glu Val Glu 130 135 140 Lys Leu Leu Leu Gin Pro Ser Pro Tyr Val Arg Lys Lys Ala He Leu 145 150 155 160 Thr Ala Val His Met He Arg Lys Aβp Pro Glu Leu Ser Gly He Phe 165 170 175 Leu Pro Pro Cyβ Thr Lys Leu Leu Arg Glu Arg His His Gly He Gin 180 185 190 Leu Gly Thr Val Thr Leu He Thr Glu Leu Cys Glu Arg Asn Pro Ala _ _ 195 200 205 Ala Leu Arg His Phe Arg Lye Val Val Pro Gin Leu Val Gin He Leu 210 215 220 Arg Thr Leu Val Thr Thr Gly Tyr Ser Thr Glu His Ser He Ser Gly 225 230 235 240 Val Ser Asp Pro Phe Leu Gin Val Gin He Leu Arg Leu Leu Arg He 245 250 255 Leu Gly Arg Asn His Glu Glu Ser Ser Glu Thr Met Asn Aβp Leu Leu 260 265 270 Ala Gin Val Ala Thr Aβn Thr Aβp Thr Ser Arg Aβn Ala Gly Asn Ala 275 280 285 Val Leu Leu Glu Thr Val Leu Thr He Met Ala He His Ser Ala Ala 290 295 300 Gly Leu Arg Val Leu Ala Val Asn He Leu Gly Arg Phe Leu Leu Aβn 305 310 315 320 Aβn Asp Lys Aβn He Arg Tyr Val Ala Leu Thr Ser Leu Leu Gin Leu 325 330 33S Val Gin Ser Asp Hie Ser Ala Val Gin Arg His Arg Ser Thr Val Val 340 345 350 Glu Cys Leu Gin Glu Thr Asp Ala Ser Leu Ser Arg Arg Ala Leu Glu 355 360 365 Leu Ser Leu Ala Leu Val Asn Ser Ser Asn Val Arg Ala Met Met Gin 370 375 380 Glu Leu Gin Ala Phe Leu Glu Ser Cyβ Pro Pro Aβp Leu Arg Ala Aβp 385 390 395 400 Cyβ Ala Ser Gly He Leu Leu Ala Ala Glu Arg Phe Ala Pro Ser Lye 405 410 415 Arg Trp His He Aβp Thr He Leu Hie Val Leu Thr Thr Ala Gly Ala 420 425 430 His Val Arg Aβp Aβp Ala Val Ala Aβn Leu Thr Gin Leu He Gly Glu 435 440 445 Ala Glu Glu Leu Hie Thr Tyr Ser Val Arg Arg Leu Tyr Ser Ala Leu 450 455 460 Ala Glu Aβp He Ser Gin Gin Pro Leu Val Gin Val Ala Ala Trp Cyβ 465 470 475 480 He Gly Glu Tyr Gly Asp Leu Leu Leu Glu Gly Asn Cys Glu Glu Thr 485 490 495 Glu Pro Phe Gin Val Glu Glu Glu Asp Val Leu Ala Leu Leu Glu Lys 500 505 510 Val Leu Gin Ser His Met Ser Leu Pro Ala Thr Arg Gly Tyr Ala He 515 520 525 Thr Ala Leu Met Lys Leu Ser Thr Arg Leu Arg Gly Asp Asn Asn Arg 530 535 540 He Arg Gin Val Val Ser He Tyr Gly Ser Cys Val Asp Leu Glu Leu 545 550 555 560 Gin Gin Arg Ala Val Glu Tyr Asn Thr Leu Phe Gin Lys Tyr Aβp His 565 570 575 Met Arg Ala Ala He Leu Glu Lys Met Pro Leu Val Glu Arg Gly Asp 580 585 590 Pro His Val Lys Glu Gly Gly Lys Glu Lys Gin Thr Glu Ala Gin Pro 595 600 605 Leu Glu Val Thr Ala Pro Ala Pro Thr Glu Pro Gin Ala Thr Lys Leu 610 615 620 Leu Aβp Leu Leu Aβp Leu Leu Gly Asp Thr Ser Glu Pro Leu Ser Ser 625 630 635 640 Gly Hie Ala Gin Hie Leu Pro Pro Gin Thr Pro Ser Pro Gly Glu Ala 645 650 655 Leu He His Leu Leu Asp Leu Pro Cys Thr Pro Pro Pro Pro Ala Pro _ . 660 665 670 He Pro Ser Val Arg Val Phe Glu Arg Glu Gly Leu Gin Leu Asp Leu 675 680 685 Ser Phe Met Arg Pro Leu Glu Thr Pro Ala Leu Leu Leu Val Thr Ala 690 695 700 Thr Thr Thr Asn Ser Ser Lys Glu Aβp Val Thr Hie Phe Val Cys Gin 705 710 715 720 Ala Ala Val Pro Lys Ser Phe Gin Leu Gin Leu Gin Ala Pro Ser Gly 725 730 735 Asn Thr He Pro Ala Gin Gly Gly Leu Pro He Thr Gin Val Phe Arg 740 745 750 He Leu Asn Pro Asn Gin Ala Pro Leu Arg Leu Lys Leu Arg Leu Thr 755 760 765 Tyr Asn His Ser Gly Gin Pro Val Gin