WO1998008948A1 - Protein targeting to glycogen - Google Patents

Protein targeting to glycogen Download PDF

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Publication number
WO1998008948A1
WO1998008948A1 PCT/US1997/014142 US9714142W WO9808948A1 WO 1998008948 A1 WO1998008948 A1 WO 1998008948A1 US 9714142 W US9714142 W US 9714142W WO 9808948 A1 WO9808948 A1 WO 9808948A1
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leu
val
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asp
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PCT/US1997/014142
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French (fr)
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Matthew Jemail Brady
John Andrew Printen
Alan Robert Saltiel
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Warner-Lambert Company
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Priority to EP97938246A priority Critical patent/EP0941324A1/en
Priority to US09/242,948 priority patent/US6252057B1/en
Priority to JP51167598A priority patent/JP2002514173A/en
Priority to AU40623/97A priority patent/AU4062397A/en
Publication of WO1998008948A1 publication Critical patent/WO1998008948A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

Definitions

  • This invention relates to isolated murine and human genomic and cDNA molecules that encode for a protein called the Protein Targeting to Glycogen (PTG) protein.
  • PTG Protein Targeting to Glycogen
  • This invention also relates to the PTG protein and to methods of increasing the amount of glycogen in a cell.
  • PPl has been shown regulate cell cycle progression (Hisamoto N., et al., Mol. Cell. Biol. 1994;14:3158; Zang S., et al., Mol. Cell. Biol., 1995; 15:2037), chromosome segregation (Francisco L., et al., Mol. Cell. Biol., 1994; 14:4731), protein synthesis (Wek R.C., et al., Mol. Cell. Biol., 1992;12:5700), and glycogen metabolism (Cannon J.F., et al., Genetics, 1994;136:485).
  • PPl In mammalian cells, PPl also has multiple physiological roles, such as regulation of glycogen metabolism (Bollen M. and Stalmans W., Crit. Rev. Biochem. Mol. Bio., 1992;27:227), protein synthesis (Cohen P., Ann. Rev. Biochem., 1989;58,453), and muscle contraction (Shenolikar S., Annu. Rev. Cell Biol., 1994;10:55). Many of the metabolic effects of insulin are thought to occur via activation of PPl (Saltiel A.R., Am. J. Physiol, 1996;33:E375).
  • two targeting subunits M and G direct the catalytic subunit of PPl, PPIC, to different subcellular locations.
  • the M subunit directs PPIC to myofibrils, acting to facilitate dephosphorylation of myosin (Dent P., et al., Eur. J. Biochem., 1992;210: 1037), whereas the G subunit localizes PPIC to both the glycogen particle and the membranes of the sarcoplasmic reticulum, where glycogen metabolizing enzymes and SR proteins serve as substrates for PPIC (Stralfors P., et al., Eur. J. Biochem., 1985;149:295; Hubbard M.J.
  • PPIC is also known to interact with proteins in nuclei, an inhibitor protein, NIPP1 (Beullins M., et al, J. Biol. Chem., 1992;267: 16538), and the
  • PPIC has also been shown to interact with the product of the tumor suppressor gene, Rb, implicating PPIC in the control of tumorigenesis and cell cycle progression (Durfee T., et al., Genes Dev., 1993;7:555).
  • Rb tumor suppressor gene
  • phosphorylation of site 1 following insulin stimulation leads to a higher affinity of PPIC for RGl, leading to activation of glycogen metabolizing enzymes by dephosphorylation, while phosphorylation of site 2 by cAMP activated protein kinase A (PKA) causes a reduced affinity and subsequent release of PPIC from the glycogen pellet.
  • PKA protein kinase A
  • G A glycogen binding subunit expressed exclusively in liver, G , was recently cloned and was found to encode a predicted protein product of only 33 kD. G was shown to also differ from RGl with respect to enzymatic activity towards various substrates and in the ability of G to serve as a substrate for PKA (Doherty M.J., et al., FEBS Lett., 1995;375:294).
  • a 3T3-L1 adipocyte cDNA 2-hybrid library was screened for PPIC interacting proteins.
  • PTG a novel glycogen binding subunit of PPIC, called PTG, that may act as a scaffold for the localization of critical enzymes in glycogen metabolism, including phosphorylase b, glycogen synthase, and phosphorylase kinase.
  • PTG is expressed predominantly in insulin-sensitive tissues and was found to mediate the hormonal control of glycogen accumulation in intact cells.
  • Figure 1 shows the sequence comparison of PTG with glycogen localizing subunits of PPIC.
  • Figure 2 shows PPIC binding and glycogen localizing activity of PTG protein.
  • Figure 3 shows the tissue distribution of PTG expression.
  • Figure 4 shows the binding of phosphorylase a, glycogen synthase, and phosphorylase kinase to PTG.
  • Figure 5 shows glycogen synthesis in CHO-IR cells overexpressing PTG.
  • the present invention provides an isolated murine protein comprising the amino acid sequence:
  • the present invention also provides an isolated human protein protein comprising the amino acid sequence; Met He Gin Val Leu Asp Pro Arg Pro Leu Thr Ser Ser Val Met Pro
  • the present invention also provides an isolated murine genomic DNA molecule comprising the sequence:
  • the present invention also provides an isolated human genomic DNA molecule comprising the sequence:
  • the present invention also provides an isolated murine cDNA molecule comprising the sequence:
  • the present invention also provides an isolated human cDNA molecule comprising the sequence:
  • PTG is a PPIC binding protein with homology to known glycogen binding proteins:
  • a 3T3-L1 adipocyte cDNA library fused to the Gal4p transcriptional activation domain was screened for proteins that could interact with a Gal4p-PPlC DNA binding domain fusion.
  • Library plasmids containing interacting proteins were identified by the ability to induce transcription of the integrated GALI-lacZ reporter. Of approximately 3.5 x 10 ⁇ primary tranformants, 27 were positive for ⁇ -galactosidase activity.
  • One class of interacting cDN As typified by clone B 1 - 1 , consistently gave the highest levels of ⁇ -galactosidase activity when plated on X-gal containing media.
  • PTG can direct PPIC localization to glycogen both in vivo and in vitro:
  • the homology of the predicted PTG protein with G j ⁇ , RGl and Gael suggests that PTG might bind simultaneously to both PPl C and glycogen.
  • a FLAG epitope tagged PTG construct (FLAG-PTG) was transiently transfected into CHO cells over-expressing the insulin receptor (CHO-IR), followed by immunoprecipitation with ⁇ FLAG antibodies and subsequent immunoblotting with ⁇ PPIC antibodies.
  • PPIC could be co-immunoprecipitated from cell lysates with the antibodies directed against FLAG-PTG ( Figure 2A), demonstrating direct association of PPIC with PTG in vivo. This association was unaffected by treatment of cells with insulin. Moreover, PTG did not appear to undergo phosphorylation in response to insulin, and was not a substrate in vitro for cAMP-dependent protein kinase, or other protein kinases in lysates from insulin stimulated cells (not shown). To determine the subcellular localization of PTG, similarly transfected CHO-IR cells were fractionated by differential centrifugation followed by SDS-PAGE and immunoblotting with ⁇ FLAG antibodies.
  • RGl is expressed in muscle tissue (diaphragm, skeletal muscle, and heart) (Tang P.M., supra., 1991), whereas GL is expressed exclusively in liver tissue (Doherty M.J., supra., 1995).
  • a rat multi-tissue northern blot (Clontech) was hybridized with a probe prepared from the cDNA insert of clone Bl-1.
  • An mRNA of approximately 2.3 kb was detected in all tissues except testis ( Figure 3A).
  • the PTG mRNA was most abundant in skeletal muscle, liver, and heart.
  • PTG transcript was also detected in RNA prepared from rat adipose tissue (not shown).
  • 3T3-L1 fibroblasts into adipocytes is correlated with a significant increase in insulin sensitivity, including the stimulation of glycogen synthesis.
  • the expression of many genes critical to insulin action is increased during adipocyte differentiation, including the insulin receptor, GLUT4 and others
  • PTG can associate with multiple proteins involved in regulating glycogen metabolism: Glycogen synthesis and breakdown is regulated by the reciprocal actions of protein phosphatases and kinases. To determine whether PTG is involved in the localization of the metabolizing enzymes, and of the kinases and phosphatases involved in their regulation, a series of in vitro binding assays were performed with a bacterially expressed PTG fusion protein. First, we examined the ability of PTG to bind to phosphorylase a, the phosphorylated, active form of the enzyme that directly catalyzes glycogen breakdown. GST-PTG bound to glutathione-Sepharose beads was incubated with [32p]-phosphorylated phosphorylase a.
  • GST-PTG efficiently bound to phosphorylase a, but did not bind an unrelated GST-fusion protein, GST-PTP1B ( Figure 4A).
  • glutathione-Sepharose bound GST-PTG was incubated with purified glycogen synthase. Glycogen synthase activity was specifically associated with GST-PTG, and not with an irrelevent GST-fusion protein, PTP1B ( Figure 4B).
  • Phosphorylase kinase converts phosphorylase from the inactive b form to the active a form. This activation can be reversed via dephosphorylation by PPIC.
  • Phosphorylase kinase can also be directly inactivated by PPl. Because of the central role of phosphorylase kinase in regulating glycogen metabolism, it is also a candidate for association with PTG.
  • GST-PTG-glutathione-Sepharose beads were incubated with 3T3-L1 adipocyte lysates, washed extensively, and assayed for phosphorylase kinase activity with [32p]- ⁇ -ATP and phosphorylase b as substrate.
  • Calcium-stimulated phosphorylase kinase activity was associated with GST-PTG, but not with GST-PTP 1 B
  • PTG overexpression cannot only increase basal glycogen synthesis, but also dramatically elevate maximally insulin-stimulated glycogen accumulation in a poorly responsive cell line to a level comparable to that observed in insulin target cells (Lazar D.F., supra., 1995). However, because the sensitivity of these transfected cells to insulin remains unchanged, and because insulin does not appear to modulate PP 1 C-PTG binding, PTG itself is not likely to be a direct target of insulin signaling.
  • DNA sequences such as PTG cDNA can be subcloned into a variety of plasmid shuttle vectors, allowing rapid amplification and manipulation of recombinant DNA sequences in bacterial and mammalian hosts.
  • Plasmid vectors used for routine manipulation of DNA such as Bluescript SK (GenBank
  • Plasmid vectors that are used to introduce desired DNA fragments into mammalian cells contain, in addition to those components required for bacterial vectors, a promoter sequence and a gene coding for resistance to eukaryotic antibiotics.
  • the promoter region is typically a viral promoter (CMV, Simian Virus 40, SV40) that directs high expression of the cloned gene in mammalian cells, and the antibiotic resistance gene is typically Neomycin phosphotransferase (Neo r ), which confers resistance to the eukaryotic antibiotic neomycin.
  • Viral (retrovirus and adenovirus) vectors typically contain all of the above mentioned components, in addition to viral sequences that allow recombinant DNA to be efficiently packaged into viral particles and infect the mammalian host cells. These types of viral vectors are widely used to introduce recombinant DNA into mammalian tissue culture cells and in gene therapy, where recombinant viral particles are used to infect tissues in vivo.
  • 3T3-L1 and 3T3-F442A cell lines derived from a variety of tissues are used as model systems to examine intracellular processes in the laboratory.
  • the 3T3-L1 and 3T3-F442A cell lines cloned from Swiss mouse embryo fibroblast 3T3 cultures (Green and Kehinde, 1974, Cell, 1 :1 13-116), differentiate into adipocytes and are useful in studying adipogenesis and insulin action (Garcia de Herreros and Birnbaum, 1989, J. Biol. Chem., 264 -.19994- 19999V
  • Other cell lines commonly used include NIH 3T3 fibroblasts (ATCC #CRL-1658), rat muscle cell line L6 (Proc. Natl. Acad. Sci.
  • CHO-K1 Chinese hamster ovary cells
  • ATCC #CRL-9618 Chinese hamster ovary cells
  • Primary cells derived from isolated mammalian tissues can also be cultured in the laboratory, however these cells have a limited lifespan in culture and usually die after 7 to 10 days.
  • Recombinant DNA can be introduced (transfected) into mammalian cells either in culture or in vivo by a number of techniques.
  • Calcium Phosphate- mediated transfection Choen and Okayama 1987, Mol. Cell. Biol.. 7:2745-2752
  • Liposome-mediated transfection Lipofectamine; Gibco-BRL
  • adenoviral -mediated gene transfer has been used to infect terminally differenciated cells and tissue (Becker et al. 1994, Methods Cell Biol.. 43:161-189), since adenoviral infection does not require actively dividing cells, as does retroviral gene transfer.
  • Retroviral-mediated gene transfer has been successfully used to correct adenosine deaminase (ADA) deffiency in humans (Blaese, et al., 1995, Science. 270:475-480; Kohn, et al., 1995. Nature Med.. 1 :1017-1023).
  • ADA adenosine deaminase
  • the present invention is also useful in that new drugs can be identified by screening librarys of chemical compounds for agonists or antagonists (inhibitors) of the PTG protein.
  • CHO cells expressing >1 x 10 ⁇ human insulin receptors were grown in alpha-minimal essential medium containing nucleotides,
  • 3T3-L1 Adipocyte 2-Hybrid Library Construction of 3T3-L1 Adipocyte 2-Hybrid Library. 3T3-L1 Fibroblasts were differentiated to adipocytes, as described previously
  • Stratogene cDNA synthesis kit First strand synthesis utilized an oligo ⁇ T-Xho ⁇ primer, whereas the 5' end was ligated to an EcoRl adapter following second strand synthesis. cDNA Fragments were then ligated unidirectionally into EcoRl/Xhol digested pGAD-GH GAL4 activation domain plasmid (Clontech, Palo Alto, CA). Ligations were electroporated into E. coli D12S to yield >2 x
  • a Gal4p- DNA binding domain (BD) fusion of PPIC was constructed by cloning the entire PPIC open reading frame (a generous gift of A. Nairn), contained within a 1.0 kb EcoRl/BamHl fragment, into the Eco ⁇ /Bam sites of pGBT9 (Clontech), creating BD-PPIC.
  • Strain Y190 was transformed first with BD-PP1C, Trp+ prototrophs selected, and then transformed with 150 ⁇ g of 3T3-L1 adipocyte library DNA.
  • Transformants were selected by plating cells on synthetic medium lacking tryptophan, leucine, and histidine (SD-Trp-Leu-His) and containing 25 mM 3-aminotriazole (ATZ). Yeast transformations were performed by the lithium acetate procedure of Geitz, et al.,
  • GST S-transferase
  • Sub-Cellular Fractionation of PPIC Activity Following a 3-hour serum deprivation in KRBH/0.5% BSA/2.5 mM glucose, Ll adipocyte cells were washed three times with ice cold PBS. Cells were scraped in homogenization buffer (50 mM HEPES, pH 7.2/2 mM EDTA 2 mg/mL glycogen/0.2% 2-ME/+ protease inhibitors). Samples were sonicated and centrifuged at 2500 x g to remove nuclei and unlysed cells. The PNS was removed, and glycogen bound PTG was added. Samples were incubated at 4°C for 60 minutes with gentle mixing.
  • homogenization buffer 50 mM HEPES, pH 7.2/2 mM EDTA 2 mg/mL glycogen/0.2% 2-ME/+ protease inhibitors
  • Lysates were subjected to centrifugation for 15 minutes at 10,000 x g and 1 hour at 100,000 x g to pellet plasma membranes and glycogen pellets, respectively. The final supernatant was called cytosol.
  • the glycogen pellets were resuspended in homogenization buffer by 10 passes through a 23 gauge needle. Protein concentrations and PPl activity were measured in the PNS, plasma membrane, glycogen pellet, and cytosolic fractions, as described previously (Lazar D.F., supra., 1995). Fractionation of pFPTG transfected CHO-IR cells into cytosol and glycogen pellet was performed similarly.
  • CHO cells expressing insulin receptor were transfected with Lipofectamine (Gibco-BRL) according to manufacturer recommendations. Typically, 1 ⁇ g of pFPTG/6 ⁇ L Lipofectamine was used per 60 mm dish to achieve 20% to 30% transfection efficiency, as determined by a CMV-lacZ reporter vector transfected in parallel.
  • CHO-IR cells transfected with pFPTG were sonicated in homogenization buffer and subjected to 14000 x g centrifugation for 10 minutes at 4°C to remove nuclei and cell debris.
  • FLAG-PTG was immunoprecipitated from the supernatant by incubation with 10 ⁇ g of ⁇ FLAG antibody (IBI) for 1 hour at 4°C.
  • Immune complexes were precipitated by incubation with Protein A G-agarose for 1 hour at 4°C and washed four times with homogenization buffer prior to the addition of SDS -sample buffer. Immunoprecipitates and subcellular fractions were separated on SDS- polyacrylamide gels and transferred to nitrocellulose.
  • Immunoblots were performed with either FLAG monoclonal antibody or with PPIC polyclonal antibody (a generous gift from Dr. J. Lawrence).
  • the primary monoclonal and polyclonal antibodies were detected with horseradish peroxidase-conjugated anti- mouse or anti-chicken IgG, respectively, and visualized by the enhanced chemiluminescence detection system (Amersham).
  • RNA was washed at 65°C in 2 x SSC/0.1% SDS for 15 minutes, then washed twice in 0.1 x SSC/0.1% SDS at 65°C for 15 minutes each time.
  • Equal loading of RNA was determined by ethidium bromide staining of rRNA and by probing for ⁇ -actin, as described above.
  • Glycogen Synthase and Glycogen Synthesis Assays Glycogen synthase activity associated with immobilized GST-PTG was determined as described previously (Lazar D.F., supra., 1995).
  • the agarose beads were washed four times with glycogen synthase buffer, brought to a final volume of 300 ⁇ L and 50 ⁇ L assayed for glycogen synthase activity by measuring the incorporation of UDP-[ ⁇ C]glucose into glycogen, both in the presence and absence of 10 mM glucose-6-phosphate (Sigma).
  • the accumulation of glycogen in intact pFPTG transfected CHO-IR cells was determined by an adaptation of the method of Lawrence J.C., et al., J. Biol. Chem., 1977;252:444 as described previously (Lazar D.F., supra., 1995).
  • Phosphorylase kinase 50 ⁇ L of GST-PTG fusion protein beads was added to 750 ⁇ L homogenization buffer containing 0.15 M NaCl, 0.1 % BSA, and 25 ⁇ g of [ 32 P]-labeled phosphorylase a. The tubes were incubated at 37°C for 20 minutes, washed four times with homogenization buffer, and proteins separated by SDS-PAGE, followed by autoradiography (Lawrence J.C., supra., 1977).
  • glycogen synthase buffer 50 mM HEPES, pH 7.8/100 mM NaF/10 mM EDTA
  • 25 ⁇ g (0.1 U) purified glycogen synthase Sigma
  • the agarose beads were washed four times with glycogen synthase buffer, brought to a final volume of 300 ⁇ L and 50 ⁇ L assayed for glycogen synthase activity (Lazar D.F., supra., 1995) by measuring the incorporation of UDP- [ ⁇ Cjglucose into glycogen, both in the presence and absence of 10 mM glucose- 6-phosphate (Sigma).
  • Phosphorylase Kinase Fifty microliters of fusion protein beads were incubated with 10 ⁇ g purified phosphorylase kinase (Gibco) in homogenization buffer plus 0.15 M NaCl and 0.1 % BS A, or with 3T3-L 1 adipocyte cell lysate, incubated 30 minutes at 4°C and washed four times with the same buffer. Ten microliter beads were assayed (Lazar D.F., supra., 1995) in 50 mM HEPES, pH 7.4, 10 mM MgCl, 1 ⁇ M okadaic acid, in the absence (1 mM EGTA) or presence
  • Murine and human genomic PTG sequences were obtained by screening the respective genomic Bacterial Artificial Chromosome (BAC) (Shizuya H, et al., Proc. Natl.
  • BAC Bacterial Artificial Chromosome
  • BAC clone 201D24 were chosen for further characterization following southern analysis with the labeled 1.0 kb cDNA fragment from clone B2-2 to confirm the presence of hybridizing DNA sequences.
  • 255E4 (5 ⁇ g) was digested with EcoRI (1 unit) at 37°C for 1 hour prior to separation of the resulting DNA fragments by electrophoresis through a 0.6% agarose gel. DNA fragments were transferred to nylon membrane (Hybond, Amersham) by capillary diffusion and probed with cDNA fragment encompassing the PTG coding sequence from clone B2-2. The transfer membrane was pre- hybridized for 1 hour in FBY hybridization buffer ( 10% PEG/1.5 x SSPE/7%
  • a 5.0 kb EcoRI fragment was found to hybridize to the Bl-1 PTG probe and was subsequently cloned into the EcoRI site of vector pBluescript II SK" (Stratogene, La Jolla, CA), creating the plasmid pJPD23.
  • Preliminary sequence analysis of subcloned 5.0 kb fragment was performed by using T3 and T7 primers complementary to vector sequences flanking the inserted fragment. Complete sequence information was obtained by synthesizing oligonucleotide primers complementary to both positive and negative strands of the inserted human genomic DNA. Sequencing was performed at The University of Michigan DNA sequencing core facility with an Appligen fluorescent dye terminator kit (Perkin- ⁇ lmer) and an ABI 8700 automated sequencer.
  • BAC clone 201D24 was subjected to southern analysis, as described for the human genomic BAC clone, except Bam ⁇ (1 unit, 37°C, 2 hours) was used to digest 5 ⁇ g of DNA. A 7.0 kb hybridizing fragment was identified and subcloned into the BamHl site of pBluescript II Sk", creating pJPD27. An overlapping 5.0 kb EcoRI fragment 3' to the PTG open reading frame was identified by restriction digest of BAC clone 201D24 (5 ⁇ g) with EcoRI (1 unit, 37°C, 2 hours), followed by southern analysis using a 0.8 kb S.stl-Ba Vll fragment from the extreme 3' of genomic DNNA of pJPD27.
  • PTG Knockout Vector To further characterize the physiological role of PTG in overall glycogen metabolism in vivo, a targeted replacement vector was constructed to delete the PTG coding sequences from a mouse genome.
  • pKO Scrambler V901 vector (Lexicon Genetics, Ine, The Woodlands, TX) forms the backbone of the targeting vector, as this vector has scrambled polylinkers, for insertion of 5' and 3' homologous genomic DNA, flanking a unique restriction site for insertion of a positive selectable marker (neo r for selection of transfected ⁇ S cells on the antibiotic G418).
  • pKO Scrambler V901 also contains a unique restriction site for the insertion of a negative selection element (Thymidine Kinase) for positive-negative selection strategies, which has been reported to increase targeting efficiency to the desired locus 2- to 20-fold (Hasty P. and Bradley A. in Gene Targeting: A Practical Approach, 1993, A.L. Joyner, ⁇ d., IRL Press, Oxford).
  • Neomycin Phosphotransferase gene under the control of the PGK promoter was excised from plasmid pKO selectN ⁇ O V800 (Lexicon Genetics, Ine, The Woodlands, TX) by digestion with the restriction enzyme Ascll (New England Biolabs, Beverly, MA) and subcloned into the unique Ascll site of pKO Scrambler V901 , creating plasmid pKO-neo.
  • a negative selection cassette containing the thymidine kinase gene under the control of the MCI promoter was subcloned into the unique Rsr ⁇ l site of pKO-neo by digestion of plasmid pKO SelectTK V800 (Lexicon Genetics, Ine, The Woodlands, TX) withforll (New England Biolabs, Beverly, MA) followed by separation and isolation of the appropriate restriction fragment (2.0 kb) by electrophoresis through a 1.0% agarose gel, creating plasmid pKO-TK/neo.
  • a 2.0 kb region of DNA 5' to the PTG coding region was amplified by Polymerase Chain Reaction (PCR) using primers 5'-CGAGGATCCTTGTCTTCTCTGCAGATG-3' (SEQ ID NO.: 7) and 5'-GCTGGTACCTGAATGAGCCAAGCAAATCCTC-3' (SEQ ID NO.: 8), which contain BamHl and Kpnl sites, respectively.
  • the amplified DNA product was then cloned into the Bgl ⁇ l-Kpnl of plasmid pKO-TK/neo, creating the plasmid pKO-TK/neo-5'.
  • the 3.5 kb 3' homology region of genomic PTG DNA was cloned into the EcoRI-S ⁇ /I sites of pKO-TK/neo-5' by first digesting pJPD27 (1 ⁇ g) with Smal (1 unit, 22°C, 2 hours), and inserting a Sail oligonucleotide linker (5'-CCGG CGACCGG-3') (S ⁇ Q ID NO.: 9), creating plasmid pJPD27 ⁇ Sma.
  • the 3.5 kb EcoRI-S ⁇ fl fragment from pJPD27 ⁇ Sma was then ligated into the EcoRI -Sa l sites of pKO-TK/Neo-5' to create the targeting vector pKO-PTG.
  • a 0.5 kb 5' DNA probe was generated by PCR amplification from plasmid pJPD27 with the T3 specific primer, complementary to DNA sequences contained within the vector pBluescript II Sk " and a primer specific to the extreme 5' region of mouse genomic DNA sequence (5'-GCAGAGAAGACAAAACCAC-3') (S ⁇ Q ID NO.: 10).
  • the 3' DNA probe was generated by digestion of plasmid p201-3' with Bam l and isolation of the resulting 0.8 kb fragment following electrophoretic separation on a 1.5% agarose gel.
  • FIG. 1 Sequence comparison of PTG with glycogen localizing subunits of PPIC.
  • BESTFIT sequence comparison program was used to align and compare the primary amino acid sequences of PTG, GL, RGl and Gael. The boxed regions represent areas of similarity and the sites of conservation are indicated by shading.
  • PPIC binds PTG in vivo.
  • pCI-neo expressing FLAG-PTG from the CMV promoter was transiently transfected into CHO-IR cells and immunoprecipitated from cell lysates with antibodies directed against the FLAG epitope. Precipitates were analyzed by SDS-PAGE on a 4% to 20% gel, transferred to nitrocellulose and blotted with ⁇ PPIC polyclonal antibodies. Immunoreactive proteins were visualized by
  • ECL Enhanced Chemiluminescence
  • PTG targets PPIC to glycogen in vitro PTG dependent localization of PPl C to the glycogen pellet was determined by incubated 3T3-L1 adipocyte cell lysates with bacterially expressed GST-PTP1B or GST-PTG prior to subcellular fractionation as above. PPIC activity in the glycogen pellet was measured as described previously (Lazar D.F., supra., 1995).
  • A PTG is expressed in insulin responsive tissues.
  • a multi-tissue northern blot (Clonetech) was hybridized overnight at 65°C with a 1.0 kb EcoRI fragment of clone Bl-1, which was labeled with [ ⁇ -32p]dCTP by random priming, and exposed to film for 24 hours.
  • B PTG expression is induced by adipocyte differentiation. 3T3-L1 fibroblast and fully differentiated 3T3-L1 adipocyte total RNA was isolated and electrophoresed (15 ⁇ g) in 1.2% agarose/2.2 M formaldehyde/ 1 x MOPS, followed by transfer to nylon membrane by capillary diffusion. The transfer membrane was hybridized and probed as in (A). Equal loading of RNA was determined by ethidium bromide staining of rRNA and by probing for ⁇ -actin transcript. Molecular size markers (kb) are indicated on the left.
  • glycogen synthase buffer 50 mM HEPES, pH 7.8/100 mM NaF/10 mM EDTA
  • 25 ⁇ L purified glycogen synthase (Sigma), followed by incubation at 4°C for 1 hour with gentle mixing.
  • the agarose beads were washed four times with glycogen synthase buffer and assayed for glycogen synthase activity.
  • C, D Phosphorylase kinase binds to PTG.
  • Fifty microliters of bacterially expressed GST-PTG bound to glutathione-agarose beads was incubated with of 3T3-L1 adipocyte cell lysate (C) or 10 ⁇ g purified phosphorylase kinase (Gibco) (D) in homogenization buffer. Samples were incubated 30 minutes at 4°C and washed four times with the same buffer.
  • Ten microliter beads were assayed for phosphorylase kinase activity using 2 ⁇ g phosphorylase b per sample in the absence (1 mM EGTA) or presence (0.5 mM) of Ca + ⁇ .
  • Complexed proteins were separated on a 10% SDS-polyacrylamide gel and radiolabeled phosphorylase a was visualized by autoradiography.
  • FIG. 1 Glycogen synthesis in CHO-IR cells overexpressing PTG.
  • CHO-IR cells were grown to 40% to 50% confluency in 6-well dishes and transiently transfected with pFLAG-PTG. Forty-eight hours after transfection, cells were serum deprived for 3 hours and glycogen accumulation in intact pFLAG-PTG or lacZ transfected cells, in the presence or absence of 100 nM insulin, was determined. Results are expressed as means of triplicate determinations, of SD, and were repeated in two separate experiments.
  • MOLECULE TYPE DNA (genomic)
  • AAGTCATTCT TTCTTTTAAC AAGCGTCACC TACTGTCACT CTAAGGACAG CATGACATTT 1920 TAAGAATTGC TTCATTT ⁇ TT GTTTCCCAAG TGGATTACTT CTCCTGAGAA GTAAAACCGG 1980
  • MOLECULE TYPE DNA (genomic)
  • TCTCTCCCAG CGACCGCCGC GGGGGCAAGG CCTGGAGCTG TGGTTCGAAT TTGTGCAGGC 2100 AGCGGGTGCT GGCTTTTAGG GTCCGCCGCC TCTCTGCCTA ATGAGCTGCA CCAGGTAGGT 2160
  • AATCGAACCC TTTTATTTCT CAGATGGGGA AACTGAGACC CCCATCACCC TCT ⁇ AGTGTT 4020 TTAAGCAATT AATAGCCTTT ACCGGCCAAG GGTAGAGGTA GACATAGAAG ATCTGATCAC 4080
  • TTAATACTGT TCTCTTTTAC TACATATGAT AGCACCTGCC TGATATCTAG TGCACTGGCT 140
  • ATCCAGGTGA TAATCCCTCT TCTTTTTGCA TTCCAGA ATG ATC CAG GTT TTA GAT 4255
  • TCA TGT CTC AAT ATA AAA CAC AAA GCC AAA TCA CAG AAT GAC TGG AAG 4447 Ser Cys Leu Asn He Lys His Lys Ala Lys Ser Gin Asn Asp Trp Lys 350 355 360 365
  • TTAGAGTCAA CAATCTTTGG CAGTCCGAGG CTGGCTAGTG GGCTCTTCCC AGAGTGGCAG 900

Abstract

The present invention provides human and murine genomic and complementary DNA and the proteins that are encoded by the DNA, which is called 'Protein Targeting to Glycogen'. Also provided is a method of increasing the amount of glycogen in a cell.

Description

PROTEIN TARGETING TO GLYCOGEN
FIELD OF THE INVENTION
This invention relates to isolated murine and human genomic and cDNA molecules that encode for a protein called the Protein Targeting to Glycogen (PTG) protein. This invention also relates to the PTG protein and to methods of increasing the amount of glycogen in a cell.
BACKGROUND OF THE INVENTION
While much emphasis in recent years has been focused on the identification and dissection of signaling pathways mediated by protein kinase cascades, it has long been recognized that protein dephosphorylation plays an essential role in regulating the activity of many enzymes involved in cell growth and metabolism (Krebs E.G. and Fischer E.H., Biochim. Biophys. Ada, 1953;20:150; Shenolikar S., Annu. Rev. Cell Biol., 1994; 10:55; Saltiel A.R., FASEB J, 1994;8:1034). Studies in evolutionarily distant organisms have demonstrated a critical role for type 1 protein phosphatase (PPl) action in modulating a wide variety of intracellular processes. In yeast, PPl has been shown regulate cell cycle progression (Hisamoto N., et al., Mol. Cell. Biol. 1994;14:3158; Zang S., et al., Mol. Cell. Biol., 1995; 15:2037), chromosome segregation (Francisco L., et al., Mol. Cell. Biol., 1994; 14:4731), protein synthesis (Wek R.C., et al., Mol. Cell. Biol., 1992;12:5700), and glycogen metabolism (Cannon J.F., et al., Genetics, 1994;136:485). In mammalian cells, PPl also has multiple physiological roles, such as regulation of glycogen metabolism (Bollen M. and Stalmans W., Crit. Rev. Biochem. Mol. Bio., 1992;27:227), protein synthesis (Cohen P., Ann. Rev. Biochem., 1989;58,453), and muscle contraction (Shenolikar S., Annu. Rev. Cell Biol., 1994;10:55). Many of the metabolic effects of insulin are thought to occur via activation of PPl (Saltiel A.R., Am. J. Physiol, 1996;33:E375). Indeed, many of the rate-limiting enzymes involved in glucose and lipid metabolism, such as glycogen synthase, hormone sensitive lipase, and pyruvate dehydrogenase are regulated by dephosphorylation. Thus, these dephosphorylations are likely to be critical to many of the metabolic effects of insulin, including stimulation of glycogen and lipid synthesis, and inhibition of lipo lysis. Given the array of physiological processes presumed to be mediated by PPl , it becomes apparent that organisms must have evolved a mechanism of regulating PPl activity to maintain substrate specificity and ensure against accidental activation of competing signaling pathways.
Early biochemical studies on the regulation of protein phosphatases lead to the hypothesis that protein phosphatases acted to constitutively oppose the action of specific protein kinases, since purified phosphatases could act on a wide variety of phosphorylated substrates in vitro. This idea has been challenged recently by the identification of tissue specific proteins that act to target Ser/Thr phosphatases to specific subcellular locations, thereby endowing phosphatases with a high degree of specificity in vivo (Hubbard M.J. and Cohen P., Trends Biochem. Sci., 1993;18:172; Mochly-Rosen D., Science, 1995;268:247). For example, in striated muscle, two targeting subunits M and G, direct the catalytic subunit of PPl, PPIC, to different subcellular locations. The M subunit directs PPIC to myofibrils, acting to facilitate dephosphorylation of myosin (Dent P., et al., Eur. J. Biochem., 1992;210: 1037), whereas the G subunit localizes PPIC to both the glycogen particle and the membranes of the sarcoplasmic reticulum, where glycogen metabolizing enzymes and SR proteins serve as substrates for PPIC (Stralfors P., et al., Eur. J. Biochem., 1985;149:295; Hubbard M.J. and Cohen P., Eur. J. Biochem., 1989;186:711 ; Hubbard M.J. and Cohen P., Eur. J. Biochem., 1990;189:243; Macdougall L.K., et al., Eur. J. Biochem., 1991; 196:725). In addition, PPIC is also known to interact with proteins in nuclei, an inhibitor protein, NIPP1 (Beullins M., et al, J. Biol. Chem., 1992;267: 16538), and the
Saccharomyces cerevisiae protein sds22+ (Stone E.M., et al., Curr. Biol., 1993;3:13). PPIC has also been shown to interact with the product of the tumor suppressor gene, Rb, implicating PPIC in the control of tumorigenesis and cell cycle progression (Durfee T., et al., Genes Dev., 1993;7:555). Thus, the targeting of PPIC to discrete subcellular locations by physically interacting proteins allows for a high degree of substrate specificity and tight control of phosphatase activity. Recently, a number of proteins that direct PPIC to the glycogen pellet have been characterized and cloned from mammals and yeast. In Saccharomyces cerevisiae, the product of the GAC1 gene is required for glycogen metabolism and physically interacts with PPIC (Stuart J.S., et al., Mol. Cell. Biol, 1994,14:896). In mammals, two tissue specific glycogen localizing subunits of PPl C have been identified. RGl, the glycogen binding subunit of skeletal muscle, encodes a protein product of 160 kD and is expressed in both heart and skeletal muscle (Tang P.M., et al., J. Biol. Chem., 1991;266:15782). Reversible phosphorylation on two closely spaced serine residues contained within the amino terminal portion of RGl (sites 1 and 2) has been implicated in regulating PPIC activity in response to hormonal stimulation (Dent P., et al, Nature, 1990;348:302). According to this hypothesis, phosphorylation of site 1 following insulin stimulation leads to a higher affinity of PPIC for RGl, leading to activation of glycogen metabolizing enzymes by dephosphorylation, while phosphorylation of site 2 by cAMP activated protein kinase A (PKA) causes a reduced affinity and subsequent release of PPIC from the glycogen pellet. A glycogen binding subunit expressed exclusively in liver, G , was recently cloned and was found to encode a predicted protein product of only 33 kD. G was shown to also differ from RGl with respect to enzymatic activity towards various substrates and in the ability of G to serve as a substrate for PKA (Doherty M.J., et al., FEBS Lett., 1995;375:294).
In an effort to identify novel PPIC localizing subunits involved in regulating insulin stimulated metabolic pathways, a 3T3-L1 adipocyte cDNA 2-hybrid library was screened for PPIC interacting proteins. We describe the isolation and characterization of a novel glycogen binding subunit of PPIC, called PTG, that may act as a scaffold for the localization of critical enzymes in glycogen metabolism, including phosphorylase b, glycogen synthase, and phosphorylase kinase. PTG is expressed predominantly in insulin-sensitive tissues and was found to mediate the hormonal control of glycogen accumulation in intact cells. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the sequence comparison of PTG with glycogen localizing subunits of PPIC.
Figure 2 shows PPIC binding and glycogen localizing activity of PTG protein.
Figure 3 shows the tissue distribution of PTG expression. Figure 4 shows the binding of phosphorylase a, glycogen synthase, and phosphorylase kinase to PTG.
Figure 5 shows glycogen synthesis in CHO-IR cells overexpressing PTG.
SUMMARY OF THE INVENTION
The present invention provides an isolated murine protein comprising the amino acid sequence:
Met Ala Met Arg He Cys Leu Ala His Ser Pro Pro Leu Lys Ser Phe Leu Gly Pro Tyr Asn Gly Phe Gin Arg Arg Asn Phe Val Asn Lys Leu Lys Pro Leu Lys Pro Cys Leu Ser Val Lys Gin Glu Ala Lys Ser Gin
Ser Glu Trp Lys Ser Pro His Asn Gin Ala Lys Lys Arg Val Val Phe Ala Asp Ser Lys Gly Leu Ser Leu Thr Ala He His Val Phe Ser Asp Leu Pro Glu Glu Pro Ala Trp Asp Leu Gin Phe Asp Leu Leu Asp Leu Asn Asp He Ser Ser Ser Leu Lys Leu His Glu Glu Lys Asn Leu Val Phe Asp Phe Pro Gin Pro Ser Thr Asp Tyr Leu Ser Phe Arg Asp Arg
Phe Gin Lys Asn Phe Val Cys Leu Glu Asn Cys Ser Leu Glu Asp Arg Thr Val Thr Gly Thr Val Lys Val Lys Asn Val Ser Phe Glu Lys Lys Val Gin Val Arg He Thr Phe Asp Thr Trp Lys Thr Tyr Thr Asp Val Asp Cys Val Tyr Met Lys Asn Val Tyr Ser Ser Ser Asp Ser Asp Thr Phe Ser Phe Ala He Asp Leu Pro Arg Val He Pro Thr Glu Glu Lys
He Glu Phe Cys He Ser Tyr His Ala Asn Gly Arg He Phe Trp Asp Asn Asn Glu Gly Gin Asn Tyr Arg He Val His Val Gin Trp Lys Pro Asp Gly Val Gin Thr Gin Val Ala Pro Lys Asp Cys Ala Phe Gin Gin Gly Pro Pro Lys Thr Glu He Glu Pro Thr Val Phe Gly Ser Pro Arg Leu Ala Ser Gly Leu Phe Pro Glu Trp Gin Ser Trp Gly Arg Val Glu Asn Leu Thr Ser Tyr Arg. (SEQ ID NO.: 2)
The present invention also provides an isolated human protein protein comprising the amino acid sequence; Met He Gin Val Leu Asp Pro Arg Pro Leu Thr Ser Ser Val Met Pro
Val Asp Val Ala Met Arg Leu Cys Leu Ala His Ser Pro Pro Val Lys Ser Phe Leu Gly Pro Tyr Asp Glu Phe Gin Arg Arg His Phe Val Asn Lys Leu Lys Pro Leu Lys Ser Cys Leu Asn He Lys His Lys Ala Lys Ser Gin Asn Asp Trp Lys Cys Ser His Asn Gin Ala Lys Lys Arg Val Val Phe Ala Asp Ser Lys Gly Leu Ser Leu Thr Ala He His Val Phe
Ser Asp Leu Pro Glu Glu Pro Ala Trp Asp Leu Gin Phe Asp Leu Leu Asp Leu Asn Asp He Ser Ser Ala Leu Lys His His Glu Glu Lys Asn Leu He Leu Asp Phe Pro Gin Pro Ser Thr Asp Tyr Leu Ser Phe Arg Ser His Phe Gin Lys Asn Phe Val Cys Leu Glu Asn Cys Ser Leu Gin Glu Arg Thr Val Thr Gly Thr Val Lys Val Lys Asn Val Ser Phe Glu
Lys Lys Val Gin He Arg He Thr Phe Asp Ser Trp Lys Asn Tyr Thr Asp Val Asp Cys Val Tyr Met Lys Asn Val Tyr Gly Gly Thr Asp Ser Asp Thr Phe Ser Phe Ala He Asp Leu Pro Pro Val He Pro Thr Glu Gin Lys He Glu Phe Cys He Ser Tyr His Ala Asn Gly Gin Val Phe Trp Asp Asn Asn Asp Gly Gin Asn Tyr Arg He Val His Val Gin Trp
Lys Pro Asp Gly Val Gin Thr Gin Met Ala Pro Gin Asp Cys Ala Phe His Gin Thr Ser Pro Lys Thr Glu Leu Glu Ser Thr He Phe Gly Ser Pro Arg Leu Ala Ser Gly Leu Phe Pro Glu Trp Gin Ser Trp Gly Arg Met Glu Asn Leu Ala Ser Tyr Arg. (SEQ ID NO.: 4) The present invention also provides an isolated murine genomic DNA molecule comprising the sequence:
GGATCCCGCTGGCCCAGGGCTCAGGGACTCTACGGCCGCCTTCC AGCACGCTCTGGTCACATCCAGCCCCGCGGTGATCACGTTCCAG GGGCAGGGGCTCGTGCCCCAGCTGCACAGTGGTTGGTCAGGGC GCACGGCCTTTGATTGGTCGGAGGGACCGGTTCACGTGATCTGG
CTTTGATAAGCTGCCTCCCGGTTGCCTGCGGTCAGTCGGCCGGC TGGACCGCGGCGCTGCATCCTCTTAAGTACTGAGTGCGAAGTTG CTCAGGGTGCGGTTGTCGCGGTCGCCGCTGCCTCTCGGTCCAAT GAACTGCACCAGGTAAGTTCAGTGCAACTCTGCGCCCGAATGG AGGGGAGCCTGCAGCTGCGGGGACTTGGGGGCTTTTGTGGTTTT GTCTTCTCTGCAGATGGGAGACTCCCAGGTTGATGGCTGAGTTG AATCGTCCCGGAGGCTTGGAAGGGATGGAATTTGAGTTGAATTT
CTTAGGCGCCTGTTTTGGGGAATCTAACATCTCAACAGTAGAAA CTGCCTTAGTGAGTCAGTTTTAGCTGGGATTCCTTTGCTGAAGT GACTCTGTTGCCTTTGAGCTCAAAGACCCAAAAAATTGGGTTGC AGCTTAGTTTTATTGATGAGGTAGTTTAGCCTGTGGCAAAAGGT AGAGGATTTTGC ATTTCTTCTTTCCCCACTTCCCCTCTCCTGGTT
TTGTCATAAAAAAATGCAGTCTTAGAGTTTGAGCAAGCTATTAT TTTTAACTCCTGGCCAGGACTTAGGAATTCCAGGGAAATAGCCC ACTACTAAAACTTTCTATAAATGCAGGAGTAAAAAAGGAAAAA AACCAAAAAAACTAAAACAAAAAGAAAAACAAGAACAGCTCA ACCAATAAAAAACGAACAACAGCTCCATCAACCAGGCAACCAA
GACCATCTCACGAAATGTTGCATCTCCAGTTGTAACTTAGTCCA AGAATTCTCCCTCCCCAAATCCTGCCTCTTAATGGTTGTGAGAC AAATAGACTTTGAGGTTTTGTTGCTGGAAGTTTGTAATCCTCTCA GAAAAACCCTAGAGCTATTAAAGTGAGTTCAGTATACATTCTTT ACAGGCCACTTTGTGATGATGACCATATACAGGGTGCTGTGCTT
AGCACTGGTATGAAGAGAATTCTTCAAGCCAGACTATATTTATG ATCAAAATATGTTATATACATATATAAACTGTTAAAGAATAAAT AATTAATAAAGCCCCAGACTCCAAAATGGCACCTACTGTATTTC ATGTTTCTTTGATTTATAAGGCTGACATTGGAGAGGAAAAACCT AATTCTGTAAGTGTGGAATTATAGTGTGGTTCAAGGGAAAGAA
AGGCCAGAAAAGTCTTGATGGATGGTTCTTTGTTTGATGCAAAC TATTGTTATTGCCTTCCTACTTGTAAATAGGACCTGCTCAACAAC AGGAAACATCATAGAAGCCCAAACCAGTAGATGCTATAATNCC ATCAGTCAAAGATAATGAACATCTTAGTGTTTNCCTTTGTAGTTT NCAGTATTTATAAATNCATATATCTTCAGTGTATTTTAAAACAG
CTCATTTNCTGGTTATCAGTTTTTAAAACTACTTTATGTTGTGTA TATATAATGTACACTGCAGGCTACAAGACAGAGCTATAGTAAA GTGGTTATTACTGGTCAGAATGAACAANTCTGTTATCCCTGCAG GTTAGCTATTCCCTTTATAGTTGGACCTGTCATGGGCATCTTTCC TATATGAACTGTCAGATTGTTTAAAGTTTTTCCTTTAGTCTGTGA GATGTTGGCTGGTGTTGCAGTTGGTCATTTGTGAAAAAATGAGG ATGACTGTGATAAAATGAAAAAGTCATTCTTTCTTTTAACAAGC
GTCACCTACTGTCACTCTAAGGACAGCATGACATTTTAAGAATT GCTTCATTTATTGTTTCCCAAGTGGATTACTTCTCCTGAGAAGTA AAACCGGTTCGAGAGCCAAAATAGGAAACAGCAGCCAGAGGG AGCGAGAGGCTGGGACTGTGATAATGGAAGAAGCTGTCTGGCC AATGGACTCTTTTGGGGGAAGCTTTAAG AACATATTTACCTTTC
TGGCTCCATGCCATGAAGCTCTACTGTAGTGGTTTTAAGTCCCC GGAATCTGAATTTTTTTTTTCTAAAGGAAAGAAACTTCTCAGGT CTTGTTGATCTGACAGGTTTAAGAACCACTGGCCCAGAACAGAG TACATAATTCCAAGAGCTGTGTCAGACTTGTTCAGATAGAGCCC TCTTGTTTCTC AGATGGAGAAACTGAATCCTCTCTGAGTGTTTCA
GGCAGTTTACACATGGGCCCAGCAGCCTGCCAAGCACAGAGCT AGACTGTAGATCTCATCACCCCAGTGCTCTCCTTTTCTCCACGTG ATAGCACCTCTCTGCACTGGAGTACTAGTGTGTGTGCATTTGGG ACCAGGGGAAGACGACTCCAGACCTCGGTGATTACCACTGTTTT TTTTTTTTTTTTCTCATTCCAGAATGATCCATGTGCTAGATCCAC
GTCCTTTGACAAGTTCCGTCATGCCCGTGGACATGGCCATGAGG ATTTGCTTGGCTCATTCACCACCTCTGAAGAGTTTCCTGGGTCCT TACAATGGTTTTCAACGAAGAAATTTTGTGAATAAATTGAAACC TTTGAAACCATGTCTCAGTGTCAAGCAGGAAGCCAAATCGCAG AGTGAGTGGAAGAGCCCACACAACCAAGCCAAGAAGCGGGTCG
TGTTTGCGGACTCCAAGGGGCTGTCACTCACTGCTATCCATGTC TTCTCCGACCTTCCAGAAGAACCAGCGTGGGACCTGCAGTTTGA TCTCTTGGACCTTAACGATATCTCCTCCAGCTTAAAACTTCACG AGGAGAAAAATTTGGTTTTTGATTTTCCCCAGCCCTCAACCGAC TACTTAAGTTTCCGGGACCGCTTTCAGAAGAACTTTGTCTGCCT
CGAGAACTGCTCTTTGGAAGATCGGACGGTGACCGGGACAGTG AAAGTGAAGAATGTGAGCTTTGAGAAGAAGGTTCAGGTCCGGA TCACCTTTGACACCTGGAAAACCTACACAGATGTGGACTGTGTC TACATGAAGAATGTTTACAGCAGCTCAGACAGCGACACCTTCTC CTTTGCAATCGACTTGCCCCGTGTCATTCCAACTGAGGAGAAAA TTGAGTTCTGCATTTCTTATCACGCTAATGGGAGGATCTTCTGG GACAACAATGAGGGTCAGAATTACAGAATTGTCCATGTGCAAT
GGAAACCTGACGGAGTGCAGACTCAGGTGGCACCCAAAGACTG TGCATTCCAACAGGGGCCCCCTAAGACTGAGATAGAGCCCACA GTCTTTGGCAGTCCAAGGCTTGCTAGCGGCCTCTTCCCAGAGTG GCAGAGCTGGGGGAGAGTGGAGAACTTGACCTCCTATCGATGA. (SEQ ID NO.: 1)
The present invention also provides an isolated human genomic DNA molecule comprising the sequence:
CTTGACCTGTCTAAGCTTTCAGTTCCTCATCTGTGAAATAAAGA GTTTGATGCCTATCACCTCCTACCTCCATAATTCTAACCATTGATGGGT CATTAAAATAAGACAATATGGTGCAGCGGTTATTGCTCTGGTATCAGC
CAGGCTCTAATCCCTGCTCTACCTGTGAGAACCTGGGCAGGTTTTTTTT TTGTTTTTTGTTTTCGAGATAGAGTCTCGCTCTGTTGCCCAGGCTGGAG TGCAGTGGTGCAATCTCAGCTCACTGCAACCTCCGCCTCCCGGGTTCA AGCGATTCTCCTGCCTCAGCCTCCAGAGTAGCTGAGAGTACAGGTGTG CACCACCATGCCCGGCTAATTTTTGTATTTTTAGTAGAGATAGGGTTTC
ACCATGTTGGCCAGGCTGGTCTTGAACTCCTGGCCTCAAGTGATCCACT GGGCAGATTTCCTGACCATTCAGTGTCTCCGTTTTCTTTTCTCTAAAAT GGGATTAATAACTGGACATATCACATAGGGTTGTTGTGAGGATTGAAT TGATAGCACATAGTGTTTGGCACAGAGTAAAGGCTCAACAAGCAGCAG CTATTCTCAATATTTTAGCTCAGGCACCAGGCGCCTTGAGGTGATAGA
GTAAAAACTCTAGCTGAGAGATCAAGTAGAAACTTGGGAACTAGCCCG GGTGGAACACAGGCACTGGGCATCGTGCTGAGTCTGTTCATTGGCACC ATCTTACTTCATCTTCAGAACGTTACTATCTCTGTTTTACACATGAGGA AACTGAGGTTAGAACTTGCCTAGTTCGGTAGCTAGTAAGTGTCAATCC AAAGACCTTCCAGCTAGTTTTGGTTGAGCTAAAGGGGCTAGAAGACCT
GCCATTAGTTAGATATTTCATTTCAAAAATAAAACCCAGGCATGAAGT CCCTTTCCCAGTGATATTCAGTGTGATTTTTTTCTTCACTCTAATAATTT TAACAATTCCACTGTTTGACAGTTGTTTAAAAGACATAGGAATTTTTGT ATATTTTAATTGACTAATGGATAGCTCAATTAGGGGAGCAAAACTAGG ATGTGGGTTTTATAAAAATAATTTAGACTTGACTTAGACATTTAATTTT ACAGTTGTAAATGATGGTCTAAAAATTCTTCAAACTAATCAAAATAAT GAAACTTCAGCGAAAGTGAGTGGCTCAGAAGGCCCATGAAACATACG
GCGTGATTTTTTAAATTTTATTTTAACATTTTGATTTCCACACCACTGCC AAAGGACGTCAGAATTGAGTAAGGGGTTTGGGTTGACTGCTGCCTCTT GACCGGCTGTATGTGTGAAAAGGGTCATTTCACTTCCGGCTTTAGTGTT CCCCGCAGGGGAGAAAATTGAAGAATAGACAGAAATACGAAGTGTCT TTTAATTAAATGCCACCTTGGTGTTTTATGGGGCTCGTATGCTTTCCTA
ACAACATTTGTTAGATAAGTTGGTAATTCCCGGCAGCTGTCTACTGTGT GGTGCATCTGTGAACTCATACTAATCGAAAAGCATGCAGCCAGTTTGG GATCGCGCAGGCTAAGGTGAGGGAGAAATGCGGATACACCGGGTAAT GAACGATATAAACATTTCAAATGCGATACACATTCGGTTTGAGCCACA TCTTCTGTGTGCAGATTCACCCGCAGTGACCCACAAAGCTATTCCC AA
GTAACAGCCGCCCCAAGCCTGAGGCACTGGCGCCCCGCCTGGGCGAG GCTGGCTGCGCTCTCTCTTGGCCGGCGCCCGCTGCATGCGGTACGTGCC TGCCCGGCCCCTAGCCCAGGGTTCCCGTTACGCGGCTGGTTCCAGCTG GCCGCGGAGTCCCAGAACCTCCCCGGGATGCCCAGATAGCTCTCTGCA CGTCTGGCCCCGGGGCGATCACGTTGCCGGGGCGAGGGCTGGCGCCCC
AGCTGGGCGCTGGTTGGTCGCGCCCTGGGGCTCGAGGCCCGGCGATTG GTCCCAGGGATCGGGTCACGTGCTTGGGAGCAGATAAGCGGCCTCTAG GCGCCGGGCCCTCAGTCTCTCCCAGCGACCGCCGCGGGGGCAAGGCCT GGAGCTGTGGTTCGAATTTGTGCAGGCAGCGGGTGCTGGCTTTTAGGG TCCGCCGCCTCTCTGCCTAATGAGCTGCACCAGGTAGGTTCGCTGCAA
CTCTGCGCGCTAGGAACACAGGGGAACGCGCAGCTGTGGGGAAGTTG GGGGGCGTTTCAGTTCTATCATCTCTGGAAATGGACACCCCAGGGGGA GGACAAGTGGACTGACTGCGTAGTTGAATCTGGCAACCGAGAGGCCTT GGAGGTGTAGAAATTTGGCTCTATTTCTTAAGCAGAGCCTATTTTAGTA ATCAGCATCTTAAAGCAGAAATTATCTTAACGTGAATCAGCTTGAGTT
AGGATTTTCTCATGGATGCGGCTGTTCTTTTGGTCCTGCACAAATGTCC CAAAGACTCGGGCAGCTGAAGTGGTGAGAACAGCACTCTGACATTGCT GGTTAGGTGGTTTAGCTTGGAGGAAAAAAATTACAGGACGACGTTTGC ATTCATTCGTCCTTCTTATCACAGTTTGCCATAGCAAAATCTCAAGAGT TTGAGCAAACGATTACTTTTAACTCTTGTCCAGGACTTAAAGTTCCAAG GAAATCACCCAAACTAAAACTGTCTTTCTATAAATGCAAAAAGTAAAA AAAAAAAAACAAAAAAACCAAAAAAAACCTCCCATAAAACTACTTTA