Glu He Phe Glu Val Aβp Aβn 770 775 780 Leu Pro Val Glu Thr Trp Gin 785 790 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2533 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGYt linear (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TCGAGCTACG TCAGGGCCGA AGCCAGGATG GTGGTGCATT CGTTGAGACT TCAGGACCTA 60 ATCGAAGAGA TTCGCGGGGC CAAGACGCAG GCCCAGGAAC GGGAGGTGAT CCAGAAGGAG 120 TGCGCCCAAA TTCGGGCCTC CTTCCGCGAT GGGGATCCCC TGCAGAGGCA TCGCCAGCTG 180 GCCAAACTGC TCTACGTCCA CATGTTGGGC TACCCCGCCC ACTTTGGACA GATGGAGTGC 240 - CTGAAACTGA TCGCCTCCCC CAGATTCACA GACAAGAGGG TGGGCTACCT TGGGGCCATG 300 CTTCTATTGG ATGAGAGGCA CGATTCCCAT CTGCTCATCA CCAACAGCAT CAAGAATGAC 360 CTGAGCCAAG GGAACCAGCC AGTTCAAGGC CTGGCCCTGT GTACTCTGAG TACCATGGGC 420 TCTGCTGAGA TGTGCCGGGA CCTAGCCCCT GAGGTGGAAA AGCTGCTTCT GCAGCCCAGT 480 CCCTATGTGC GGAAGAAGGC TATTTTGACT GCAGTGCACA TGATCCGGAA GGACCCTGAG 540 CTCTCCGGCA TCTTCCTCCC ACCTTGTACC AAACTGCTTC GTGAGCGTCA TCATGGCATC 600 CAGCTGGGCA CAGTCACGCT GATCACGGAG CTCTGTGAAA GAAACCCTGC AGCCCTCAGG 660 CACTTTCGCA AGGTAGTGCC GCAGCTGGTA CAGATCCTCC GGACTCTGGT GACTACAGGA 720 TACTCCACGG AGCACAGCAT CTCTGGAGTC AGCGACCCCT TCTTGCAGGT CCAGATACTC 780 CGCCTACTTC GGATCCTGGG ACGGAACCAT GAAGAAAGTA GCGAGACCAT GAACGACTTG 840 CTGGCCCAGG TCGCCACCAA CACAGACACC AGCCGAAATG CAGGCAACGC TGTCCTGTTG 900 GAAACAGTGC TTACCATCAT GGCCATCCAC TCTGCTGCTG GCCTCCGGGT TCTAGCTGTT 960 AACATTCTTG GTCGTTTCTT GCTCAACAAT GACAAGAATA TTAGGTATGT GGCTCTGACA 1020 TCATTGCTGC AGCTGGTGCA GTCTGACCAC AGTGCTGTAC AACGTCACCG GTCCACTGTG 1080 GTGGAGTGTC TACAGGAAAC GGACGCCTCC CTTAGCAGGC GGGCCCTGGA GCTGAGCCTG 1140 GCTCTGGTGA ACAGCTCCAA TGTACGAGCC ATGATGCAGG AGCTGCAGGC CTTTCTGβAG 1200 TCCTGCCCCC CTGATCTTCG GGCTGATTGT GCCTCAGGCA TTCTGTTGGC TGCGGAGAGG 1260 TTTGCTCCCA GCAAGCGATG GCACATAGAC ACCATCCTGC ACGTGCTGAC CACGGCAGGA 1320 GCCCATGTGA GGGATGACGC AGTGGCCAAC CTGACCCAGC TGATTGGAGA GGCCGAGGAG 1380 CTβCATACCT ACTCTGTGCG CCGTCTCTAC AGTGCCTTAG CAGAGGATAT CTCCCAGCAA 1440 CCACTGGTTC AGGTGGCAGC CTGGTGCATT GGCGAGTATG GGGACCTCCT GCTGGAAGGT 1500 AACTGTGAGG AGACGGAGCC TTTTCAGGTG GAAGAAGAGG ACGTGCTAGC ACTGCTGGAA 1560 . . AAGGTGCTGC AGTCCCATAT GTCCCTGCCA GCCACTCGGG GCTACGCCAT CACAGCCCTC 1620 ATGAAGCTGA GCACCCGACT CCGGGGAGAC AACAATCGTA TTCGCCAGGT GGTGTCCATC 1680 TACGGGAGCT GTGTGGACTT AGAGCTGCAA CAGCGGGCTG TGGAGTATAA CACACTCTTC 1740 CAGAAGTACG ACCACATGAG AGCCGCCATC CTAGAAAAGA TGCCTCTTGT AGAGCGTGGT 1800 GACCCCCACG TTAAAGAGGG AGGGAAGGAG AAGCAAACGG AAGCCCAGCC CTTGGAAGTG 1860 ACAGCCCCTG CCCCCACAGA ACCCCAGGCC ACCAAACTCT TAGATCTACT GGATCTCCTG 1920 GGTGACACTT CAGAGCCTCT CTCTTCTGGG CATGCCCAGC ATCTTCCTCC TCAGACTCCT 1980 TCCCCAGGGG AAGCCTTAAT TCATCTCCTT GACCTTCCCT GTACACCGCC ACCCCCAGCT 2040 CCCATCCCCA GTGTCAGAGT GTTTGAGCGT GAGGGCCTAC AGCTGGATCT TTCTTTCATG 2100 CGGCCCTTGG AGACCCCTGC TTTGCTCTTA GTCACTGCCA CCACCACCAA CTCCTCAAAG 2160 GAGCATGTTA CCCACTTCGT TTGCCAGGCA GCTGTGCCCA AGAGTTTCCA GCTGCAGTTA 2220 CAGGCCCCCA GTGGGAACAC AATTCCAGCT CAGGGTGGTC TTCCCATCAC CCAGGTCTTC 2280 AGAATCCTCA ATCCTAACCA GGCACCTTTG CGACTTAAGC TGCGCCTCAC CTACAACCAC 2340 TCTGGCCAGC CAGTACAGGA