AATAGCTTCTCCAGACATAGCTTAGCAGAAGANTTCTCTAAAAATCCT GCCTATTAACTATTATTAGACCCACAAATATAGCTTTAGCTTTCATTTG TTTGTTNTAAGTTTGCAGATCTCCCAGAAAAACCCCAGAGCTAACACA GTAAATTCTGCGAGTGTTATTACACACTTTTGTGATAATGACCACTTGC ATACATGTTTAGAGCTGCTGTGAGGAGAGTTACTAAAGCCAGACTGAG
AAATGTCGTGTACAGTATACACACACCTCTTACTTGTAAGGCTAAGAT AGGGAAAAAAATCTTAATACCATAAGCTTGGAAATATATGATGAGGGC TAAAGGTCAGAGAAAAGTCTTCTTTATAGATGCTTCTTGGTTTAATATT GCTGAGCATAGTCATGTTTAAAACTTTAAATGGTTTTATTGTCTTTCTA CTTATAAATGTCTATTAGAA AATGCC AAAAAAGAACAAAAACG AAAA
TAGATAATCTATAATCCTATCACCCAGAAATAATAATTATTAAATTATT AGGAAAGGTGTATTTCCTATAGAGTTTTTCAATATTTATAAGTTTGTAT ATATAAAATGTATATTTTAAAACACTCCAACTTTCAGGTAATCAGTTTT TCCACTTAAATGTGGACTTGTCATGGGCATCTCTTTAGGTGAATTATCA ATTATATAGTTTTTAAGTGCATATGAATTGTTGGCTTGTATTTCAGTGG
TTATTTGTGAAAAATAAGAGCATGATAATCAAAGTGCAAAGATGATTC TTTGACTTCTTCTCTAGCCTTCTCACTTTCAAAACTGCATGTTATTTTTT TTTTTCAAGTGAATTACCTTACCAGAGAAGTGTCAATCAATTTAGCAGC AAAATAAGCCAACGTAGCCAGAGGGAGCAGAGGGTCTGGAACTGTGG CTCCTGAACCTGTCTGGTCATTAGAATCACCTGGGAAGCTTTAAGAAC
ATACCCATCCCTTGGCCCTAGCCCCAGAAGTTCTGCCTCAGTAGTTCTG AGTCCCAGGAATTGGAAAGAAAGAAGAAAGAGAAAGAGAGAGAGAG AGGAAGAAAGGAAGGAAGGAGGGAAGGAGGAAAGGAGGAAAGACAA GAAAGAAAGAAAATGAATTCCCTAGACATAGTGACCAGACAGGTTTG AGGACCACTGGTCCAGAACAGAGCACACAGTTCTCAAGGCTGCCTTGG
AGATAATCAAATCGAACCCTTTTATTTCTCAGATGGGGAAACTGAGAC CCCCATCACCCTCTAAGTGTTTTAAGCAATTAATAGCCTTTACCGGCCA AGGGTAGAGGTAGACATAGAAGATCTGATCACTTAATACTGTTCTCTT TTACTACATATGATAGCACCTGCCTGATATCTAGTGCACTGGCTATAAT TCAGTCAGCACAAAAATAGTACATATGTATTTGGCACTGGGGAAGAGC ATTTCCGATCCAGGTGATAATCCCTCTTCTTTTTGCATTCCAGAATGAT CCAGGTTTTAGATCCACGTCCTTTGACAAGTTCGGTCATGCCCGTGGAT
GTGGCCATGAGGCTTTGCTTGGCACATTCACCACCTGTGAAGAGTTTCC TGGGCCCGTACGATGAATTTCAACGACGACATTTTGTGAATAAATTAA AGCCCCTGAAATCATGTCTCAATATAAAACACAAAGCCAAATCACAGA ATGACTGGAAGTGCTCACACAACCAAGCCAAGAAGCGCGTTGTGTTTG CTGACTCC AAGGGCCTCTCTCTCACTGCGATCCATGTCTTCTCCGACCT
CCCAGAAGAACCAGCGTGGGATCTGCAGTTTGATCTCTTGGACCTTAA TGATATCTCCTCTGCCTTAAAACACCACGAGGAGAAAAACTTGATTTT AGATTTCCCTCAGCCTTCAACCGATTACTTAAGTTTCCGGAGCCACTTT CAGAAGAACTTTGTCTGTCTGGAGAACTGCTCGTTGCAAGAGCGAACA GTGACAGGGACTGTTAAAGTCAAAAATGTGAGTTTTGAGAAGAAAGTT
CAGATCCGTATCACTTTCGATTCTTGGAAAAACTACACTGACGTAGAC TGTGTCTATATGAAAAATGTGTATGGTGGCACAGATAGTGATACCTTCT CATTTGCCATTGACTTACCCCCTGTCATTCCAACTGAGCAGAAAATTGA GTTCTGCATTTCTTACCATGCTAATGGGCAAGTCTTTTGGGACAACAAT GATGGTCAGAATTATAGAATTGTTCATGTTCAATGGAAGCCTGATGGG
GTGCAGACACAGATGGCACCCCAGGACTGTGCATTCCACCAGACGTCT CCTAAGACAGAGTTAGAGTCAACAATCTTTGGCAGTCCGAGGCTGGCT AGTGGGCTCTTCCCAGAGTGGCAGAGCTGGGGGAGAATGGAGAACTT GGCCTCTTATCGATGAATTAAGCAACAATGTAACTGGTCTTGACTTGTC ATATTCCCCCATGCAATCCTAGGTCTGTATTGCTCAATTTTAGGAAGCC
TTTGCTACTCCATCAGTAGGTTTAGATTTGAGCTTTTGAAACCTGGCTA TGGAAAAGAAAGACACTTGAGAATTTATGTTGGGGTCTGTACAGATAA ATGCTAACCCAATTTGGCTTTGAAGGATCAAGTAACAGGTTGAAAACT ATTTTTATAAAGGTAATACTTTTTCAGTTCCCTTCTTCCTTCCCTCTCAA TCCACTAGCTTTCATGTTGGGCAAGGAAAAGTTGAGGAAGGATGGCTG
ATGGTGATGGAAAGCTATGTTAATGGTATGAGGAATGTGTGAAAAGTA TACACAAAGGGCTCTGAAGCTCAAGTCAGAGGAGTGGGAGGTCTGATC ATTGTTGGTGGAAAAACGTAAGGTTATTTTGTGTTTTTAAGTTGGTTTT ACAATTCTTTCCTGGGGAAATTATTTCTGGAGGGGAAAAAGATCCATT CTACGTATCCTTGTGGAGAAAAGCTAAATAACCTTTAAGAATGTGGGT GGTATTGGAGAAAGAAGATGAATTATAGCTCCGGAGAATCAAGATCT. (SEQ ID NO.: 3)
The present invention also provides an isolated murine cDNA molecule comprising the sequence:
ATGGCCATGAGGATTTGCTTGGCTCATTCACCACCTCTGAAGAG TTTCCTGGGTCCTTACAATGGTTTTCAACGAAGAAATTTTGTGAATAAA TTGAAACCTTTGAAACCATGTCTCAGTGTC AAGCAGGAAGCCAAATCG
CAGAGTGAGTGGAAGAGCCCACACAACCAAGCCAAGAAGCGGGTCGT GTTTGCGGACTCCAAGGGGCTGTCACTCACTGCTATCCATGTCTTCTCC GACCTTCCAGAAGAACCAGCGTGGGACCTGCAGTTTGATCTCTTGGAC CTTAACGATATCTCCTCCAGCTTAAAACTTCACGAGGAGAAAAATTTG GTTTTTGATTTTCCCC AGCCCTC AACCGACTACTTAAGTTTCCGGGACC
GCTTTCAGAAGAACTTTGTCTGCCTCGAGAACTGCTCTTTGGAAGATCG GACGGTGACCGGGACAGTGAAAGTGAAGAATGTGAGCTTTGAGAAGA AGGTTCAGGTCCGGATCACCTTTGACACCTGGAAAACCTACACAGATG TGGACTGTGTCTACATGAAGAATGTTTACAGCAGCTCAGACAGCGACA CCTTCTCCTTTGCAATCGACTTGCCCCGTGTCATTCCAACTGAGGAGAA
AATTGAGTTCTGCATTTCTTATCACGCTAATGGGAGGATCTTCTGGGAC AACAATGAGGGTCAGAATTACAGAATTGTCCATGTGCAATGGAAACCT GACGGAGTGCAGACTCAGGTGGCACCCAAAGACTGTGCATTCCAACA GGGGCCCCCTAAGACTGAGATAGAGCCCACAGTCTTTGGCAGTCCAAG GCTTGCTAGCGGCCTCTTCCCAGAGTGGCAGAGCTGGGGGAGAGTGGA
GAACTTGACCTCCTATCGATGA. (SEQ ID NO.: 5)
The present invention also provides an isolated human cDNA molecule comprising the sequence:
ATGATCCAGGTTTTAGATCCACGTCCTTTGACAAGTTCGGTCAT GCCCGTGGATGTGGCCATGAGGCTTTGCTTGGCACATTCACCACCTGT
GAAGAGTTTCCTGGGCCCGTACGATGAATTTCAACGACGACATTTTGT GAATAAATTAAAGCCCCTGAAATCATGTCTCAATATAAAACACAAAGC CAAATCACAGAATGACTGGAAGTGCTCACACAACCAAGCCAAGAAGC GCGTTGTGTTTGCTGACTCCAAGGGCCTCTCTCTCACTGCGATCCATGT CTTCTCCGACCTCCCAGAAGAACCAGCGTGGGATCTGCAGTTTGATCT CTTGGACCTTAATGATATCTCCTCTGCCTTAAAACACCACGAGGAGAA AAACTTGATTTTAGATTTCCCTCAGCCTTCAACCGATTACTTAAGTTTC
CGGAGCCACTTTCAGAAGAACTTTGTCTGTCTGGAGAACTGCTCGTTG CAAGAGCGAACAGTGACAGGGACTGTTAAAGTCAAAAATGTGAGTTTT GAGAAGAAAGTTCAGATCCGTATCACTTTCGATTCTTGGAAAAACTAC ACTGACGTAGACTGTGTCTATATGAAAAATGTGTATGGTGGCACAGAT AGTGATACCTTCTC ATTTGCCATTGACTTACCCCCTGTC ATTCCAACTG
AGCAGAAAATTGAGTTCTGCATTTCTTACCATGCTAATGGGCAAGTCTT TTGGGACAACAATGATGGTCAGAATTATAGAATTGTTCATGTTCAATG GAAGCCTGATGGGGTGCAGACACAGATGGCACCCCAGGACTGTGCATT CCACCAGACGTCTCCTAAGACAGAGTTAGAGTCAACAATCTTTGGCAG TCCGAGGCTGGCTAGTGGGCTCTTCCCAGAGTGGCAGAGCTGGGGGAG
AATGGAGAACTTGGCCTCTTATCGATGA. (SEQ ID NO.: 6)
DETAILED DESCRIPTION OF THE INVENTION
PTG is a PPIC binding protein with homology to known glycogen binding proteins: A 3T3-L1 adipocyte cDNA library fused to the Gal4p transcriptional activation domain was screened for proteins that could interact with a Gal4p-PPlC DNA binding domain fusion. Library plasmids containing interacting proteins were identified by the ability to induce transcription of the integrated GALI-lacZ reporter. Of approximately 3.5 x 10^ primary tranformants, 27 were positive for β-galactosidase activity. One class of interacting cDN As, typified by clone B 1 - 1 , consistently gave the highest levels of β-galactosidase activity when plated on X-gal containing media. Induction of β-galactosidase activity was dependent upon coexpression of BD-PP1C, since β-galactosidase activity was not observed when non-specific gene fusions were used. Partial DNA sequence from the GAL4 fusion junction followed by a BLAST search revealed that the cDNA contained in clone Bl-1 was homologous to the hepatic glycogen binding subunit (GL) cloned from rat liver (Doherty M.J., supra., 1995), both in nucleotide and predicted amino acid sequence. Sequencing of an additional clone (B2-2) from the same class provided a probable translational initiation site (Kozak M., J. Cell Biol, 1989; 108:229). The PPIC interacting cDNA contained in clones Bl-1 and B2-2 has been assigned the name PTG.
Analysis of the predicted primary amino acid sequence of PTG revealed significant homology with other proteins previously shown to localize PPIC to the glycogen particle (Figure 1). Sequence comparison of the predicted protein product of PTG to known glycogen binding subunits of PPl C indicates that PTG is most homologous to GL (42% Identity; 60% similarity), with less striking homology to the skeletal muscle protein RGl, and Gael, the yeast glycogen binding subunit (Stuart J.S., supra., 1994) (26% identity; 49% similarity and 27% identity; 50% similarity, respectively). Interestingly, the residues corresponding to phosphorylation site one and two of RGl, which were implicated in hormonal control of PPIC activity (Dent P., supra., 1992), are not conserved in the predicted PTG protein.
PTG can direct PPIC localization to glycogen both in vivo and in vitro: The homology of the predicted PTG protein with Gj^, RGl and Gael suggests that PTG might bind simultaneously to both PPl C and glycogen. We tested this hypothesis directly by evaluating these associations in both in vivo and in vitro assays. A FLAG epitope tagged PTG construct (FLAG-PTG) was transiently transfected into CHO cells over-expressing the insulin receptor (CHO-IR), followed by immunoprecipitation with αFLAG antibodies and subsequent immunoblotting with αPPIC antibodies. PPIC could be co-immunoprecipitated from cell lysates with the antibodies directed against FLAG-PTG (Figure 2A), demonstrating direct association of PPIC with PTG in vivo. This association was unaffected by treatment of cells with insulin. Moreover, PTG did not appear to undergo phosphorylation in response to insulin, and was not a substrate in vitro for cAMP-dependent protein kinase, or other protein kinases in lysates from insulin stimulated cells (not shown). To determine the subcellular localization of PTG, similarly transfected CHO-IR cells were fractionated by differential centrifugation followed by SDS-PAGE and immunoblotting with αFLAG antibodies. FLAG-PTG in the 14 K x g supernatant was found to localize exclusively to the glycogen-enriched cell fraction (Figure 2B). Immunoblotting of these same samples with αPPIC antibodies showed that overexpression of FLAG-PTG caused a dramatic translocation of PPIC from the cytosol into the glycogen pellet (Figure 2C). Binding of PPIC and targeting to the glycogen fraction was also observed in vitro. Addition of a GST-PTG fusion protein to 3T3-L1 adipocyte lysates caused an 8-fold increase in the PPl activity sedimenting with the glycogen pellet, whereas the addition of a irrelevant GST fusion protein (enzymatically inactive tyrosine phosphatase PTP1B) had no effect on the localization of phosphatase activity to the glycogen pellet (Figure 2D). These data demonstrate that PTG can simultaneously and specifically associate with both PPIC and glycogen. PTG is expressed in insulin responsive tissues: Although many tissues and tissue culture cell lines express insulin receptors, the metabolic actions of insulin occur mainly in fat, liver, and muscle cells. Other PPIC -targeting proteins are expressed in a tissue specific manner. RGl is expressed in muscle tissue (diaphragm, skeletal muscle, and heart) (Tang P.M., supra., 1991), whereas GL is expressed exclusively in liver tissue (Doherty M.J., supra., 1995). To determine the tissue distribution of PTG expression, a rat multi-tissue northern blot (Clontech) was hybridized with a probe prepared from the cDNA insert of clone Bl-1. An mRNA of approximately 2.3 kb was detected in all tissues except testis (Figure 3A). However, the PTG mRNA was most abundant in skeletal muscle, liver, and heart. PTG transcript was also detected in RNA prepared from rat adipose tissue (not shown).
The differentiation of 3T3-L1 fibroblasts into adipocytes is correlated with a significant increase in insulin sensitivity, including the stimulation of glycogen synthesis. The expression of many genes critical to insulin action is increased during adipocyte differentiation, including the insulin receptor, GLUT4 and others
(Reed B.C., et al., Proc. Natl. Acad. Sci., USA, 1977;74:4876; Rubin C.S., et al., J Biol Chem., 1978;253:7570; de Herreros A.G. and Birnbaum M.J., J. Biol. Chem., 1989;264: 19994), although PPIC levels remain constant (Brady M., et al., in preparation). To determine if PTG expression is correlated with the observed increase in insulin sensitivity, northern analysis was performed on RNA isolated from both 3T3-L1 fibroblasts and fully differentiated adipocytes. A hybridizing mRNA species of approximately 2.5 kb was observed in 3T3-L1 adipocytes, whereas it is weakly expressed in pre-adipocytes (Figure 3B). Thus, PTG is expressed mainly in liver, muscle, and fat tissues in which the regulation of glycogen synthesis plays an important part in insulin action. The tissue distribution of PTG is therefore significantly different from that of previously described glycogen binding subunits of PPIC (Tavy P.M., 1991 and Doherty, M.J., 1995).
PTG can associate with multiple proteins involved in regulating glycogen metabolism: Glycogen synthesis and breakdown is regulated by the reciprocal actions of protein phosphatases and kinases. To determine whether PTG is involved in the localization of the metabolizing enzymes, and of the kinases and phosphatases involved in their regulation, a series of in vitro binding assays were performed with a bacterially expressed PTG fusion protein. First, we examined the ability of PTG to bind to phosphorylase a, the phosphorylated, active form of the enzyme that directly catalyzes glycogen breakdown. GST-PTG bound to glutathione-Sepharose beads was incubated with [32p]-phosphorylated phosphorylase a. After extensive washing, bound proteins were analyzed by SDS-PAGE followed by autoradiography. GST-PTG efficiently bound to phosphorylase a, but did not bind an unrelated GST-fusion protein, GST-PTP1B (Figure 4A). To determine whether PTG could also target the anabolic enzyme involved in glycogen synthesis, glutathione-Sepharose bound GST-PTG was incubated with purified glycogen synthase. Glycogen synthase activity was specifically associated with GST-PTG, and not with an irrelevent GST-fusion protein, PTP1B (Figure 4B).
Phosphorylase kinase converts phosphorylase from the inactive b form to the active a form. This activation can be reversed via dephosphorylation by PPIC.
Phosphorylase kinase can also be directly inactivated by PPl. Because of the central role of phosphorylase kinase in regulating glycogen metabolism, it is also a candidate for association with PTG. GST-PTG-glutathione-Sepharose beads were incubated with 3T3-L1 adipocyte lysates, washed extensively, and assayed for phosphorylase kinase activity with [32p]-γ-ATP and phosphorylase b as substrate. Calcium-stimulated phosphorylase kinase activity (Krebs E.G., et al., Biochem., 1964;3 : 1022) was associated with GST-PTG, but not with GST-PTP 1 B
(Figure 4C). To confirm these results, the binding of phosphorylase kinase to PTG was also assayed using purified enzyme. As seen in Figure 4D, the purified protein also specifically bound to the PTG-glutathione Sepharose beads. Interestingly, neither PP2A nor cAMP-dependent protein kinase in Ll -adipocyte lysates bound to GST-PTG (data not shown), demonstrating the specificity of binding for both phosphatases and kinases to PTG. Taken together, these results imply that PTG can complex with each of the key proteins involved in regulating glycogen metabolism, although it is unclear whether all of the glycogen metabolizing enzymes are bound to a single PTG molecule, or if individual binding sites are shared between one or more proteins.
Overexpression of PTG increases insulin stimulated glycogen synthesis: The data presented above indicate that PTG can target to glycogen the two enzymes that are critical in controlling its metabolism, glycogen synthase, and phosphorylase a, as well as the major proteins that mediate the hormonal regulation of these enzymes, PPIC and phosphorylase kinase. These findings prompted us to examine whether overexpression of PTG could cause an increase in both basal and insulin-stimulated glycogen synthesis. CHO-IR cells express no detectable PTG transcript or protein (data not shown), and have a low basal rate of glycogen synthesis, which increases approximately 1.5- to 2-fold upon insulin treatment (Figure 5). Overexpression of PTG in the CHO-IR cells caused a 7-fold increase in the basal rate of glycogen synthesis. Exposure of these cells to insulin produced another 2-fold increase, with total glycogen synthesis increased over 10-fold. It should be noted that only 20% efficiency of transfection was achieved in these experiments, suggesting that the 10-fold increase in maximal glycogen synthesis by PTG is significantly underestimated. These results demonstrate that
PTG overexpression cannot only increase basal glycogen synthesis, but also dramatically elevate maximally insulin-stimulated glycogen accumulation in a poorly responsive cell line to a level comparable to that observed in insulin target cells (Lazar D.F., supra., 1995). However, because the sensitivity of these transfected cells to insulin remains unchanged, and because insulin does not appear to modulate PP 1 C-PTG binding, PTG itself is not likely to be a direct target of insulin signaling.
DNA sequences, such as PTG cDNA can be subcloned into a variety of plasmid shuttle vectors, allowing rapid amplification and manipulation of recombinant DNA sequences in bacterial and mammalian hosts. Plasmid vectors used for routine manipulation of DNA, such as Bluescript SK (GenBank
#X52328, Stratagene, La Jolla, CA), typically contain: 1) bacterial origin of replication, usually ColEl , 2)t Antibiotic resistance gene to select for transformed bacteria (usually Ampr, for selection on Amplicillin), 3) Multicloning site containing unique restriction enzyme sites to facilitate insertion of desired DNA fragments. Plasmid vectors that are used to introduce desired DNA fragments into mammalian cells contain, in addition to those components required for bacterial vectors, a promoter sequence and a gene coding for resistance to eukaryotic antibiotics. The promoter region is typically a viral promoter (CMV, Simian Virus 40, SV40) that directs high expression of the cloned gene in mammalian cells, and the antibiotic resistance gene is typically Neomycin phosphotransferase (Neor), which confers resistance to the eukaryotic antibiotic neomycin. Viral (retrovirus and adenovirus) vectors typically contain all of the above mentioned components, in addition to viral sequences that allow recombinant DNA to be efficiently packaged into viral particles and infect the mammalian host cells. These types of viral vectors are widely used to introduce recombinant DNA into mammalian tissue culture cells and in gene therapy, where recombinant viral particles are used to infect tissues in vivo.
Many mammalian cell lines derived from a variety of tissues are used as model systems to examine intracellular processes in the laboratory. For example, the 3T3-L1 and 3T3-F442A cell lines, cloned from Swiss mouse embryo fibroblast 3T3 cultures (Green and Kehinde, 1974, Cell, 1 :1 13-116), differentiate into adipocytes and are useful in studying adipogenesis and insulin action (Garcia de Herreros and Birnbaum, 1989, J. Biol. Chem., 264 -.19994- 19999V Other cell lines commonly used include NIH 3T3 fibroblasts (ATCC #CRL-1658), rat muscle cell line L6 (Proc. Natl. Acad. Sci. USA 61:477-483, 1968) and Chinese hamster ovary cells, CHO-K1 (ATCC #CRL-9618). Primary cells derived from isolated mammalian tissues can also be cultured in the laboratory, however these cells have a limited lifespan in culture and usually die after 7 to 10 days.
Recombinant DNA can be introduced (transfected) into mammalian cells either in culture or in vivo by a number of techniques. Calcium Phosphate- mediated transfection (Chen and Okayama 1987, Mol. Cell. Biol.. 7:2745-2752) is used with cells in tissue culture, however some cell types transfect at very low efficiency. Liposome-mediated transfection (Lipofectamine; Gibco-BRL) (Feigner, et. al., 1987, Proc. Natl. Acad. Sci. USA 84:7413-7417.) has been used to transfect cells resistant calcium phosphate mediated transfection. Virtually all cell types, including primary cells in vitro and in vivo, can be infected with recombinant retrovirus, making this method of DNA delivery particularly useful
(Miller, et al., 1993, Methods in Enzymology, 217:581-599). Likewise, adenoviral -mediated gene transfer has been used to infect terminally differenciated cells and tissue (Becker et al. 1994, Methods Cell Biol.. 43:161-189), since adenoviral infection does not require actively dividing cells, as does retroviral gene transfer.
Human gene therapy has relied upon the ability of recombinant virus, both retrovirus and adenovirus, to deliver desired genes to the appropriate tissues in vivo. The expression of foreign genes using adenovirus vectors was first developed over a decade ago, and since then, adenovirus vectors have proven useful in studies of gene therapy and vaccine development, as well as in basic biology. In gene-therapy studies, effective gene transfer using adenovirus vectors has been demonstrated (Engelhardt, et al., 1994, Proc. Natl. Acad. Sci. USA. 91 :6196-6200). Retroviral-mediated gene transfer has been successfully used to correct adenosine deaminase (ADA) deffiency in humans (Blaese, et al., 1995, Science. 270:475-480; Kohn, et al., 1995. Nature Med.. 1 :1017-1023).
The manufacture of animals having one or more endogenous genes deleted is well known to these skilled in the art. See, for example, U.S. Patent Numbers 5,487,992 and 5,464,764, herein incorporated by reference, teach how to make animals having a gene deleted (also called a knock-out animal). In particular, U.S. Patent Numbers 5,464,764 and 5,487,992 teach how to make knock-out mice. Mice having a PTG knock-out are useful as animal models for increasing glycogen in cells, and are specifically useful as a model for diabetes.
The present invention is also useful in that new drugs can be identified by screening librarys of chemical compounds for agonists or antagonists (inhibitors) of the PTG protein.
The experimental presented below is intended to illustrate particular embodiments of the invention and is not intended to limit the specification, including the claims, in any manner.
EXPERIMENTAL
The examples and procedures set forth below are intended to illustrate particular embodiments of the invention and are not intended to limit the specification, including the claims, in any manner.
The following abbreviations are used herein:
A Adenosine
C Cytosine
G Guanosine
T Thymine
U Uracil
Ala Alanine
Arg Arginine
Asn Asparagine
Asp Aspartic Acid
Cys Cysteine
Glu Glutamic Acid
Gin Glutamine
Gly Glycine His Histidine
He Isoleucine
Leu Leucine
Lys Lysine
Met Methionine
Phe Phenylalanine
Pro Proline
Ser Serine
Thr Threonine
Trp Tryptophan
Tyr Tyrosine
Val Valine
PTG Protein targeting to glycogen
CHO-IR Chinese hamster ovary cell expressing human insulin receptors cAMP Cyclic adenosine monophosphate kD Kilodaltons
DNA Deoxyribonucleic acid cDNA Complimentary deoxyribonucleic acid
SDS-page Sodium dodecyl sulfate-polyacrylamide gel electrophonesis
GST Glutathione S-transferase mRNA Messenger ribonucleic acid
BSA Bovine serum albumin
KPBH Krebs-ringer phosphate-buffered saline
HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid])
PNS Post nuclear supernatant
EDTA Ethylenediaminetetraacetic acid
CMV Cytomyalovirus
RNA Ribonucleic acid
MOPS 4-Morpholine propane sulfonic acid
PEG Polyethylene glycol
SSPE Standard saline phosphate EDTA SDS Sodium dodecylsulfate rRNA Ribosomal ribonucleic acid
EGTA Ethylene glycol-bis (β-aminoethylether)-N,N,N',N'-tetraacetic acid
ATP Adenosine triphosphate UDP Uridine 5'-diphospho-α-D-glucopyranose
SD Standard deviation
Cell Culture. Propagation and differentiation of 3T3-L1 fibroblasts has been described previously (Waters S.B., et al., Mol Cell Biol, 1995; 15:2791).
Chinese hamster ovary (CHO) cells expressing >1 x 10^ human insulin receptors (IRs) were grown in alpha-minimal essential medium containing nucleotides,
100 U of penicillin/m L, 100 μg of streptomycin/mL, 10% fetal bovine serum, and 250 μg G418/mL. Cells were incubated at 37°C in an 8% CO2-air gas mixture. Prior to experiments involving insulin stimulation, CHO-IR cells were incubated in serum free medium for 3 hours, followed by the addition of 100 nmol insulin for an additional 15 minutes.
Construction of 3T3-L1 Adipocyte 2-Hybrid Library. 3T3-L1 Fibroblasts were differentiated to adipocytes, as described previously
(Lazar D.V., et al., J. Biol. Chem., 1995;270:20801), and poly-(A+) mRNA obtained via the Messenger RNA Isolation kit (Stratogene, La Jolla, CA). Five micrograms of poly-(A+) mRNA was used to synthesize cDNA with the
Stratogene cDNA synthesis kit. First strand synthesis utilized an oligo άT-Xho\ primer, whereas the 5' end was ligated to an EcoRl adapter following second strand synthesis. cDNA Fragments were then ligated unidirectionally into EcoRl/Xhol digested pGAD-GH GAL4 activation domain plasmid (Clontech, Palo Alto, CA). Ligations were electroporated into E. coli D12S to yield >2 x
1 θ6 individual transformants.
2-Hybrid Screen. A Gal4p- DNA binding domain (BD) fusion of PPIC was constructed by cloning the entire PPIC open reading frame (a generous gift of A. Nairn), contained within a 1.0 kb EcoRl/BamHl fragment, into the Eco Λ/Bam sites of pGBT9 (Clontech), creating BD-PPIC. Strain Y190 was transformed first with BD-PP1C, Trp+ prototrophs selected, and then transformed with 150 μg of 3T3-L1 adipocyte library DNA. Transformants were selected by plating cells on synthetic medium lacking tryptophan, leucine, and histidine (SD-Trp-Leu-His) and containing 25 mM 3-aminotriazole (ATZ). Yeast transformations were performed by the lithium acetate procedure of Geitz, et al.,
Nucleic Acids Res., 1992;20:1425. Colonies that appeared after 5 days of incubation at 30°C were patched onto SD-Trp-Leu-His plates and then replica plated onto M63GV-Trp-Leu-His media containing 5-bromo-4-chloro- 3-indolylphosphate-β-D-galacto pyranoside (X-gal; Gibco-BRL) for preliminary determination of β-galactosidasc activity (Printen J.A. and Sprague G.F. Jr.,
Genetics, 1994;138:609). Of approximately 3.5 x 105 total transformants, 64 His4" prototrophic colonies were recovered, of which 27 were β-galactosidase positive. Plasmids containing interacting cDNAs were rescued from strain Y 190 by transforming into E. coli HB101 and plating onto M9 minimal media lacking leucine. HB101 contains a leuB deletion that can be complimented by the yeast
LEU2 gene, thereby selecting for E. coli transformants carrying only the library plasmid. When individual Leu+ HB101 transformants were analyzed, it was found that from a single plasmid rescue, 2 to 3 different plasmids were recovered, of which only a subset would be β-galactosidase positive upon retransformation into Y190 containing Gal4-PPlC. Apparently, individual yeast cells are able to take up more than one plasmid in the conditions used during the library transformation. This problem was circumvented by growing the yeast preparations in SD-Trp-Leu- His + 25 mM ATZ broth during plasmid rescue.
DNA Sequencing of PTG. Preliminary sequence information of all Gal4p-PPl interacting cDNAs was obtained by double-stranded sequencing using an oligonucleotide complimentary to the 3' end of the GAL4-AD coding region of pGAD-GH. Sequencing was performed with an Appligen fluorescent dye terminator kit (Perkin-Elmer) and an ABI 8700 automated sequencer. The entire DNA sequence of clone Bl-1 was obtained by subcloning a 1.0 kb EcoRI fragment of clone Bl-1 into the EcoRI site of pCRH (Invitrogen) and sequencing both strands using both T7, SP6, and custom primers. Some sequencing was performed at the University of Michigan DNA sequencing core facility. DNA and protein sequence analysis was performed using BLAST and BESTFIT (Genetics Computer Group, Madison, WI).
GST-PTG Fusion Protein Production. In order to facilitate rapid purification of PTG protein for use in biochemical studies, a glutathione-
S-transferase (GST) -PTG fusion was constructed. A 1.0 Kb EcoRI fragment from clone Bl-1, encoding residues 8-293, was subcloned into the EcoRI site of pGΕX-5X-3 expression vector (Pharmacia). Fusion protein was produced by IPTG induction of IL cultures of E. coli BL21(DE3)LysS and purified by affinity chromatography on glutathione-agarose beads.
Sub-Cellular Fractionation of PPIC Activity: Following a 3-hour serum deprivation in KRBH/0.5% BSA/2.5 mM glucose, Ll adipocyte cells were washed three times with ice cold PBS. Cells were scraped in homogenization buffer (50 mM HEPES, pH 7.2/2 mM EDTA 2 mg/mL glycogen/0.2% 2-ME/+ protease inhibitors). Samples were sonicated and centrifuged at 2500 x g to remove nuclei and unlysed cells. The PNS was removed, and glycogen bound PTG was added. Samples were incubated at 4°C for 60 minutes with gentle mixing. Lysates were subjected to centrifugation for 15 minutes at 10,000 x g and 1 hour at 100,000 x g to pellet plasma membranes and glycogen pellets, respectively. The final supernatant was called cytosol. The glycogen pellets were resuspended in homogenization buffer by 10 passes through a 23 gauge needle. Protein concentrations and PPl activity were measured in the PNS, plasma membrane, glycogen pellet, and cytosolic fractions, as described previously (Lazar D.F., supra., 1995). Fractionation of pFPTG transfected CHO-IR cells into cytosol and glycogen pellet was performed similarly.
Transient Transfection Studies. To facilitate in vivo studies of PTG action, an expression vector containing an epitope tagged version of PTG was constructed. The FLAG-epitope (N-DYKDDDDK-C) (IBI) was introduced into pCI-neo (Promega) by ligating complementary ohgonucleotides into Nhel/EcoRl digested vector. A 1.0 EcoRI fragment from clone Bl-1 was cloned in-frame at the
EcoRI site of the resulting plasmid, producing plasmid pFPTG. The FLAG-PTG fiision is expressed from the strong CMV enhancer/promoter. CHO cells expressing insulin receptor (CHO-IR) were transfected with Lipofectamine (Gibco-BRL) according to manufacturer recommendations. Typically, 1 μg of pFPTG/6 μL Lipofectamine was used per 60 mm dish to achieve 20% to 30% transfection efficiency, as determined by a CMV-lacZ reporter vector transfected in parallel.
Immunoprecipitation and Immunoblotting. CHO-IR cells transfected with pFPTG were sonicated in homogenization buffer and subjected to 14000 x g centrifugation for 10 minutes at 4°C to remove nuclei and cell debris. FLAG-PTG was immunoprecipitated from the supernatant by incubation with 10 μg of αFLAG antibody (IBI) for 1 hour at 4°C. Immune complexes were precipitated by incubation with Protein A G-agarose for 1 hour at 4°C and washed four times with homogenization buffer prior to the addition of SDS -sample buffer. Immunoprecipitates and subcellular fractions were separated on SDS- polyacrylamide gels and transferred to nitrocellulose. Immunoblots were performed with either FLAG monoclonal antibody or with PPIC polyclonal antibody (a generous gift from Dr. J. Lawrence). The primary monoclonal and polyclonal antibodies were detected with horseradish peroxidase-conjugated anti- mouse or anti-chicken IgG, respectively, and visualized by the enhanced chemiluminescence detection system (Amersham).
Northern Blot Analysis: Total RNA was isolated from 3T3-L1 fibroblast cells and fully differentiated 3T3-L1 adipocyte cells by the acid guanidinium thiocyanate-phenol-chloroform extraction method (Rnasol; Biotex Laboratories). RNA samples (15 μg) were electrophoresed in 1.2% agarose/2.2 M formaldehyde/ 1 x MOPS and transferred to nylon membrane (Hybond;
Amersham) by capillary diffusion. The transfer membrane was pre-hybridized for
1 hour in FBY hybridization buffer (10% PEG/1.5 x SSPE/7% SDS) and hybridized overnight at 65°C with the 1.0 kb EcoRI fragment of clone Bl-1,
32 which was gel purified and labeled with [α- PjdCTP by random priming (sp act.
>1 x 10 cpm/μg DNA). Following hybridization, the blot was washed at 65°C in 2 x SSC/0.1% SDS for 15 minutes, then washed twice in 0.1 x SSC/0.1% SDS at 65°C for 15 minutes each time. Equal loading of RNA was determined by ethidium bromide staining of rRNA and by probing for β-actin, as described above. Glycogen Synthase and Glycogen Synthesis Assays. Glycogen synthase activity associated with immobilized GST-PTG was determined as described previously (Lazar D.F., supra., 1995). Briefly, 100 μL of GST-PTG bound to glutathione-agarose beads was resuspended in 725 μL of glycogen synthase buffer (50 mM HEPES, pH 7.8/100 M NaF/10 mM EDTA) plus 25 μL (0.1 U) purified glycogen synthase (Sigma), followed by incubation at 4°C for 1 hour with gentle mixing. The agarose beads were washed four times with glycogen synthase buffer, brought to a final volume of 300 μL and 50 μL assayed for glycogen synthase activity by measuring the incorporation of UDP-[^C]glucose into glycogen, both in the presence and absence of 10 mM glucose-6-phosphate (Sigma). The accumulation of glycogen in intact pFPTG transfected CHO-IR cells was determined by an adaptation of the method of Lawrence J.C., et al., J. Biol. Chem., 1977;252:444 as described previously (Lazar D.F., supra., 1995).
In Vitro Binding Assays. Phosphorylase kinase: 50 μL of GST-PTG fusion protein beads was added to 750 μL homogenization buffer containing 0.15 M NaCl, 0.1 % BSA, and 25 μg of [32P]-labeled phosphorylase a. The tubes were incubated at 37°C for 20 minutes, washed four times with homogenization buffer, and proteins separated by SDS-PAGE, followed by autoradiography (Lawrence J.C., supra., 1977). One hundred microliters of GST-PTG bound to glutathione-agarose beads was resuspended in 725 μL of glycogen synthase buffer (50 mM HEPES, pH 7.8/100 mM NaF/10 mM EDTA) plus 25 μg (0.1 U) purified glycogen synthase (Sigma), followed by incubation at 4°C for 1 hour with gentle mixing. The agarose beads were washed four times with glycogen synthase buffer, brought to a final volume of 300 μL and 50 μL assayed for glycogen synthase activity (Lazar D.F., supra., 1995) by measuring the incorporation of UDP- [^Cjglucose into glycogen, both in the presence and absence of 10 mM glucose- 6-phosphate (Sigma).
Phosphorylase Kinase: Fifty microliters of fusion protein beads were incubated with 10 μg purified phosphorylase kinase (Gibco) in homogenization buffer plus 0.15 M NaCl and 0.1 % BS A, or with 3T3-L 1 adipocyte cell lysate, incubated 30 minutes at 4°C and washed four times with the same buffer. Ten microliter beads were assayed (Lazar D.F., supra., 1995) in 50 mM HEPES, pH 7.4, 10 mM MgCl, 1 μM okadaic acid, in the absence (1 mM EGTA) or presence