GATCTTTGAG GTGGATAACT TGCCTGTGGA GACGTGGCAG 2400 TAACCGACTG TGGTCAGTGT CTGGCCTGGG TGTCTCCAGG CTCCTGGTGT TCAAGGAACG 2460 GAAATAAAGA CCCACGTAAA TGGCGAAGTA AACTTTATTT AAAGGCGACβ TCAGGGCCCT 2520 GACGTAGCTC GAG 2533 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Val Glu Arg Gly Aβp Pro Hie Val Lye Glu Gly Gly Lye Glu Lys Gin 1 5 10 IS Thr Glu Ala Gin Pro Gly Ser Gly Ser 20 25 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 189 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Lys Leu Val Glu Ala He Glu Lys Lys Gly Leu Glu Cys Ser Thr Leu 1 5 10 15 Tyr Pro Thr Gin Ser Ser Ser Aβn Pro Ala Glu Leu Arg Gin Leu Leu 20 25 30 Aβp Cyβ Asp Thr Ala Ser Leu Asp Leu Glu Met Phe Asp Val His Val 35 40 45 Leu Ala Aβp Ala Phe Lye Arg Tyr Leu Leu Asp Leu Pro Aβn Pro Val 50 55 60 He Pro Val Ala Val Ser Ser Glu Leu He Ser Leu Ala Pro Glu Val 65 70 75 80 Gin Ser Ser Glu Glu Tyr He Gin Leu He Lys Lys Leu He Arg Ser 85 90 95 Pro Ser Leu Pro His Gin Tyr Trp Leu Thr Leu Gin Tyr Leu Leu Lys 100 105 110 His Phe Phe Lye Leu Ser Gin Thr Ser Ser Lye Aβn Leu Leu Asn Ala 115 120 125 Arg Val Leu Ser Glu Leu Phe Ser Pro Leu Leu Phe Arg Phe Pro Ala 130 135 140 Ala Ser Ser Glu Aβn Thr Glu His Leu He Lys He He Glu He Leu 145 150 155 160 He Ser Thr Glu Trp Aβn Glu Arg Gin Pro Ala Pro Ala Leu Pro Pro 165 170 175 Lye Pro Pro Lys Pro Thr Thr Val Ala Asn Aβn Gly Met 180 185

Claims: - - CLAIMS I. An isolated and purified mammalian γU-adaptin protein which binds to the Bcr homology domain of phosphatidylinositol 3-kinase. 2. The protein of claim 1 which is at least 85 % identical to the amino acid sequence shown in SEQ ID NO: ID NO: 1. 3. An isolated and purified protein comprising the amino acid sequence as shown in SEQ ID NO: 1. 4. A fusion protein which comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond, wherein the first protein segment consists of at least eight contiguous amino acids of a mammalian γH-adaptin protein 5. The fusion protein of claim 4 wherein the at least dght contiguous amino acids are selected from the amino acid sequence shown in SEQ ID NO: ID NO:l. 6. The fusion protein of claim 4 which binds to a p85 subunit of phosphatidylinositol 3-kinase. 7. An isolated polypeptide which consists of at least eight contiguous amino adds of a mammalian γπ-adaptin protein, wherein said protein binds to the Bcr homology domain of phosphatidylinositol 3-kinase. 8. The polypeptide of claim 7 which has a sequence of at least dght contiguous amino acids selected from the sequence shown in SEQ ID NO: 1. 9. The isolated polypeptide of claim 7 which binds to a p85 subunit of phosphatidylinositol 3-kinase. 10. An isolated preparation of antibodies which specifically binds to a mammalian γπ-adaptin protein, wherein said protein binds to a Bcr homology domain as shown in SEQ ID NO:4. I I . The preparation of claim 10 wherein the mammalian γπ-adaptin protein has the sequence shown in SEQ ID NO: 1. . - 12. The preparation of antibodies of claim 10 wherein the antibodies are monoclonal. 