(0.5 mM) of Ca++. Two micrograms phosphorylase b, 20 μM cold ATP, and 2 μCi [32p]-g-ATP per tube was added and allowed to incubate at 37°C for
5 minutes. At the end of the incubation period, SDS-sample buffer was added and the proteins separated by SDS-PAGE on a 10 % gel.
Isolation of Human and Mouse Genomic PTG Sequence. Murine and human genomic PTG sequences were obtained by screening the respective genomic Bacterial Artificial Chromosome (BAC) (Shizuya H, et al., Proc. Natl.
Acad. Sci., USA, 1992;89:8794; Kim U-J, et al, Genomics, 1996;34:213) library by hybridization to a [32p]-labeled 1.0 kb cDNA fragment from clone B2-2. Hybridization of probe DNA to filters spotted with BAC DNA library was performed by Research Genetics, Huntsville, AL. The source of the mouse genomic DNA is the cell line, CJ7 (Swiatek P. J. and Gridley T., Gene and Dev. ,
1993;7:2071), derived from mouse strain 129SV. The sources of human DNA are the cell line 978SK and human sperm. Screening of the human BAC library resulted in the identification of three hybridization positive BAC clones (103 D21, 117C9, 255E4) and four positive clones from the mouse BAC library (201D24, 211P10, 219K2, 427F20). Human genomic BAC clone 255E4 and mouse genomic
BAC clone 201D24 were chosen for further characterization following southern analysis with the labeled 1.0 kb cDNA fragment from clone B2-2 to confirm the presence of hybridizing DNA sequences.
Characterization of Human Genomic PTG DNA Sequences. To identify DNA fragments containing the PTG coding region, human BAC clone
255E4 (5 μg) was digested with EcoRI (1 unit) at 37°C for 1 hour prior to separation of the resulting DNA fragments by electrophoresis through a 0.6% agarose gel. DNA fragments were transferred to nylon membrane (Hybond, Amersham) by capillary diffusion and probed with cDNA fragment encompassing the PTG coding sequence from clone B2-2. The transfer membrane was pre- hybridized for 1 hour in FBY hybridization buffer ( 10% PEG/1.5 x SSPE/7%
SDS) and hybridized overnight at 65°C with the 1.0 kb EcoRI fragment of clone Bl-1, which was gel purified and labeled with [α-32p]dCTP by random priming (sp act. >1 x 10^ cpm/mg DNA). Following hybridization, the blot was washed at 65°C in 2 x SSC/0.1% SDS for 15 minutes, then washed twice in 0.1 x SSC/0.1% SDS at 65°C for 15 minutes each time. A 5.0 kb EcoRI fragment was found to hybridize to the Bl-1 PTG probe and was subsequently cloned into the EcoRI site of vector pBluescript II SK" (Stratogene, La Jolla, CA), creating the plasmid pJPD23. Preliminary sequence analysis of subcloned 5.0 kb fragment was performed by using T3 and T7 primers complementary to vector sequences flanking the inserted fragment. Complete sequence information was obtained by synthesizing oligonucleotide primers complementary to both positive and negative strands of the inserted human genomic DNA. Sequencing was performed at The University of Michigan DNA sequencing core facility with an Appligen fluorescent dye terminator kit (Perkin-Εlmer) and an ABI 8700 automated sequencer. Assignment of the human PTG open reading frame continued within the 5.0 kb genomic sequence is based upon sequence comparison with the mouse PTG cDNA from the 2-hybrid clone B2-2 and upon favorable translational initiation sequences surrounding the putative initiating methionine (Kozak M, J. Cell Biol, 1989; 108:229). Characterization of Mouse Genomic PTG DNA Sequences. The mouse
BAC clone 201D24 was subjected to southern analysis, as described for the human genomic BAC clone, except BamΑλ (1 unit, 37°C, 2 hours) was used to digest 5 μg of DNA. A 7.0 kb hybridizing fragment was identified and subcloned into the BamHl site of pBluescript II Sk", creating pJPD27. An overlapping 5.0 kb EcoRI fragment 3' to the PTG open reading frame was identified by restriction digest of BAC clone 201D24 (5 μg) with EcoRI (1 unit, 37°C, 2 hours), followed by southern analysis using a 0.8 kb S.stl-Ba Vll fragment from the extreme 3' of genomic DNNA of pJPD27. This fragment was isolated from the agarose gel and ligated into the EcoRI site of pBluescript II Sk", creating plasmid p201-3'. Sequence information of the additional 2.0 kb genomic DNA 3' to the BamHl site of pJPD27 was obtained as described above for the human genomic PTG sequence.
Construction of PTG Knockout Vector. To further characterize the physiological role of PTG in overall glycogen metabolism in vivo, a targeted replacement vector was constructed to delete the PTG coding sequences from a mouse genome. pKO Scrambler V901 vector (Lexicon Genetics, Ine, The Woodlands, TX) forms the backbone of the targeting vector, as this vector has scrambled polylinkers, for insertion of 5' and 3' homologous genomic DNA, flanking a unique restriction site for insertion of a positive selectable marker (neor for selection of transfected ΕS cells on the antibiotic G418). pKO Scrambler V901 also contains a unique restriction site for the insertion of a negative selection element (Thymidine Kinase) for positive-negative selection strategies, which has been reported to increase targeting efficiency to the desired locus 2- to 20-fold (Hasty P. and Bradley A. in Gene Targeting: A Practical Approach, 1993, A.L. Joyner, Εd., IRL Press, Oxford). A positive selection cassette containing the
Neomycin Phosphotransferase gene under the control of the PGK promoter was excised from plasmid pKO selectNΕO V800 (Lexicon Genetics, Ine, The Woodlands, TX) by digestion with the restriction enzyme Ascll (New England Biolabs, Beverly, MA) and subcloned into the unique Ascll site of pKO Scrambler V901 , creating plasmid pKO-neo. A negative selection cassette containing the thymidine kinase gene under the control of the MCI promoter was subcloned into the unique Rsrϊl site of pKO-neo by digestion of plasmid pKO SelectTK V800 (Lexicon Genetics, Ine, The Woodlands, TX) withforll (New England Biolabs, Beverly, MA) followed by separation and isolation of the appropriate restriction fragment (2.0 kb) by electrophoresis through a 1.0% agarose gel, creating plasmid pKO-TK/neo. A 2.0 kb region of DNA 5' to the PTG coding region was amplified by Polymerase Chain Reaction (PCR) using primers 5'-CGAGGATCCTTGTCTTCTCTGCAGATG-3' (SEQ ID NO.: 7) and 5'-GCTGGTACCTGAATGAGCCAAGCAAATCCTC-3' (SEQ ID NO.: 8), which contain BamHl and Kpnl sites, respectively. The amplified DNA product was then cloned into the Bglϊl-Kpnl of plasmid pKO-TK/neo, creating the plasmid pKO-TK/neo-5'. The 3.5 kb 3' homology region of genomic PTG DNA was cloned into the EcoRI-Sα/I sites of pKO-TK/neo-5' by first digesting pJPD27 (1 μg) with Smal (1 unit, 22°C, 2 hours), and inserting a Sail oligonucleotide linker (5'-CCGG CGACCGG-3') (SΕQ ID NO.: 9), creating plasmid pJPD27ΔSma. The 3.5 kb EcoRI-Sαfl fragment from pJPD27ΔSma was then ligated into the EcoRI -Sa l sites of pKO-TK/Neo-5' to create the targeting vector pKO-PTG.
A 0.5 kb 5' DNA probe was generated by PCR amplification from plasmid pJPD27 with the T3 specific primer, complementary to DNA sequences contained within the vector pBluescript II Sk" and a primer specific to the extreme 5' region of mouse genomic DNA sequence (5'-GCAGAGAAGACAAAACCAC-3') (SΕQ ID NO.: 10). The 3' DNA probe was generated by digestion of plasmid p201-3' with Bam l and isolation of the resulting 0.8 kb fragment following electrophoretic separation on a 1.5% agarose gel.
DETAILED DESCRIPTION OF THE FIGURES
Figure 1. Sequence comparison of PTG with glycogen localizing subunits of PPIC. BESTFIT sequence comparison program was used to align and compare the primary amino acid sequences of PTG, GL, RGl and Gael. The boxed regions represent areas of similarity and the sites of conservation are indicated by shading.
Figure 2. PPIC binding and glycogen localizing activity of PTG protein.
(A) PPIC binds PTG in vivo. pCI-neo expressing FLAG-PTG from the CMV promoter was transiently transfected into CHO-IR cells and immunoprecipitated from cell lysates with antibodies directed against the FLAG epitope. Precipitates were analyzed by SDS-PAGE on a 4% to 20% gel, transferred to nitrocellulose and blotted with αPPIC polyclonal antibodies. Immunoreactive proteins were visualized by
Enhanced Chemiluminescence (ECL).
(B) PTG partitions to the glycogen pellet. Lysates from pFLAG-PTG transfected or untransfected CHO-IR cells were fractionated by centrifugation into 14 K x g supernatant (14,000 x g, 15 min, supernatant), cytosol (100,000 x g, 1 hr, supernatant) or glycogen pellet (100,000 x g, 1 hr, pellet), and proteins were subjected to SDS-PAGE and immunoblotted with αFLAG antibodies.
(C) PTG overexpression causes translocation of PPIC to the glycogen pellet. Lysates from pFLAG-PTG transfected or untransfected CHO-IR cells were fractionated by centrifugation and immunoblotted as in (A) to determine relative levels of PPIC contained in the various fractions. All fractions were normalized for protein concentration prior to loading.
(D) PTG targets PPIC to glycogen in vitro. PTG dependent localization of PPl C to the glycogen pellet was determined by incubated 3T3-L1 adipocyte cell lysates with bacterially expressed GST-PTP1B or GST-PTG prior to subcellular fractionation as above. PPIC activity in the glycogen pellet was measured as described previously (Lazar D.F., supra., 1995).
Figure 3. Tissue distribution of PTG expression.
(A) PTG is expressed in insulin responsive tissues. A multi-tissue northern blot (Clonetech) was hybridized overnight at 65°C with a 1.0 kb EcoRI fragment of clone Bl-1, which was labeled with [α-32p]dCTP by random priming, and exposed to film for 24 hours. (B) PTG expression is induced by adipocyte differentiation. 3T3-L1 fibroblast and fully differentiated 3T3-L1 adipocyte total RNA was isolated and electrophoresed (15 μg) in 1.2% agarose/2.2 M formaldehyde/ 1 x MOPS, followed by transfer to nylon membrane by capillary diffusion. The transfer membrane was hybridized and probed as in (A). Equal loading of RNA was determined by ethidium bromide staining of rRNA and by probing for β-actin transcript. Molecular size markers (kb) are indicated on the left.
Figure 4. Binding of phosphorylase a, glycogen synthase, and phosphorylase kinase to PTG.
(A) PTG binds to phosphorylase a. [32p]-labeled phosphorylase a (25 μg) was incubated with the indicated fusion protein immobilized to glutathione-agarose beads for 1 hour at 4°C in homogenization buffer (50 mM HEPES, pH 7.2/ 2 mM EDTA/2 mg/mL glycogen/0.2% 2-ME/0.1 mM PMSF/1 mM benzamidine/10 μg/mL aprotinin), followed by washing four times with homogenization buffer prior to the addition of SDS sample buffer. Proteins were separated by SDS-PAGE and the gel exposed to film.
(B) PTG binds glycogen synthase. One hundred microliters of GST-PTG bound to glutathione-agarose beads was resuspended in
725 μL of glycogen synthase buffer (50 mM HEPES, pH 7.8/100 mM NaF/10 mM EDTA) plus 25 μL purified glycogen synthase (Sigma), followed by incubation at 4°C for 1 hour with gentle mixing. The agarose beads were washed four times with glycogen synthase buffer and assayed for glycogen synthase activity.
(C, D) Phosphorylase kinase binds to PTG. Fifty microliters of bacterially expressed GST-PTG bound to glutathione-agarose beads was incubated with of 3T3-L1 adipocyte cell lysate (C) or 10 μg purified phosphorylase kinase (Gibco) (D) in homogenization buffer. Samples were incubated 30 minutes at 4°C and washed four times with the same buffer. Ten microliter beads were assayed for phosphorylase kinase activity using 2 μg phosphorylase b per sample in the absence (1 mM EGTA) or presence (0.5 mM) of Ca+÷. Complexed proteins were separated on a 10% SDS-polyacrylamide gel and radiolabeled phosphorylase a was visualized by autoradiography.
Figure 5. Glycogen synthesis in CHO-IR cells overexpressing PTG. CHO-IR cells were grown to 40% to 50% confluency in 6-well dishes and transiently transfected with pFLAG-PTG. Forty-eight hours after transfection, cells were serum deprived for 3 hours and glycogen accumulation in intact pFLAG-PTG or lacZ transfected cells, in the presence or absence of 100 nM insulin, was determined. Results are expressed as means of triplicate determinations, of SD, and were repeated in two separate experiments.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Brady, Matthew J Printen, John A Saltiel, Alan R Warner-Lambert Company, (Outside USA)
(ii) TITLE OF INVENTION: Protein Targeting to Glycogen
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Warner-Lambert Company
(B) STREET: 201 Tabor Road
(C) CITY: Morris Plains
(D) STATE: NJ
(E) COUNTRY: US
(F) ZIP: 07950
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/025,107
(B) FILING DATE: 30-AUG-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Ashbrook, Charles W
(C) REFERENCE/DOCKET NUMBER: 5485-01-CA
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 313 996-5215
(B) TELEFAX: 313 996-1553
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3461 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2577..3461
(XT ) SEQUENCE DESCRIPTION: SEQ ID NO:l: GGATCCCGCT GGCCCAGGGC TCAGGGACTC TACGGCCGCC TTCCAGCACG CTCTGGTCAC 60
ATCCAGCCCC GCGGTGATCA CGTTCCAGGG GCAGGGGCTC GTGCCCCAGC TGCACAGTGG 120
TTGGTCAGGG CGCACGGCCT TTGATTGGTC GGAGGGACCG GTTCACGTGA TCTGGCTTTG 180
ATAAGCTGCC TCCCGGTTGC CTGCGGTCAG TCGGCCGGCT GGACCGCGGC GCTGCATCCT 240
CTTAAGTACT GAGTGCGAAG TTGCTCAGGG TGCGGTTGTC GCGGTCGCCG CTGCCTCTCG 300
GTCCAATGAA CTGCACCAGG TAAGTTCAGT GCAACTCTGC GCCCGAATGG AGGGGAGCCT 360
GCAGCTGCGG GGACTTGGGG GCTTTTGTGG TTTTGTCTTC TCTGCAGATG GGAGACTCCC 420
AGGTTGATGG CTGAGTTGAA TCGTCCCGGA GGCTTGGAAG GGATGGAATT TGAGTTGAAT 480
TTCTTAGGCG CCTGTTTTGG GGAATCTAAC ATCTCAACAG TAGAAACTGC CTTAGTGAGT 540
CAGTTTTAGC TGGGATTCCT TTGCTGAAGT GACTCTGTTG CCTTTGAGCT CAAAGACCCA 600
AAAAATTGGG TTGCAGCTTA GTTTTATTGA TGAGGTAGTT TAGCCTGTGG CAAAAGGTAG 660
AGGATTTTGC ATTTCTTCTT TCCCCACTTC CCCTCTCCTG GTTTTGTCAT AAAAAAATGC 720
AGTCTTAGAG TTTGAGCAAG CTATTATTTT TAACTCCTGG CCAGGACTTA GGAATTCCAG 780
GGAAATAGCC CACTACTAAA ACTTTCTATA AATGCAGGAG TAAAAAAGGA AAAAAACCAA 840
AAAAACTAAA ACAAAAAGAA AAACAAGAAC AGCTCAACCA ATAAAAAACG AACAACAGCT 900
CCATCAACCA GGCAACCAAG ACCATCTCAC GAAATGTTGC ATCTCCAGTT GTAACTTAGT 960
CCAAGAATTC TCCCTCCCCA AATCCTGCCT CTTAATGGTT GTGAGACAAA TAGACTTTGA 1020
GGTTTTGTTG CTGGAAGTTT GTAATCCTCT CAGAAAAACC CTAGAGCTAT TAAAGTGAGT 1080
TCAGTATACA TTCTTTACAG GCCACTTTGT GATGATGACC ATATACAGGG TGCTGTGCTT 1140
AGCACTGGTA TGAAGAGAAT TCTTCAAGCC AGACTATATT TATGATCAAA ATATGTTATA 1200
TACATATATA AACTGTTAAA GAATAAATAA TTAATAAAGC CCCAGACTCC AAAATGGCAC 1260
CTACTG ATT TCATGTTTCT TTGATTTATA AGGCTGACAT TGGAGAGGAA AAACCTAATT 1320
CTGTAAGTGT GGAATTATAG TGTGGTTCAA GGGAAAGAAA GGCCAGAAAA GTCTTGATGG 1380
ATGGTTCTTT GTTTGATGCA AACTATTGTT ATTGCCTTCC TACTTGTAAA TAGGACCTGC 1440
TCAACAACAG GAAACATCAT AGAAGCCCAA ACCAGTAGAT GCTATAATCC ATCAGTCAAA 1500
GATAATGAAC ATCTTAGTGT TTCCTTTGTA GTTTCAGTAT TTATAAATCA TATATCTTCA 1560
GTGTATTTTA AAACAGCTCA TTTCTGGTTA TCAGTTTTTA AAACTACTTT ATGTTGTGTA 1620
TATATAATGT ACACTGCAGG CTACAAGACA GAGCTATAGT AAAGTGGTTA TTACTGGTCA 1680
GAATGAACAA TCTGT ATCC CTGCAGGTTA GCTATTCCCT TTATAGTTGG ACCTGTCATG 1740
GGCATCTTTC CTATATGAAC TGTCAGATTG TTTAAAGTTT TTCCTTTAGT CTGTGAGATG 1800
TTGGCTGGTG TTGCAGTTGG TCATTTGTGA AAAAATGAGG ATGACTGTGA TAAAATGAAA 1860
AAGTCATTCT TTCTTTTAAC AAGCGTCACC TACTGTCACT CTAAGGACAG CATGACATTT 1920 TAAGAATTGC TTCATTTΛTT GTTTCCCAAG TGGATTACTT CTCCTGAGAA GTAAAACCGG 1980
TTCGAGAGCC AAAATAGGAA ACAGCAGCCA GAGGGAGCGA GAGGCTGGGA CTGTGATAAT 2040
GGAAGAAGCT GTCTGGCCAA TGGACTCTTT TGGGGGAAGC TTTAAGAACA TATTTACCTT 2100
TCTGGCTCCA TGCCATGAAG CTCTACTGTA GTGGTTTTAA GTCCCCGGAA TCTGAATTTT 2160
TTTTTTCTAA AGGAAAGAAA CTTCTCAGGT CTTGTTGATC TGACAGGTTT AAGAACCACT 2220
GGCCCAGAAC AGAGTACATA ATTCCAAGAG CTGTGTCAGA CTTGTTCAGA TAGAGCCCTC 2280
TTGTTTCTCA GATGGAGAAA CTGAATCCTC TCTGAGTGTT TCAGGCAGTT TACACATGGG 2340
CCCAGCAGCC TGCCAAGCAC AGAGCTAGAC TGTAGATCTC ATCACCCCAG TGCTCTCCTT 2400
TTCTCCACGT GATAGCACCT CTCTGCACTG GAGTACTAGT GTGTGTGCAT TTGGGACCAG 2460
GGGAAGACGA CTCCAGACCT CGGTGATTAC CACTGTTTTT ττττττττττ TCTCATTCCA 2520
GAATGATCCA TGTGCTAGAT CCACGTCCTT TGACAAGTTC CGTCATGCCC GTGGAC 2576
ATG GCC ATG AGG ATT TGC TTG GCT CAT TCA CCA CCT CTG AAG AGT TTC 2624 Met Ala Met Arg He Cys Leu Ala His Ser Pro Pro Leu Lys Ser Phe 1 5 10 15
CTG GGT CCT TAC AAT GGT TTT CAA CGA AGA AAT TTT GTG AAT AAA TTG 2672 Leu Gly Pro Tyr Asn Gly Phe Gin Arg Arg Asn Phe Val Asn Lys Leu 20 25 30
AAA CCT TTG AAA CCA TGT CTC AGT GTC AAG CAG GAA GCC AAA TCG CAG 2720 Lys Pro Leu Lys Pro Cys Leu Ser Val Lys Gin Glu Ala Lys Ser Gin 35 40 45
AGT GAG TGG AAG AGC CCA CAC AAC CAA GCC AAG AAG CGG GTC GTG TTT 2768 Ser Glu Trp Lys Ser Pro His Asn Gin Ala Lys Lys Arg Val Val Phe 50 55 60