13. The preparation of antibodies of claim 10 wherein the antibodies are purified from an animal antiserum. 14. The preparation of antibodies of claim 10 wherein the antibodies are affinity purified. 15. A subgenomic polynucleotide which encodes a mammalian γπ- adaptin protein, wherein said protein binds to a Bcr homology domain of a p85 subunit of phosphatidylinositol 3-kinase. 16. The polynucleotide of claim 15 which encodes a niammalian γπ- adaptin protein as shown in SEQ ID NO: 1. 17. The subgenomic polynucleotide of claim 15 which is intron-free. 18. The subgenomic polynucleotide of claim 15 which comprises the sequence shown in SEQ ID NO:2. 19. A vector comprising the polynucleotide of claim 15. 20. A vector comprising the polynucleotide of claim 16. 21. A vector comprising the polynucleotide of claim 17. 22. A vector comprising the polynucleotide of claim 18. 23. A recombinant host cell which comprises the vector of claim 19. 24. A recombinant host cell which comprises the vector of claim 20. 25. A recombinant host cell which comprises the vector of claim 21. 26. A recombinant host cell which comprises the vector of claim 22. 27. An isolated polynucleotide which encodes the fusion protein of claim 4. 28. An isolated subgenomic polynucleotide which encodes the polypeptide of claim 7. 29. A method of screening for agents useful in inhibiting mitogenesis, comprising the steps of: . - contacting a test compound with a mammalian γπ-adaptin and a protein comprising a Bcr homology domain, wherein the protein binds to the γπ- adaptin in the absence of the test compound; determining the amount of at least one of said γπ-adaptin and said protein which is bound or unbound in the presence of the test compound, wherein a test compound which decreases the amount bound or increases the amount unbound, of at least one of said γπ-adaptin and said protein is an agent useful for inhibiting mitogenesis. 30. The method of claim 29 wherein the γπ-adaptin and the protein are prebound prior to the step of contacting. 31. The method of claim 29 wherein the test compound is contacted with one of the γH-adaptin and the protein prior to the step of contacting. 32. The method of claim 29 wherein the Bcr homology domain is that of the p85 subunit of phosphatidylinositol 3-kinase. 33. The method of claim 29 wherein the protein comprising a Bcr homology domain is the p85 subunit of phosphatidylinositol 3-kinase. 34. The method of claim 29 wherein the protein comprising a Bcr homology domain is the phosphatidylinositol 3-kinase. 35. The method of claim 29 wherein the protein comprising a Bcr homology domain is selected from the group consisting of: rho-GAP, n- chimerin, pl90, Bem2, Bem3, 3BP1, Bcr, and phosphatidylinositol 3-kinase. 36. The method of claim 29 wherein the Bcr homology domain is selected from the group consisting of the Bcr homology domain of: rho-GAP, n- chimerin, pl90, Bem2, Bem3, 3BP1, Bcr, and phosphatidylinositol 3-kinase. 37. The method of claim 29 wherein one member selected from the group consisting of γπ-adaptin and the protein is bound to a solid support. 