GCG GAC TCC AAG GGG CTG TCA CTC ACT GCT ATC CAT GTC TTC TCC GAC 2816 Ala Asp Ser Lys Gly Leu Ser Leu Thr Ala He His Val Phe Ser Asp 65 70 75 80
CTT CCA GAA GAA CCA GCG TGG GAC CTG CAG TTT GAT CTC TTG GAC CTT 2864 Leu Pro Glu Glu Pro Ala Trp Asp Leu Gin Phe Asp Leu Leu Asp Leu 85 90 95
AAC GAT ATC TCC TCC AGC TTA AAA CTT CAC GAG GAG AAA AAT TTG GTT 2912 Asn Asp He Ser Ser Ser Leu Lys Leu His Glu Glu Lys Asn Leu Val 100 105 110
TTT GAT TTT CCC CAG CCC TCA ACC GAC TAC TTA AGT TTC CGG GAC CGC 2960 Phe Asp Phe Pro Gin Pro Ser Thr Asp Tyr Leu Ser Phe Arg Asp Arg 115 120 125
TTT CAG AAG AAC TTT GTC TGC CTC GAG AAC TGC TCT TTG GAA GAT CGG 3008 Phe Gin Lys Asn Phe Val Cys Leu Glu Asn Cys Ser Leu Glu Asp Arg 130 135 140
ACG GTG ACC GGG ACA GTG AAA GTG AAG AAT GTG AGC TTT GAG AAG AAG 3056 Thr Val Thr Gly Thr Val Lys Val Lys Asn Val Ser Phe Glu Lys Lys 145 150 155 160
GTT CAG GTC CGG ATC ACC TTT GAC ACC TGG AAA ACC TAC ACA GAT GTG 3104 Val Gin Val Arg He Thr Phe Asp Thr Trp Lys Thr Tyr Thr Asp Val 165 170 175
GAC TGT GTC TAC ATG AAG AAT GTT TAC AGC AGC TCA GAC AGC GAC ACC 3152 Asp Cys Val Tyr Met Lys Asn Val Tyr Ser Ser Ser Asp Ser Asp Thr 180 185 190
TTC TCC TTT GCA ATC GAC TTG CCC CGT GTC ATT CCA ACT GAG GAG AAA 3200 Phe Ser Phe Ala He Asp Leu Pro Arg Val He Pro Thr Glu Glu Lys 195 200 205
ATT GAG TTC TGC ATT TCT TAT CAC GCT AAT GGG AGG ATC TTC TGG GAC 3248 He Glu Phe Cys He Ser Tyr His Ala Asn Gly Arg He Phe Trp Asp 210 215 220
AAC AAT GAG GGT CAG AAT TAC AGA ATT GTC CAT GTG CAA TGG AAA CCT 3296 Asn Asn Glu Gly Gin Asn Tyr Arg He Val His Val Gin Trp Lys Pro 225 230 235 240
GAC GGA GTG CAG ACT CAG GTG GCA CCC AAA GAC TGT GCA TTC CAA CAG 3344 Asp Gly Val Gin Thr Gin Val Ala Pro Lys Asp Cys Ala Phe Gin Gin 245 250 255
GGG CCC CCT AAG ACT GAG ATA GAG CCC ACA GTC TTT GGC AGT CCA AGG 3392 Gly Pro Pro Lys Thr Glu He Glu Pro Thr Val Phe Gly Ser Pro Arg 260 265 270
CTT GCT AGC GGC CTC TTC CCA GAG TGG CAG AGC TGG GGG AGA GTG GAG 3440 Leu Ala Ser Gly Leu Phe Pro Glu Trp Gin Ser Trp Gly Arg Val Glu 275 280 285
AAC TTG ACC TCC TAT CGA TGA 3461
Asn Leu Thr Ser Tyr Arg * 290 295
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 295 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Ala Met Arg He Cys Leu Ala His Ser Pro Pro Leu Lys Ser Phe 1 5 10 15
Leu Gly Pro Tyr Asn Gly Phe Gin Arg Arg Asn Phe Val Asn Lys Leu 20 25 30
Lys Pro Leu Lys Pro Cys Leu Ser Val Lys Gin Glu Ala Lys Ser Gin 35 40 45
Ser Glu Trp Lys Ser Pro His Asn Gin Ala Lys Lys Arg Val Val Phe 50 55 60
Ala Asp Ser Lys Gly Leu Ser Leu Thr Ala He His Val Phe Ser Asp 65 70 75 80
Leu Pro Glu Glu Pro Ala Trp Asp Leu Gin Phe Asp Leu Leu Asp Leu 85 90 95 Asn Asp He Ser Ser Ser Leu Lys Leu His Glu Glu Lys Asn Leu Val 100 105 110
Phe Asp Phe Pro Gin Pro Ser Thr Asp Tyr Leu Ser Phe Arg Asp Arg 115 120 125
Phe Gin Lys Asn Phe Val Cys Leu Glu Asn Cys Ser Leu Glu Asp Arg 130 135 140
Thr Val Thr Gly Thr Val Lys Val Lys Asn Val Ser Phe Glu Lys Lys 145 150 155 160
Val Gin Val Arg He Thr Phe Asp Thr Trp Lys Thr Tyr Thr Asp Val 165 170 175
Asp Cys Val Tyr Met Lys Asn Val Tyr Ser Ser Ser Asp Ser Asp Thr 180 185 190
Phe Ser Phe Ala He Asp Leu Pro Arg Val He Pro Thr Glu Glu Lys 195 200 205
He Glu Phe Cys He Ser Tyr His Ala Asn Gly Arg He Phe Trp Asp 210 215 220
Asn Asn Glu Gly Gin Asn Tyr Arg He Val His Val Gin Trp Lys Pro 225 230 235 240
Asp Gly Val Gin Thr Gin Val Ala Pro Lys Asp Cys Ala Phe Gin Gin 245 250 255
Gly Pro Pro Lys Thr Glu He Glu Pro Thr Val Phe Gly Ser Pro Arg 260 265 270
Leu Ala Ser Gly Leu Phe Pro Glu Trp Gin Ser Trp Gly Arg Val Glu 275 280 285
Asn Leu Thr Ser Tyr Arg * 290 295
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5789 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 4238..5176
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CTTGACCTGT CTAAGCTTTC AGTTCCTCAT CTGTGAAATA AAGAGTTTGA TGCCTATCAC 60
CTCCTACCTC CATAATTCTA ACCATTGATG GGTCATTAAA ATAAGACAAT ATGGTGCAGC 120
GGTTATTGCT CTGGTATCAG CCAGCCTCTA ATCCCTGCTC TACCTGTGAG AΛCCTGGGCA 180 GGTTTTTTTT TTGTTTTTTG TTTTCGAGAT AGAGTCTCGC TCTGTTGCCC AGGCTGGAGT 240
GCAGTGGTGC AATCTCAGCT CACTGCAACC TCCGCCTCCC GGGTTCAAGC GATTCTCCTG 300
CCTCAGCCTC CAGAGTAGCT GAGAGTACAG GTGTGCACCA CCATGCCCGG CTAATTTTTG 360
TATTTTTAGT AGAGATAGGG TTTCACCATG TTGGCCAGGC TGGTCTTGAA CTCCTGGCCT 420
CAAGTGATCC ACTGGGCAGA TTTCCTGACC ATTCAGTGTC TCCGTTTTCT TTTCTCTAAA 480
ATGGGATTAA TAACTGGACA TATCACATAG GGTTGTTGTG AGGATTGAAT TGATAGCACA 540
TAGTGTTTGG CACAGAGTAA AGGCTCAACA AGCAGCAGCT ATTCTCAATA TTTTAGCTCA 600
GGCACCAGGC GCCTTGAGGT GATAGAGTAA AAACTCTAGC TGAGAGATCA AGTAGAAACT 660
TGGGAACTAG CCCGGGTGGA ACACAGGCAC TGGGCATCGT GCTGAGTCTG TTCATTGGCA 720
CCATCTTACT TCATCTTCAG AACGTTACTA TCTCTGTTTT ACACATGAGG AAACTGAGGT 780
TAGAACTTGC CTAGTTCGGT AGCTAGTAAG TGTCAATCCA AAGACCTTCC AGCTAGTTTT 840
GGTTGAGCTA AAGGGGCTAG AAGACCTGCC ATTAGTTAGA TATTTCATTT CAAAAATAAA 900
ACCCAGGCAT GAAGTCCCTT TCCCAGTGAT ATTCAGTGTG ATTTTTTTCT TCACTCTAAT 960
AATTTTAACA ATTCCACTGT TTGACAGTTG TTTAAAAGAC ATAGGAATTT TTGTATATTT 1020
TAATTGACTA ATGGATAGCT CAATTAGGGG AGCAAAACTA GGATGTGGGT TTTATAAAAA 1080
TAATTTAGAC TTGACTTAGA CATTTAATTT TACAGTTGTA AATGATGGTC TAAAAATTCT 1140
TCAAACTAAT CAAAATAATG AAACTTCAGC GAAAGTGAGT GGCTCAGAAG GCCCATGAAA 1200
CATACGGCGT GATTTTTTAA ATTTTATTTT AACATTTTGA TTTCCACACC ACTGCCAAAG 1260
GACGTCAGAA TTGAGTAAGG GGTTTGGGTT GACTGCTGCC TCTTGACCGG CTGTATGTGT 1320
GAAAAGGGTC ATTTCACTTC CGGCTTTAGT GTTCCCCGCA GGGGAGAAAA TTGAAGAATA 1380
GACAGAAATA CGAAGTGTCT TTTAATTAAA TGCCACCTTG GTGTTTTATG GGGCTCGTAT 1440
GCTTTCCTAA CAACATTTGT TAGATAAGTT GGTAATTCCC GGCAGCTGTC TACTGTGTGG 1500
TGCATCTGTG AACTCATACT AATCGAAAAG CATGCAGCCA GTTTGGGATC GCGCAGGCTA 1560
AGGTGAGGGA GAAATGCGGA TACACCGGGT AATGAACGAT ATAAACATTT CAAATGCGAT 1620
ACACATTCGG TTTGAGCCAC ATCTTCTGTG TGCAGATTCA CCCGCAGTGA CCCACAAAGC 1680
TATTCCCAAG TAACAGCCGC CCCAAGCCTG AGGCACTGGC GCCCCGCCTG GGCGAGGCTG 1740
GCTGCGCTCT CTCTTGGCCG GCGCCCGCTG CATGCGG AC GTGCCTGCCC GGCCCCTAGC 1800
CCAGGGTTCC CGTTACGCGG CTGGTTCCAG CTGGCCGCGG AGTCCCAGAA CCTCCCCGGG 1860
ATGCCCAGAT AGCTCTCTGC ACGTCTGGCC CCGGGGCGAT CACGTTGCCG GGGCGAGGGC 1920
TGGCGCCCCA GCTGGGCGCT GGTTGGTCGC GCCCTGGGGC TCGAGGCCCG GCGATTGGTC 1980
CCAGGGATCG GGTCACGTGC TTGGGAGCAG ATAAGCGGCC TCTAGGCGCC GGGCCCTCAG 2040
TCTCTCCCAG CGACCGCCGC GGGGGCAAGG CCTGGAGCTG TGGTTCGAAT TTGTGCAGGC 2100 AGCGGGTGCT GGCTTTTAGG GTCCGCCGCC TCTCTGCCTA ATGAGCTGCA CCAGGTAGGT 2160
TCGCTGCAAC TCTGCGCGCT AGGAACACAG GGGAACGCGC AGCTGTGGGG AAGTTGGGGG 2220
GCGTTTCAGT TCTATCATCT CTGGAAATGG ACACCCCAGG GGGAGGACAA GTGGACTGAC 2280
TGCGTAGTTG AATCTGGCAA CCGAGAGGCC TTGGAGGTGT AGAAATTTGG CTCTATTTCT 2340
TAAGCAGAGC CTATTTTAGT AATCAGCATC TTAAAGCAGA AATTATCTTA ACGTGAATCA 2400
GCTTGAGTTA GGATTTTCTC ATGGATGCGG CTGTTCTTTT GGTCCTGCAC AAATGTCCCA 2460
AAGACTCGGG CAGCTGAAGT GGTGAGAACA GCACTCTGAC ATTGCTGGTT AGGTGGTTTA 2520
GCTTGGAGGA AAAAAATTAC AGGACGACGT TTGCATTCAT TCGTCCTTCT TATCACAGTT 2580
TGCCATAGCA AAATCTCAAG AGTTTGAGCA AACGATTACT TTTAACTCTT GTCCAGGACT 2640
TAAAGTTCCA AGGAAATCAC CCAAACTAAA ACTGTCTTTC TATAAATGCA AAAAGTAAAA 2700
AAAAAAAAAC AAAAAAACCA AAAAAAACCT CCCATAAAAC TACTTTAAAT AGCTTCTCCA 2760
GACATAGCTT AGCAGAAGAT TCTCTAAAAA TCCTGCCTAT TAACTATTAT TAGACCCACA 2820
AATATAGCTT TAGCTTTCAT TTGTTTGTTT AAGTTTGCAG ATCTCCCAGA AAAACCCCAG 2880
AGCTAACACA GTAAATTCTG CGAGTGTTAT TACACACTTT TGTGATAATG ACCACTTGCA 2940
TACATGTTTA GAGCTGCTGT GAGGAGAGTT ACTAAAGCCA GACTGAGAAA TGTCGTGTAC 3000
AGTATACACA CACCTCTTAC TTGTAAGGCT AAGATAGGGA AAAAAATCTT AATACCATAA 3060
GCTTGGAAAT ATATGATGAG GGCTAAAGGT CAGAGAAAAG TCTTCTTTAT AGATGCTTCT 3120
TGGTTTAATA TTGCTGAGCA TAGTCATGTT TAAAACTTTA AATGGTTTTA TTGTCTTTCT 3180
ACTTATAAAT GTCTATTAGA AAATGCCAAA AAAGAACAAA AACGAAAATA GATAATCTAT 3240
AATCCTATCA CCCAGAAATA ATAATTATTA AATTATTAGG AAAGGTGTAT TTCCTATAGA 3300
GTTTTTCAAT ATTTATAAGT TTGTATATAT AAAATGTATA TTTTAAAACA CTCCAACTTT 3360
CAGGTAATCA GTTTTTCCAC TTAAATGTGG ACTTGTCATG GGCATCTCTT TAGGTGAATT 3420
ATCAATTATA TAGTTTTTAA GTGCATATGA ATTGTTGGCT TGTATTTCAG TGGTTATTTG 3480
TGAAAAATAA GAGCATGATA ATCAAAGTGC AAAGATGATT CTTTGACTTC TTCTCTAGCC 3540
TTCTCACTTT CAAAACTGCA TGTTATTTTT TTTTTTCAAG TGAATTACCT TACCAGAGAA 3600
GTGTCAATCA ATTTAGCAGC AAAATAAGCC AACGTAGCCA GAGGGAGCAG AGGGTCTGGA 3660
ACTGTGGCTC CTGAACCTGT CTGGTCATTA GAATCACCTG GGAAGCTTTA AGAACATACC 3720
CATCCCTTGG CCCTAGCCCC AGAAGTTCTG CCTCAGTAGT TCTGAGTCCC AGGAATTGGA 3780
AAGAAAGAAG AAAGAGAAAG AGAGAGAGAG AGGAAGAAAG GAAGGAAGGA GGGAAGGAGG 3840
AAAGGAGGAA AGACAAGAAA GAAAGAAAAT GAATTCCCTA GACATAGTGA CCAGACAGGT 3900
TTGAGGACCA CTGGTCCAGA ACAGAGCACA CAGTTCTCAA GGCTGCCTTG GAGATAATCA 3960
AATCGAACCC TTTTATTTCT CAGATGGGGA AACTGAGACC CCCATCACCC TCTΛAGTGTT 4020 TTAAGCAATT AATAGCCTTT ACCGGCCAAG GGTAGAGGTA GACATAGAAG ATCTGATCAC 4080
TTAATACTGT TCTCTTTTAC TACATATGAT AGCACCTGCC TGATATCTAG TGCACTGGCT 140
ATAATTCAGT CAGCACAAAA ATAGTACATA TGTATTTGGC ACTGGGGAAG AGCATTTCCG 4200
ATCCAGGTGA TAATCCCTCT TCTTTTTGCA TTCCAGA ATG ATC CAG GTT TTA GAT 4255
Met He Gin Val Leu Asp 300
CCA CGT CCT TTG ACA AGT TCG GTC ATG CCC GTG GAT GTG GCC ATG AGG 4303 Pro Arg Pro Leu Thr Ser Ser Val Met Pro Val Asp Val Ala Met Arg 305 310 315
CTT TGC TTG GCA CAT TCA CCA CCT GTG AAG AGT TTC CTG GGC CCG TAC 4351 Leu Cys Leu Ala His Ser Pro Pro Val Lys Ser Phe Leu Gly Pro Tyr 320 325 330
GAT GAA TTT CAA CGA CGA CAT TTT GTG AAT AAA TTA AAG CCC CTG AAA 4399 Asp Glu Phe Gin Arg Arg His Phe Val Asn Lys Leu Lys Pro Leu Lys 335 340 345
TCA TGT CTC AAT ATA AAA CAC AAA GCC AAA TCA CAG AAT GAC TGG AAG 4447 Ser Cys Leu Asn He Lys His Lys Ala Lys Ser Gin Asn Asp Trp Lys 350 355 360 365
TGC TCA CAC AAC CAA GCC AAG AAG CGC GTT GTG TTT GCT GAC TCC AAG 4495 Cys Ser His Asn Gin Ala Lys Lys Arg Val Val Phe Ala Asp Ser Lys 370 375 380
GGC CTC TCT CTC ACT GCG ATC CAT GTC TTC TCC GAC CTC CCA GAA GAA 4543 Gly Leu Ser Leu Thr Ala He His Val Phe Ser Asp Leu Pro Glu Glu 385 390 395
CCA GCG TGG GAT CTG CAG TTT GAT CTC TTG GAC CTT AAT GAT ATC TCC 4591 Pro Ala Trp Asp Leu Gin Phe Asp Leu Leu Asp Leu Asn Asp He Ser 400 405 410
TCT GCC TTA AAA CAC CAC GAG GAG AAA AAC TTG ATT TTA GAT TTC CCT 4639 Ser Ala Leu Lys His His Glu Glu Lys Asn Leu He Leu Asp Phe Pro 415 420 425
CAG CCT TCA ACC GAT TAC TTA AGT TTC CGG AGC CAC TTT CAG AAG AAC 4687 Gin Pro Ser Thr Asp Tyr Leu Ser Phe Arg Ser His Phe Gin Lys Asn 430 435 440 445
TTT GTC TGT CTG GAG AAC TGC TCG TTG CAA GAG CGA ACA GTG ACA GGG 4735 Phe Val Cys Leu Glu Asn Cys Ser Leu Gin Glu Arg Thr Val Thr Gly 450 455 460
ACT GTT AAA GTC AAA AAT GTG AGT TTT GAG AAG AAA GTT CAG ATC CGT 4783 Thr Val Lys Val Lys Asn Val Ser Phe Glu Lys Lys Val Gin He Arg 465 470 475
ATC ACT TTC GAT TCT TGG AAA AAC TAC ACT GAC GTA GAC TGT GTC TAT 4831 He Thr Phe Asp Ser Trp Lys Asn Tyr Thr Asp Val Asp Cys Val Tyr 480 485 490
ATG AAA AAT GTG TAT GGT GGC ACA GAT AGT GAT ACC TTC TCA TTT GCC 4879 Met Lys Asn Val Tyr Gly Gly Thr Asp Ser Asp Thr Phe Ser Phe Ala 495 500 505
ATT GAC TTA CCC CCT GTC ATT CCA ACT GAG CAG AAA ATT GAG TTC TGC 4927 He Asp Leu Pro Pro Val He Pro Thr Glu Gin Lys He Glu Phe Cys 510 515 520 525
ATT TCT TAC CAT GCT AAT GGG CAA GTC TTT TGG GAC AAC AAT GAT GGT 4975 He Ser Tyr His Ala Asn Gly Gin Val Phe Trp Asp Asn Asn Asp Gly 530 535 540
CAG AAT TAT AGA ATT GTT CAT GTT CAA TGG AAG CCT GAT GGG GTG CAG 5023 Gin Asn Tyr Arg He Val His Val Gin Trp Lys Pro Asp Gly Val Gin 545 550 555
ACA CAG ATG GCA CCC CAG GAC TGT GCA TTC CAC CAG ACG TCT CCT AAG 5071 Thr Gin Met Ala Pro Gin Asp Cys Ala Phe His Gin Thr Ser Pro Lys 560 565 570
ACA GAG TTA GAG TCA ACA ATC TTT GGC AGT CCG AGG CTG GCT AGT GGG 5119 Thr Glu Leu Glu Ser Thr He Phe Gly Ser Pro Arg Leu Ala Ser Gly 575 580 585
CTC TTC CCA GAG TGG CAG AGC TGG GGG AGA ATG GAG AAC TTG GCC TCT 5167 Leu Phe Pro Glu Trp Gin Ser Trp Gly Arg Met Glu Asn Leu Ala Ser 590 595 600 605
TAT CGA TGA ATTAAGCAAC AATGTAACTG GTCTTGACTT GTCATATTCC 5216
Tyr Arg *
CCCATGCAAT CCTAGGTCTG TATTGCTCAA TTTTAGGAAG CCTTTGCTAC TCCATCAGTA 5276
GGTTTAGATT TGAGCTTTTG AAACCTGGCT ATGGAAAAGA AAGACACTTG AGAATTTATG 5336
TTGGGGTCTG TACAGATAAA TGCTAACCCA ATTTGGCTTT GAAGGATCAA GTAACAGGTT 5396
GAAAACTATT TTTATAAAGG TAATACTTTT TCAGTTCCCT TCTTCCTTCC CTCTCAATCC 5456
ACTAGCTTTC ATGTTGGGCA AGGAAAAGTT GAGGAAGGAT GGCTGATGGT GATGGAAAGC 5516
TATGTTAATG GTATGAGGAA TGTGTGAAAA GTATACACAA AGGGCTCTGA AGCTCAAGTC 5576
AGAGGAGTGG GAGGTCTGAT CATTGTTGGT GGAAAAACGT AAGGTTATTT TGTGTTTTTA 5636
AGTTGGTTTT ACAATTCTTT CCTGGGGAAA TTATTTCTGG AGGGGAAAAA GATCCATTCT 5696
ACGTATCCTT GTGGAGAAAA GCTAAATAAC CTTTAAGAAT GTGGGTGGTA TTGGAGAAAG 5756
AAGATGAATT ATAGCTCCGG AGAATCAAGA TCT 5789
(2) INFORMATICM FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 313 amino acids (3) TYPE: amino acid ,'D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :
Met He Gin Val Leu Asp Pro Arg Pro Leu Thr Ser Ser Val Met Pro 1 5 10 15
Val Asp Val Ala Met Arg Leu Cys Leu Ala His Ser Pro Pro Val Lys 20 25 30 Ser Phe Leu Gly Pro Tyr Asp Glu Phe Gin Arg Arg His Phe Val Asn 35 40 45
Lys Leu Lys Pro Leu Lys Ser Cys Leu Asn He Lys His Lys Ala Lys 50 55 60
Ser Gin Asn Asp Trp Lys Cys Ser His Asn Gin Ala Lys Lys Arg Val 65 70 75 80
Val Phe Ala Asp Ser Lys Gly Leu Ser Leu Thr Ala He His Val Phe 85 90 95
Ser Asp Leu Pro Glu Glu Pro Ala Trp Asp Leu Gin Phe Asp Leu Leu 100 105 110
Asp Leu Asn Asp He Ser Ser Ala Leu Lys His His Glu Glu Lys Asn 115 120 125
Leu He Leu Asp Phe Pro Gin Pro Ser Thr Asp Tyr Leu Ser Phe Arg 130 135 140
Ser His Phe Gin Lys Asn Phe Val Cys Leu Glu Asn Cys Ser Leu Gin 145 150 155 160
Glu Arg Thr Val Thr Gly Thr Val Lys Val Lys Asn Val Ser Phe Glu 165 170 175
Lys Lys Val Gin He Arg He Thr Phe Asp Ser Trp Lys Asn Tyr Thr 180 185 190
Asp Val Asp Cys Val Tyr Met Lys Asn Val Tyr Gly Gly Thr Asp Ser 195 200 205
Asp Thr Phe Ser Phe Ala He Asp Leu Pro Pro Val He Pro Thr Glu 210 215 220
Gin Lys He Glu Phe Cys He Ser Tyr His Ala Asn Gly Gin Val Phe 225 230 235 240
Trp Asp Asn Asn Asp Gly Gin Asn Tyr Arg He Val His Val Gin Trp 245 250 255
Lys Pro Asp Gly Val Gin Thr Gin Met Ala Pro Gin Asp Cys Ala Phe 260 265 270
His Gin Thr Ser Pro Lys Thr Glu Leu Glu Ser Thr He Phe Gly Ser 275 280 285
Pro Arg Leu Ala Ser Gly Leu Phe Pro Glu Trp Gin Ser Trp Gly Arg 290 295 300
Met Glu Asn Leu Ala Ser Tyr Arg * 305 310
(2) INFORMATION FOR SEQ ID NO: 5:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 885 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
ATGGCCATGA GGATTTGCTT GGCTCATTCA CCACCTCTGA AGAGTTTCCT GGGTCCTTAC 60
AATGGTTTTC AACGAAGAAA TTTTGTGAAT AAATTGAAAC CTTTGAAACC ATGTCTCAGT 120
GTCAAGCAGG AAGCCAAATC GCAGAGTGAG TGGAAGAGCC CACACAACCA AGCCAAGAAG 180
CGGGTCGTGT TTGCGGACTC CAAGGGGCTG TCACTCACTG CTATCCATGT CTTCTCCGAC 240
CTTCCAGAAG AACCAGCGTG GGACCTGCAG TTTGATCTCT TGGACCTTAA CGATATCTCC 300
TCCAGCTTAA AACTTCACGA GGAGAAAAAT TTGGTTTTTG ATTTTCCCCA GCCCTCAACC 360
GACTΛCTTAA GTTTCCGGGA CCGCTTTCAG AAGAACTTTG TCTGCCTCGA GAACTGCTCT 420
TTGGAAGATC GGACGGTGAC CGGGACAGTG AAAGTGAAGA ATGTGAGCTT TGAGAAGAAG 480
GTTCAGGTCC GGATCACCTT TGACACCTGG AAAACCTACA CAGATGTGGA CTGTGTCTAC 540
ATGAAGAATG TTTACAGCAG CTCAGACAGC GACACCTTCT CCTTTGCAAT CGACTTGCCC 600
CGTGTCATTC CAACTGAGGA GAAAATTGAG TTCTGCATTT CTTATCACGC TAATGGGAGG 660
ATCTTCTGGG ACAACAATGA GGGTCAGAAT TACAGAATTG TCCATGTGCA ATGGAAACCT 720
GACGGAGTGC AGACTCAGGT GGCACCCAAA GACTGTGCAT TCCAACAGGG GCCCCCTAAG 780
ACTGAGATAG AGCCCACAGT CTTTGGCAGT CCAAGGCTTG CTAGCGGCCT CTTCCCAGAG 840
TGGCAGAGCT GGGGGAGAGT GGAGAACTTG ACCTCCTATC GATGA 885 (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 939 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ATGATCCAGG TTTTAGATCC ACGTCCTTTG ACAAGTTCGG TCATGCCCGT GGATGTGGCC 60
ATGAGGCTTT GCTTGGCACA TTCACCACCT GTGAAGAGTT TCCTGGGCCC GTACGATGAA 120
TTTCAACGAC GACATTTTGT GAATAAATTA AAGCCCCTGA AATCATGTCT CAATATAAAA 180
CACAAAGCCA AATCACAGAA TGACTGGAAG TGCTCACACA ACCAAGCCAA GAAGCGCGTT 240
GTGTTTGCTG ACTCCAAGGG CCTCTCTCTC ACTGCGATCC ATGTCTTCTC CGACCTCCCA 300
GAAGAACCAG CGTGGGATCT GCAGTTTGAT CTCTTGGACC TTAATGATAT CTCCTCTGCC 360
TTAAAACACC ACGAGGAGAA AAACTTGATT TTAGATTTCC CTCAGCCTTC AACCGATTAC 420 TTAAGTTTCC GGAGCCACTT TCAGAAGAAC TTTGTCTGTC TGGAGAACTG CTCGTTGCAA 480
GAGCGAACAG TGACAGGGAC TGTTAAAGTC AAAAATGTGA GTTTTGAGAA GAAAGTTCAG 540
ATCCGTATCA CTTTCGATTC TTGGAAAAAC TACACTGACG TAGACTGTGT CTATATGAAA 600
AATGTGTATG GTGGCACAGA TAGTGATACC TTCTCATTTG CCATTGACTT ACCCCCTGTC 660
ATTCCAACTG AGCAGAAAAT TGAGTTCTGC ATTTCTTACC ATGCTAATGG GCAAGTCTTT 720
TGGGACAACA ATGATGGTCA GAATTATAGA ATTGTTCATG TTCAATGGAA GCCTGATGGG 780
GTGCAGACAC AGATGGCACC CCAGGACTGT GCATTCCACC AGACGTCTCC TAAGACAGAG 840
TTAGAGTCAA CAATCTTTGG CAGTCCGAGG CTGGCTAGTG GGCTCTTCCC AGAGTGGCAG 900
AGCTGGGGGA GAATGGAGAA CTTGGCCTCT TATCGATGA 939 (2) INFORMATION FOR SEQ ID NO: 7:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CGAGGATCCT TGTCTTCTCT GCAGATG 27
(2) INFORMATION FOR SEQ ID NO: 8:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GCTGGTACCT GAATGAGCCA AGCAAATCCT C 31
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i i ) MOLECULE TYPE : DNA ( genomic )

Claims

-45/ 1 - (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : CCGGTCGACC GG 12 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GCAGAGAAGA CAAAACCAC 19 -46- CLAIMS What is claimed is:
1. An isolated murine protein comprising the amino acid sequence:
Met Ala Met Arg He Cys Leu Ala His Ser Pro Pro Leu Lys Ser Phe Leu Gly Pro Tyr Asn Gly Phe Gin Arg Arg Asn Phe Val Asn Lys Leu
Lys Pro Leu Lys Pro Cys Leu Ser Val Lys Gin Glu Ala Lys Ser Gin Ser Glu Trp Lys Ser Pro His Asn Gin Ala Lys Lys Arg Val Val Phe Ala Asp Ser Lys Gly Leu Ser Leu Thr Ala lie His Val Phe Ser Asp Leu Pro Glu Glu Pro Ala Trp Asp Leu Gin Phe Asp Leu Leu Asp Leu Asn Asp He Ser Ser Ser Leu Lys Leu His Glu Glu Lys Asn Leu Val
Phe Asp Phe Pro Gin Pro Ser Thr Asp Tyr Leu Ser Phe Arg Asp Arg Phe Gin Lys Asn Phe Val Cys Leu Glu Asn Cys Ser Leu Glu Asp Arg Thr Val Thr Gly Thr Val Lys Val Lys Asn Val Ser Phe Glu Lys Lys Val Gin Val Arg He Thr Phe Asp Thr Trp Lys Thr Tyr Thr Asp Val Asp Cys Val Tyr Met Lys Asn Val Tyr Ser Ser Ser Asp Ser Asp Thr
Phe Ser Phe Ala He Asp Leu Pro Arg Val He Pro Thr Glu Glu Lys He Glu Phe Cys He Ser Tyr His Ala Asn Gly Arg He Phe Trp Asp Asn Asn Glu Gly Gin Asn Tyr Arg He Val His Val Gin Trp Lys Pro Asp Gly Val Gin Thr Gin Val Ala Pro Lys Asp Cys Ala Phe Gin Gin Gly Pro Pro Lys Thr Glu He Glu Pro Thr Val Phe Gly Ser Pro Arg
Leu Ala Ser Gly Leu Phe Pro Glu Trp Gin Ser Trp Gly Arg Val Glu Asn Leu Thr Ser Tyr Arg. (SEQ ID NO.: 2)
2. An isolated human protein protein comprising the amino acid sequence: Met He Gin Val Leu Asp Pro Arg Pro Leu Thr Ser Ser Val Met Pro Val Asp Val Ala Met Arg Leu Cys Leu Ala His Ser Pro Pro Val Lys
Ser Phe Leu Gly Pro Tyr Asp Glu Phe Gin Arg Arg His Phe Val Asn Lys Leu Lys Pro Leu Lys Ser Cys Leu Asn He Lys His Lys Ala Lys Ser Gin Asn Asp Trp Lys Cys Ser His Asn Gin Ala Lys Lys Arg Val Val Phe Ala Asp Ser Lys Gly Leu Ser Leu Thr Ala He His Val Phe -47- Ser Asp Leu Pro Glu Glu Pro Ala Trp Asp Leu Gin Phe Asp Leu Leu Asp Leu Asn Asp He Ser Ser Ala Leu Lys His His Glu Glu Lys Asn Leu He Leu Asp Phe Pro Gin Pro Ser Thr Asp Tyr Leu Ser Phe Arg Ser His Phe Gin Lys Asn Phe Val Cys Leu Glu Asn Cys Ser Leu Gin Glu Arg Thr Val Thr Gly Thr Val Lys Val Lys Asn Val Ser Phe Glu
Lys Lys Val Gin He Arg He Thr Phe Asp Ser Trp Lys Asn Tyr Thr Asp Val Asp Cys Val Tyr Met Lys Asn Val Tyr Gly Gly Thr Asp Ser Asp Thr Phe Ser Phe Ala He Asp Leu Pro Pro Val He Pro Thr Glu Gin Lys He Glu Phe Cys He Ser Tyr His Ala Asn Gly Gin Val Phe Trp Asp Asn Asn Asp Gly Gin Asn Tyr Arg He Val His Val Gin Trp
Lys Pro Asp Gly Val Gin Thr Gin Met Ala Pro Gin Asp Cys Ala Phe His Gin Thr Ser Pro Lys Thr Glu Leu Glu Ser Thr He Phe Gly Ser Pro Arg Leu Ala Ser Gly Leu Phe Pro Glu Trp Gin Ser Trp Gly Arg Met Glu Asn Leu Ala Ser Tyr Arg.
(SEQ ID NO.: 3)
An isolated murine genomic DNA molecule comprising the sequence:
GGATCCCGCTGGCCCAGGGCTCAGGGACTCTACGGCCGCCTTCC AGCACGCTCTGGTCACATCCAGCCCCGCGGTGATCACGTTCCAG GGGCAGGGGCTCGTGCCCCAGCTGCACAGTGGTTGGTCAGGGC GCACGGCCTTTGATTGGTCGGAGGGACCGGTTCACGTGATCTGG CTTTGATAAGCTGCCTCCCGGTTGCCTGCGGTCAGTCGGCCGGC
TGGACCGCGGCGCTGCATCCTCTTAAGTACTGAGTGCGAAGTTG CTCAGGGTGCGGTTGTCGCGGTCGCCGCTGCCTCTCGGTCCAAT GAACTGCACCAGGTAAGTTCAGTGCAACTCTGCGCCCGAATGG AGGGGAGCCTGCAGCTGCGGGGACTTGGGGGCTTTTGTGGTTTT GTCTTCTCTGCAGATGGGAGACTCCCAGGTTGATGGCTGAGTTG
AATCGTCCCGGAGGCTTGGAAGGGATGGAATTTGAGTTGAATTT CTTAGGCGCCTGTTTTGGGGAATCTAACATCTCAACAGTAGAAA CTGCCTTAGTGAGTCAGTTTTAGCTGGGATTCCTTTGCTGAAGT GACTCTGTTGCCTTTGAGCTCAAAGACCCAAAAAATTGGGTTGC AGCTTAGTTTTATTGATGAGGTAGTTTAGCCTGTGGCAAAAGGT
AGAGGATTTTGCATTTCTTCTTTCCCCACTTCCCCTCTCCTGGTT -48- TTGTCATAAAAAAATGCAGTCTTAGAGTTTGAGCAAGCTATTAT TTTTAACTCCTGGCCAGGACTTAGGAATTCCAGGGAAATAGCCC ACTACTAAAACTTTCTATAAATGCAGGAGTAAAAAAGGAAAAA AACCAAAAAAACTAAAACAAAAAGAAAAACAAGAACAGCTCA ACCAATAAAAAACGAACAACAGCTCCATCAACCAGGCAACCAA
GACCATCTCACGAAATGTTGCATCTCCAGTTGTAACTTAGTCCA AGAATTCTCCCTCCCCAAATCCTGCCTCTTAATGGTTGTGAGAC AAATAGACTTTGAGGTTTTGTTGCTGGAAGTTTGTAATCCTCTCA GAAAAACCCTAGAGCTATTAAAGTGAGTTCAGTATACATTCTTT AC AGGCCACTTTGTGATGATGACC ATATACAGGGTGCTGTGCTT
AGCACTGGTATGAAGAGAATTCTTCAAGCCAGACTATATTTATG ATCAAAATATGTTATATACATATATAAACTGTTAAAGAATAAAT AATTAATAAAGCCCCAGACTCCAAAATGGCACCTACTGTATTTC ATGTTTCTTTGATTTATAAGGCTGACATTGGAGAGGAAAAACCT AATTCTGTAAGTGTGG AATTATAGTGTGGTTC AAGGGAAAGAA
AGGCCAGAAAAGTCTTGATGGATGGTTCTTTGTTTGATGCAAAC TATTGTTATTGCCTTCCTACTTGTAAATAGGACCTGCTCAACAAC AGGAAACATCATAGAAGCCCAAACCAGTAGATGCTATAATNCC ATCAGTCAAAGATAATGAACATCTTAGTGTTTNCCTTTGTAGTTT NCAGTATTTATAAATNCATATATCTTCAGTGTATTTTAAAACAG
CTCATTTNCTGGTTATCAGTTTTTAAAACTACTTTATGTTGTGTA TATATAATGTACACTGCAGGCTACAAGACAGAGCTATAGTAAA GTGGTTATTACTGGTCAGAATGAACAANTCTGTTATCCCTGCAG GTTAGCTATTCCCTTTATAGTTGGACCTGTCATGGGCATCTTTCC TATATGAACTGTCAGATTGTTTAAAGTTTTTCCTTTAGTCTGTGA
GATGTTGGCTGGTGTTGCAGTTGGTCATTTGTGAAAAAATGAGG ATGACTGTGATAAAATGAAAAAGTCATTCTTTCTTTTAACAAGC GTCACCTACTGTCACTCTAAGGACAGCATGACATTTTAAGAATT GCTTCATTTATTGTTTCCCAAGTGGATTACTTCTCCTGAGAAGTA AAACCGGTTCGAGAGCCAAAATAGGAAACAGCAGCCAGAGGG
AGCGAGAGGCTGGGACTGTGATAATGGAAGAAGCTGTCTGGCC AATGGACTCTTTTGGGGGAAGCTTTAAGAACATATTTACCTTTC -49- TGGCTCCATGCCATGAAGCTCTACTGTAGTGGTTTTAAGTCCCC GGAATCTGAATTTTTTTTTTCTAAAGGAAAGAAACTTCTCAGGT CTTGTTGATCTGACAGGTTTAAGAACCACTGGCCCAGAACAGAG TACATAATTCCAAGAGCTGTGTCAGACTTGTTCAGATAGAGCCC TCTTGTTTCTCAGATGGAGAAACTGAATCCTCTCTGAGTGTTTCA
GGCAGTTTACACATGGGCCCAGCAGCCTGCCAAGCACAGAGCT AGACTGTAGATCTCATCACCCCAGTGCTCTCCTTTTCTCCACGTG ATAGCACCTCTCTGCACTGGAGTACTAGTGTGTGTGCATTTGGG ACCAGGGGAAGACGACTCCAGACCTCGGTGATTACCACTGTTTT TTTTTTTTTT TTCTC ATTCCAGAATGATCCATGTGCTAGATCC AC
GTCCTTTGACAAGTTCCGTCATGCCCGTGGAC ATGGCCATGAGGATTTGCTTGGCTCATTCACCACCTCTGAAGAG TTTCCTGGGTCCTTACAATGGTTTTCAACGAAGAAATTTTGTGA ATAAATTGAAACCTTTGAAACCATGTCTCAGTGTCAAGCAGGAA GCCAAATCGCAGAGTGAGTGGAAGAGCCCACACAACCAAGCCA
AGAAGCGGGTCGTGTTTGCGGACTCCAAGGGGCTGTCACTCACT GCTATCCATGTCTTCTCCGACCTTCCAGAAGAACCAGCGTGGGA CCTGCAGTTTGATCTCTTGGACCTTAACGATATCTCCTCCAGCTT AAAACTTCACGAGGAGAAAAATTTGGTTTTTGATTTTCCCCAGC CCTCAACCGACTACTTAAGTTTCCGGGACCGCTTTCAGAAGAAC
TTTGTCTGCCTCGAGAACTGCTCTTTGGAAGATCGGACGGTGAC CGGGACAGTGAAAGTGAAGAATGTGAGCTTTGAGAAGAAGGTT CAGGTCCGGATCACCTTTGACACCTGGAAAACCTACACAGATGT GGACTGTGTCTACATGAAGAATGTTTACAGCAGCTCAGACAGC GACACCTTCTCCTTTGCAATCGACTTGCCCCGTGTCATTCCAACT
GAGGAGAAAATTGAGTTCTGCATTTCTTATCACGCTAATGGGAG GATCTTCTGGGACAACAATGAGGGTCAGAATTACAGAATTGTCC ATGTGCAATGGAAACCTGACGGAGTGCAGACTCAGGTGGCACC CAAAGACTGTGCATTCCAACAGGGGCCCCCTAAGACTGAGATA GAGCCCACAGTCTTTGGCAGTCCAAGGCTTGCTAGCGGCCTCTT
CCCAGAGTGGCAGAGCTGGGGGAGAGTGGAGAACTTGACCTCC TATCGATGA. (SEQ ID NO.: 1) -50-
4. An isolated human genomic DNA molecule comprising the sequence:
CTTGACCTGTCTAAGCTTTCAGTTCCTCATCTGTGAAATAAAGA GTTTGATGCCTATCACCTCCTACCTCCATAATTCTAACCATTGAT GGGTCATTAAAATAAGACAATATGGTGCAGCGGTTATTGCTCTG GTATCAGCCAGGCTCTAATCCCTGCTCTACCTGTGAGAACCTGG
GCAGGTTTTTTTTTTGTTTTTTGTTTTCGAGATAGAGTCTCGCTCT GTTGCCCAGGCTGGAGTGCAGTGGTGCAATCTCAGCTCACTGCA ACCTCCGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCC AGAGTAGCTGAGAGTACAGGTGTGCACCACCATGCCCGGCTAA TTTTTGTATTTTTAGTAGAGATAGGGTTTC ACC ATGTTGGCCAGG
CTGGTCTTGAACTCCTGGCCTCAAGTGATCCACTGGGCAGATTT CCTGACCATTCAGTGTCTCCGTTTTCTTTTCTCTAAAATGGGATT AATAACTGGACATATCACATAGGGTTGTTGTGAGGATTGAATTG ATAGCACATAGTGTTTGGCACAGAGTAAAGGCTCAACAAGCAG CAGCTATTCTCAATAT TTTAGCTCAGGCACCAGGCGCCTTGAGG
TGATAGAGTAAAAACTCTAGCTGAGAGATCAAGTAGAAACTTG GGAACTAGCCCGGGTGGAACACAGGCACTGGGCATCGTGCTGA GTCTGTTCATTGGCACCATCTTACTTCATCTTCAGAACGTTACTA TCTCTGTTTTACACATGAGGAAACTGAGGTTAGAACTTGCCTAG TTCGGTAGCTAGTAAGTGTCAATCCAAAGACCTTCCAGCTAGTT
TTGGTTGAGCTAAAGGGGCTAGAAGACCTGCCATTAGTTAGATA TTTCATTTCAAAAATAAAACCCAGGCATGAAGTCCCTTTCCCAG TGATATTCAGTGTGATTTTTTTCTTCACTCTAATAATTTTAACAA TTCCACTGTTTGACAGTTGTTTAAAAGACATAGGAATTTTTGTAT ATTTTAATTGACTAATGGATAGCTCAATTAGGGGAGCAAAACTA
GGATGTGGGTTTTATAAAAATAATTTAGACTTGACTTAGACATT TAATTTTACAGTTGTAAATGATGGTCTAAAAATTCTTCAAACTA ATCAAAATAATGAAACTTCAGCGAAAGTGAGTGGCTCAGAAGG CCCATGAAACATACGGCGTGATTTTTTAAATTTTATTTTAACATT TTGATTTCCACACCACTGCCAAAGGACGTCAGAATTGAGTAAGG
GGTTTGGGTTGACTGCTGCCTCTTGACCGGCTGTATGTGTGAAA -51- AGGGTCATTTCACTTCCGGCTTTAGTGTTCCCCGCAGGGGAGAA AATTGAAGAATAGACAGAAATACGAAGTGTCTTTTAATTAAATG CCACCTTGGTGTTTTATGGGGCTCGTATGCTTTCCTAACAACATT TGTTAGATAAGTTGGTAATTCCCGGCAGCTGTCTACTGTGTGGT GCATCTGTGAACTCATACTAATCGAAAAGCATGCAGCCAGTTTG
GGATCGCGCAGGCTAAGGTGAGGGAGAAATGCGGATACACCGG GTAATGAACGATATAAACATTTCAAATGCGATACACATTCGGTT TGAGCCACATCTTCTGTGTGCAGATTCACCCGCAGTGACCCACA AAGCTATTCCCAAGTAACAGCCGCCCCAAGCCTGAGGCACTGG CGCCCCGCCTGGGCGAGGCTGGCTGCGCTCTCTCTTGGCCGGCG
CCCGCTGCATGCGGTACGTGCCTGCCCGGCCCCTAGCCCAGGGT TCCCGTTACGCGGCTGGTTCCAGCTGGCCGCGGAGTCCCAGAAC CTCCCCGGGATGCCCAGATAGCTCTCTGCACGTCTGGCCCCGGG GCGATCACGTTGCCGGGGCGAGGGCTGGCGCCCCAGCTGGGCG CTGGTTGGTCGCGCCCTGGGGCTCGAGGCCCGGCGATTGGTCCC
AGGGATCGGGTCACGTGCTTGGGAGCAGATAAGCGGCCTCTAG GCGCCGGGCCCTCAGTCTCTCCCAGCGACCGCCGCGGGGGCAA GGCCTGGAGCTGTGGTTCGAATTTGTGCAGGCAGCGGGTGCTGG CTTTTAGGGTCCGCCGCCTCTCTGCCTAATGAGCTGCACCAGGT AGGTTCGCTGCAACTCTGCGCGCTAGGAACACAGGGGAACGCG
CAGCTGTGGGGAAGTTGGGGGGCGTTTCAGTTCTATCATCTCTG GAAATGGACACCCCAGGGGGAGGACAAGTGGACTGACTGCGTA GTTGAATCTGGCAACCGAGAGGCCTTGGAGGTGTAGAAATTTG GCTCTATTTCTTAAGCAGAGCCTATTTTAGTAATCAGCATCTTAA AGCAGAAATTATCTTAACGTGAATCAGCTTGAGTTAGGATTTTC
TCATGGATGCGGCTGTTCTTTTGGTCCTGCACAAATGTCCCAAA GACTCGGGCAGCTGAAGTGGTGAGAACAGCACTCTGACATTGC TGGTTAGGTGGTTTAGCTTGGAGGAAAAAAATTACAGGACGAC GTTTGCATTCATTCGTCCTTCTTATCACAGTTTGCCATAGCAAAA TCTCAAGAGTTTGAGCAAACGATTACTTTTAACTCTTGTCCAGG
ACTTAAAGTTCCAAGGAAATCACCCAAACTAAAACTGTCTTTCT ATAAATGCAAAAAGTAAAAAAAAAAAAACAAAAAAACCAAAA -52- AAAACCTCCCATAAAACTACTTTAAATAGCTTCTCCAGACATAG CTTAGCAGAAGANTTCTCTAAAAATCCTGCCTATTAACTATTAT TAGACCCACAAATATAGCTTTAGCTTTCATTTGTTTGTTNTAAGT TTGCAGATCTCCCAGAAAAACCCCAGAGCTAACACAGTAAATT CTGCGAGTGTTATTACACACTTTTGTGATAATGACCACTTGCAT