38. The method of claim 29 wherein one member selected from the group consisting of γll-adaptin and the protein is radiolabded. 39. A method for screening test compounds to identify agents useful for inhibiting mitogenesis, comprising the steps of: - (a) contacting a cell with a test compound, wherein the cell comprises: i) a first fusion protein comprising (1) a DNA binding domain and (2) all or a portion of a mammalian γH-adaptin, wherein the portion is capable of binding to p85 subunit of phosphatidylinositol 3-kinase; ii) a second fusion protein comprising (1) a transcriptional activating domain and (2) all or a portion of a p85 subunit of phosphatidylinositol 3-kinase, said portion comprising a Bcr homology domain of p85 subunit of phosphatidylinositol 3-kinase consisting of amino acids 134-322 as shown in SEQ ID NO: ID NO: 4, wherein the interaction of the first and second fusion proteins reconstitutes a sequence-specific transcriptional activating factor; and iii) a reporter gene comprising a DNA sequence to which the DNA binding domain of the first fusion protein specifically binds; and (b) measuring the expression of the reporter gene, wherein a test compound that decreases the expression of the reporter gene is a potential inhibitor of mitogenesis. 40. The method of claim 39 wherein the portion of mammalian γH- adaptin consists of a contiguous sequence of amino acids selected from the amino add sequence shown in SEQ ID NO: 1. 41. A method for screening test compounds to identify agents useful for inhibiting mitogenesis, comprising the steps of: (a) contacting a cell with a test compound, wherein the cell comprises: i) a first fusion protein comprising (1) a DNA binding domain and (2) all or a portion of a p85 subunit of phosphatidylinositol 3- kinase, said portion comprising a Bcr homology domain of p85 subunit of phosphatidylinositol 3-kinase consisting of amino acids 134-322 as shown in SEQ ID NO: ID NO: 4; ii) a second fusion protein comprising (1) a transcriptional activating domain and (2) all or a portion of a mammalian γπ-adaptin, wherein - - the portion is capable of binding to p85 subunit of phosphatidylinositol 3-kinase; wherein the interaction of the first and second fusion proteins reconstitutes a sequence-specific transcriptional activating factor; and iii) a reporter gene comprising a DNA sequence to which the DNA binding domain of the first fusion protein specifically binds; and (b) measuring the expression of the reporter gene, a test compound that decreases the expression of the reporter gene being a potential inhibitor of mitogenesis. 42. The method of claim 41 wherein the portion of mammalian γH- adaptin consists of a contiguous sequence of amino acids selected from the amino acid sequence shown in SEQ ID NO: ID NO: 1.

Inventors: CEN HUI, WILLIAMS LEWIS, CEN, HUI, WILLIAMS, LEWIS
Applicants: CHIRON CORP, CHIRON CORPORATION
Application year: 1997
Main classification: C12N15
IPC codes: C07K14, C12N9, C12N15