ACATGTTTAGAGCTGCTGTGAGGAGAGTTACTAAAGCCAGACTG AGAAATGTCGTGTACAGTATACACACACCTCTTACTTGTAAGGC TAAGATAGGGAAAAAAATCTTAATACCATAAGCTTGGAAATAT ATGATGAGGGCTAAAGGTCAGAGAAAAGTCTTCTTTATAGATGC TTCTTGGTTTAATATTGCTGAGCATAGTCATGTTTAAAACTTTAA
ATGGTTTTATTGTCTTTCTACTTATAAATGTCTATTAGAAAATGC CAAAAAAGAACAAAAACGAAAATAGATAATCTATAATCCTATC ACCCAGAAATAATAATTATTAAATTATTAGGAAAGGTGTATTTC CTATAGAGTTTTTCAATATTTATAAGTTTGTATATATAAAATGTA TATTTTAAAAC ACTCC AACTTTCAGGTAATCAGTTTTTCCACTTA
AATGTGGACTTGTCATGGGCATCTCTTTAGGTGAATTATCAATT ATATAGTTTTTAAGTGCATATGAATTGTTGGCTTGTATTTCAGTG GTTATTTGTGAAAAATAAGAGCATGATAATCAAAGTGCAAAGA TGATTCTTTGACTTCTTCTCTAGCCTTCTCACTTTCAAAACTGCA TGTTATTTTTTTTTTTCAAGTGAATTACCTTACCAGAGAAGTGTC
AATCAATTTAGCAGCAAAATAAGCCAACGTAGCCAGAGGGAGC AGAGGGTCTGGAACTGTGGCTCCTGAACCTGTCTGGTCATTAGA ATCACCTGGGAAGCTTTAAGAACATACCCATCCCTTGGCCCTAG CCCCAGAAGTTCTGCCTCAGTAGTTCTGAGTCCCAGGAATTGGA AAGAAAGAAGAAAGAGAAAGAGAGAGAGAGAGGAAGAAAGG
AAGGAAGGAGGGAAGGAGGAAAGGAGGAAAGACAAGAAAGA AAGAAAATGAATTCCCTAGACATAGTGACCAGACAGGTTTGAG GACCACTGGTCCAGAACAGAGCACACAGTTCTCAAGGCTGCCTT GGAGATAATCAAATCGAACCCTTTTATTTCTCAGATGGGGAAAC TGAGACCCCCATCACCCTCTAAGTGTTTTAAGCAATTAATAGCC
TTTACCGGCCAAGGGTAGAGGTAGACATAGAAGATCTGATCAC TTAATACTGTTCTCTTTTACTACATATGATAGCACCTGCCTGATA -53- TCTAGTGCACTGGCTATAATTCAGTCAGCACAAAAATAGTACAT ATGTATTTGGCACTGGGGAAGAGCATTTCCGATCCAGGTGATAA TCCCTCTTCTTTTTGCATTCCAGAATGATCCAGGTTTTAGATCCA CGTCCTTTGACAAGTTCGGTCATGCCCGTGGATGTGGCCATGAG GCTTTGCTTGGCACATTCACCACCTGTGAAGAGTTTCCTGGGCC
CGTACGATGAATTTCAACGACGACATTTTGTGAATAAATTAAAG CCCCTGAAATCATGTCTCAATATAAAACACAAAGCCAAATCAC AGAATGACTGGAAGTGCTCACACAACCAAGCCAAGAAGCGCGT TGTGTTTGCTGACTCCAAGGGCCTCTCTCTCACTGCGATCCATGT CTTCTCCGACCTCCCAGAAGAACCAGCGTGGGATCTGCAGT TTG
ATCTCTTGGACCTTAATGATATCTCCTCTGCCTTAAAACACCAC GAGGAGAAAAACTTGATTTTAGATTTCCCTCAGCCTTCAACCGA TTACTTAAGTTTCCGGAGCCACTTTCAGAAGAACTTTGTCTGTCT GGAGAACTGCTCGTTGCAAGAGCGAACAGTGACAGGGACTGTT AAAGTCAAAAATGTGAGTTTTGAGAAGAAAGTTCAGATCCGTA
TCACTTTCGATTCTTGGAAAAACTACACTGACGTAGACTGTGTC TATATGAAAAATGTGTATGGTGGCACAGATAGTGATACCTTCTC ATTTGCCATTGACTTACCCCCTGTCATTCCAACTGAGCAGAAAA TTGAGTTCTGCATTTCTTACCATGCTAATGGGCAAGTCTTTTGGG ACAACAATGATGGTCAGAATTATAGAATTGTTCATGTTCAATGG
AAGCCTGATGGGGTGCAGACACAGATGGCACCCCAGGACTGTG CATTCCACCAGACGTCTCCTAAGACAGAGTTAGAGTCAACAATC TTTGGCAGTCCGAGGCTGGCTAGTGGGCTCTTCCCAGAGTGGCA GAGCTGGGGGAGAATGGAGAACTTGGCCTCTTATCGATGAATT AAGCAACAATGTAACTGGTCTTGACTTGTCATATTCCCCCATGC
AATCCTAGGTCTGTATTGCTCAATTTTAGGAAGCCTTTGCTACTC CATCAGTAGGTTTAGATTTGAGCTTTTGAAACCTGGCTATGGAA AAGAAAGACACTTGAGAATTTATGTTGGGGTCTGTACAGATAA ATGCTAACCCAATTTGGCTTTGAAGGATCAAGTAACAGGTTGAA AACTATTTTTATAAAGGTAATACTTTTTCAGTTCCCTTCTTCCTT
CCCTCTCAATCCACTAGCTTTCATGTTGGGCAAGGAAAAGTTGA GGAAGGATGGCTGATGGTGATGGAAAGCTATGTTAATGGTATG -54- AGGAATGTGTGAAAAGTATACACAAAGGGCTCTGAAGCTCAAG TCAGAGGAGTGGGAGGTCTGATCATTGTTGGTGGAAAAACGTA AGGTTATTTTGTGTTTTTAAGTTGGTTTTACAATTCTTTCCTGGG GAAATTATTTCTGGAGGGGAAAAAGATCCATTCTACGTATCCTT GTGGAGAAAAGCTAAATAACCTTTAAGAATGTGGGTGGTATTG
GAGAAAGAAGATGAATTATAGCTCCGGAGAATCAAGATCT. (SEQ ID NO.: 3)
5. An isolated murine cDNA molecule comprising the sequence:
ATGGCCATGAGGATTTGCTTGGCTCATTCACCACCTCTGAAGAG TTTCCTGGGTCCTTACAATGGTTTTCAACGAAGAAATTTTGTGA
ATAAATTGAAACCTTTGAAACCATGTCTCAGTGTCAAGCAGGAA GCCAAATCGCAGAGTGAGTGGAAGAGCCCACACAACCAAGCCA AGAAGCGGGTCGTGTTTGCGGACTCCAAGGGGCTGTCACTCACT GCTATCCATGTCTTCTCCGACCTTCCAGAAGAACCAGCGTGGGA CCTGCAGTTTGATCTCTTGGACCTTAACGATATCTCCTCCAGCTT
AAAACTTCACGAGGAGAAAAATTTGGTTTTTGATTTTCCCCAGC CCTCAACCGACTACTTAAGTTTCCGGGACCGCTTTCAGAAGAAC TTTGTCTGCCTCGAGAACTGCTCTTTGGAAGATCGGACGGTGAC CGGGACAGTGAAAGTGAAGAATGTGAGCTTTGAGAAGAAGGTT CAGGTCCGGATCACCTTTGACACCTGGAAAACCTACACAGATGT
GGACTGTGTCTACATGAAGAATGTTTACAGCAGCTCAGACAGC GACACCTTCTCCTTTGCAATCGACTTGCCCCGTGTCATTCCAACT GAGGAGAAAATTGAGTTCTGCATTTCTTATCACGCTAATGGGAG GATCTTCTGGGACAACAATGAGGGTCAGAATTACAGAATTGTCC ATGTGCAATGGAAACCTGACGGAGTGCAGACTCAGGTGGCACC
CAAAGACTGTGCATTCCAACAGGGGCCCCCTAAGACTGAGATA GAGCCCACAGTCTTTGGCAGTCCAAGGCTTGCTAGCGGCCTCTT CCCAGAGTGGCAGAGCTGGGGGAGAGTGGAGAACTTGACCTCC TATCGATGA. (SEQ ID NO.: 5) -55-
6. An isolated human cDNA molecule comprising the sequence: ATGATCCAGGTTTTAGATCCACGTCCTTTGACAAGTTCGGTCAT GCCCGTGGATGTGGCCATGAGGCTTTGCTTGGCACATTCACCAC CTGTGAAGAGTTTCCTGGGCCCGTACGATGAATTTCAACGACGA CATTTTGTGAATAAATTAAAGCCCCTGAAATCATGTCTCAATAT
AAAACACAAAGCCAAATCACAGAATGACTGGAAGTGCTCACAC AACCAAGCCAAGAAGCGCGTTGTGTTTGCTGACTCCAAGGGCCT CTCTCTCACTGCGATCCATGTCTTCTCCGACCTCCCAGAAGAAC CAGCGTGGGATCTGCAGTTTGATCTCTTGGACCTTAATGATATC TCCTCTGCCTTAAAAC ACC ACGAGGAG AAAAACTTGATTTTAGA
TTTCCCTCAGCCTTCAACCGATTACTTAAGTTTCCGGAGCCACTT TCAGAAGAACTTTGTCTGTCTGGAGAACTGCTCGTTGCAAGAGC GAACAGTGACAGGGACTGTTAAAGTCAAAAATGTGAGTTTTGA GAAGAAAGTTCAGATCCGTATCACTTTCGATTCTTGGAAAAACT AC ACTGACGTAGACTGTGTCTATATGAAAAATGTGTATGGTGGC
ACAGATAGTGATACCTTCTCATTTGCCATTGACTTACCCCCTGTC ATTCCAACTGAGCAGAAAATTGAGTTCTGCATTTCTTACCATGC TAATGGGCAAGTCTTTTGGGACAACAATGATGGTCAGAATTATA GAATTGTTCATGTTCAATGGAAGCCTGATGGGGTGCAGACACAG ATGGCACCCCAGGACTGTGCATTCCACCAGACGTCTCCTAAGAC
AGAGTTAGAGTCAACAATCTTTGGCAGTCCGAGGCTGGCTAGTG GGCTCTTCCCAGAGTGGCAGAGCTGGGGGAGAATGGAGAACTT GGCCTCTTATCGATGA. (SEQ ID NO.: 6)
7. An expression vector that contains a human genomic DNA molecule comprising the sequence of Claim 4.
8. An expression vector that contains a murine genomic DNA molecule comprising the sequence of Claim 3.
9. An expression vector that contains a human cDNA molecule comprising the sequence of Claim 6. -56-
10. An expression vector that contains a murine cDNA molecule comprising the sequence of Claim 5.
11. A host cell containing an introduced murine genomic DNA molecule comprising the sequence of Claim 3.
12. A host cell containing an introduced human genomic DNA molecule comprising the sequence of Claim 4.
13. A host cell containing an introduced human cDNA molecule comprising the sequence of Claim 6.
14. A host cell containing an introduced murine cDNA molecule comprising the sequence of Claim 5.
15. A method of increasing the amount of glycogen in a cell, the method comprising: a. Administering to the cell an expression vector containing a human genomic DNA molecule comprising the sequence of Claim 4; and b. Allowing sufficient time for the human genomic DNA molecule to be expressed.
16. A method of increasing the amount of glycogen in a cell, the method comprising: a. Administering to the cell an expression vector containing a murine genomic DNA molecule comprising the sequence of Claim 3 ; and b. Allowing sufficient time for the murine genomic DNA molecule to be expressed.
17. A method of increasing the amount of glycogen in a cell, the method comprising: a. Administering to the cell an expression vector containing a human cDNA molecule comprising the sequence of Claim 6; and
PCT/US1997/014142 1996-08-30 1997-08-22 Protein targeting to glycogen WO1998008948A1 (en)

Priority Applications (4)

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EP97938246A EP0941324A1 (en) 1996-08-30 1997-08-22 Protein targeting to glycogen
US09/242,948 US6252057B1 (en) 1996-08-30 1997-08-22 Protein targeting to glycogen
JP51167598A JP2002514173A (en) 1996-08-30 1997-08-22 Glycogen targeting proteins
AU40623/97A AU4062397A (en) 1996-08-30 1997-08-22 Protein targeting to glycogen

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US2510796P 1996-08-30 1996-08-30
US60/025,107 1996-08-30
US5524397P 1997-08-12 1997-08-12
US60/055,243 1997-08-12

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Cited By (1)

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EP0846763A2 (en) * 1996-12-05 1998-06-10 Smithkline Beecham Corporation A protein phosphatase 1 binding protein, R5

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BRADY, MATTHEW J. ET AL: "Role of protein targeting to glycogen (PTG) in the regulation of protein phosphatase -1 activity", J. BIOL. CHEM. (1997), 272(32), 20198-20204 CODEN: JBCHA3;ISSN: 0021-9258, 1997, XP002047742 *
JOHNSON, DEBORAH F. ET AL: "Identification of protein - phosphatase -1- binding domains on the glycogen and myofibrillar targeting subunits", EUR. J. BIOCHEM. (1996), 239(2), 317-325 CODEN: EJBCAI;ISSN: 0014-2956, 1996, XP002047740 *
MARTIN J. DOHERTY ET AL.: "Amino acid sequence and expression of the hepatic glycogen-binding (Gl)-subunit of protein phosphatase-1", FEBS LETTERS, vol. 375, no. 3, 20 November 1995 (1995-11-20), AMSTERDAM NL, pages 294 - 298, XP002047738 *
NYOMBA, BULANGU L. ET AL: "Immunoreactive glycogen - binding subunit of protein phosphatase -1 in human skeletal muscle", J. CLIN. ENDOCRINOL. METAB. (1994), 79(2), 485-8 CODEN: JCEMAZ;ISSN: 0021-972X, 1994, XP002047739 *
PRINTEN, JOHN A. ET AL: "PTG, a protein phosphatase 1- binding protein with a role in glycogen metabolism", SCIENCE (WASHINGTON, D. C.) (1997), 275(5305), 1475-1478 CODEN: SCIEAS;ISSN: 0036-8075, 1997, XP002047741 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0846763A2 (en) * 1996-12-05 1998-06-10 Smithkline Beecham Corporation A protein phosphatase 1 binding protein, R5
EP0846763A3 (en) * 1996-12-05 2000-01-19 Smithkline Beecham Corporation A protein phosphatase 1 binding protein, R5
US6297359B1 (en) 1996-12-05 2001-10-02 Smithkline Beecham Corporation, Et Al. Protein phosphatase 1 binding protein, R5

Also Published As

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EP0941324A1 (en) 1999-09-15
AU4062397A (en) 1998-03-19

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