| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | December 28, 1999 |
| PATENT TITLE |
Glycine transporter |
| PATENT ABSTRACT |
Provided are, among other things, nucleic acid sequences encoding the GlyT1d form of glycine transporter, vectors, methods of producing the transporter, and methods for identifying bioactive agents. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | April 11, 1997 |
| PATENT REFERENCES CITED |
Kim et al. Cloning of the Human Glycine Transporter Type I Mol. Pharmacol. 45: 608-617 1994. Olivares et al. Carboxyl Terminus of the Glycine Transporter GIYTI is Necessary for Corred Processing of the Protein J. Biol. Chem. 269: 28400-28404 1994. Shi et al. Stable Inducible Expression of a Functional Rat Liver Organic Anion Transport Protein in Hela Cells J. Biol. Chem. 270: 25591-25595 1995. Johnson and Ascher, Nature, 325: 529-531, 1987. Fletcher et al., Glycine Neurotransmission, Otterson and Storm-Mathisen, eds., 1990, pp. 193-219. Smith et al., Neuron, 8: 927-935, 1992. Liu et al., J. Biol. Chem., 208:22802-22809, 1993. Jursky and Nelson, J. Neurochemistry, 64: 1026-1033, 1995. Uhl, Trends in Neuroscience, 15:205-208, 1992. Clark and Amara, BioEssays, 15:323-332, 1993. Yaksh, Pain, 111-123, 1989. Truong et al., Movement Disorders, 3:77-87, 1988. Becker, FASEB Journal 4:2767-2774, 1990. Lopez-Corcuera et al., J. Biol. Chem., 266:24809-24814, 1991. Liu et al., FEBS Letters, 305:110-114, 1992. Bannon et al., J. Neurochem., 54:706-708, 1990. Guastella et al., Science, 249:1303-1306, 1990. Grimwood et al., Molecular Pharmacology, 49:923-930, 1992. Kim et al., Molecular Pharmacology, 45:608-617, 1994. Lu et al., Proc. Nat'l Acad. Sci. USA, 88:6289-6292, 1991. White et al., J. Neurochemistry, 35:503-512, 1989. Becker et al., J. Neuroscience, 6:1358-1364, 1986. Adams et al., J. Neuroscience, 15:2524-2532, 1995. Zafra et al., Neuroscience, 15:3952-3969, 1995. Zafra et al., Eur. Neuroscience, 7:1342-1352, 1995. Goebel, D.J., Mol. Brain Res., 40:139-142, 1996. Jursky et al., J. Neurochem, 67:336-344, 1996. Borowsky et al., Neuron, 10:851-863, 1993. Guastella et al., Proc. Nat'l. Acad. Sci. USA 89:7189-7193, 1992. Blakely et al., Proc. Nat'l Acad., Sci. USA 85:9846-9850, 1988. Lebo et al in "Cold Spring Harbor Symposium on Quantitative Biology" vol. L1 Molecular Biology of Homosapens, Cold Spring Harbor Laboratory 1986, pp. 169-176. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed: 1. An isolated nucleic acid segment comprising a nucleic acid sequence encoding a glycine transporter protein, wherein the encoded glycine transporter protein has a protein sequence of SEQ ID NO: 2 or a protein sequence having six or fewer amino acid changes relative to SEQ ID NO: 2. 2. An isolated nucleic acid segment comprising a nucleic acid sequence encoding a glycine transporter protein, wherein the encoded protein has a protein sequence of SEQ ID NO: 2 or a protein sequence having four or fewer amino acid changes relative to SEQ ID NO: 2. 3. The isolated nucleic acid segment of claim 2, wherein the encoded protein has a protein sequence of SEQ ID NO: 2 or a protein sequence having two or fewer amino acid changes relative to SEQ ID NO: 2. 4. The isolated nucleic acid segment of claim 2, wherein the encoded protein has a protein sequence of SEQ ID NO: 2 or a protein sequence having one or fewer amino acid changes relative to SEQ ID NO: 2. 5. An isolated nucleic acid segment comprising a nucleic acid sequence encoding a glycine transporter isoform having the protein sequence of SEQ ID 2. 6. The isolated nucleic acid segment of claim 5, wherein the encoding nucleic acid sequence has the following sequence from SEQ ID 1. ATGGTAGGAA AAGGTGCCAA AGGGATGCTG GTGACGCTTC TCCCTGTTCA GAGATCCTTC 60 - TTCCTGCCAC CCTTTTCTGG AGCCACTCCC TCTACTTCCC TAGCAGAGTC TGTCCTCAAA 120 - GTCTGGCATG GGGCCTACAA CTCTGGTCTC CTTCCCCAAC TCATGGCCCA GCACTCCCTA 180 - GCCATGGCCC AGAATGGTGC TGTGCCCAGC GAGGCCACCA AGAGGGACCA GAACCTCAAA 240 - CGGGGCAACT GGGGCAACCA GATCGAGTTT GTACTGACGA GCGTGGGCTA TGCCGTGGGC 300 - CTGGGCAATG TCTGGCGCTT CCCATACCTC TGCTATCGCA ACGGGGGAGG CGCCTTCATG 360 - TTCCCCTACT TCATCATGCT CATCTTCTGC GGGATCCCCC TCTTCTTCAT GGAGCTCTCC 420 - TTCGGCCAGT TTGCAAGCCA GGGGTGCCTG GGGGTCTGGA GGATCAGCCC CATGTTCAAA 480 - GGAGTGGGCT ATGGTATGAT GGTGGTGTCC ACCTACATCG GCATCTACTA CAATGTGGTC 540 - ATCTGCATCG CCTTCTACTA CTTCTTCTCG TCCATGACGC ACGTGCTGCC CTGGGCCTAC 600 - TGCAATAACC CCTGGAACAC GCATGACTGC GCCGGTGTAC TGGACGCCTC CAACCTCACC 660 - AATGGCTCTC GGCCAGCCGC CTTGCCCAGC AACCTCTCCC ACCTGCTCAA CCACAGCCTC 720 - CAGAGGACCA GCCCCAGCGA GGAGTACTGG AGGCTGTACG TGCTGAAGCT GTCAGATGAC 780 - ATTGGGAACT TTGGGGAGGT GCGGCTGCCC CTCCTTGGCT GCCTCGGTGT CTCCTGGTTG 840 - GTCGTCTTCC TCTGCCTCAT CCGAGGGGTC AAGTCTTCAG GGAAAGTGGT GTACTTCACG 900 - GCCACGTTCC CCTACGTGGT GCTGACCATT CTGTTTGTCC GCGGAGTGAC CCTGGAGGGA 960 - GCCTTTGACG GCATCATGTA CTACCTAACC CCGCAGTGGG ACAAGATCCT GGAGGCCAAG1020 - GTGTGGGGTG ATGCTGCCTC CCAGATCTTC TACTCACTGG CGTGCGCGTG GGGAGGCCTC1080 - ATCACCATGG CTTCCTACAA CAAGTTCCAC AATAACTGTT ACCGGGACAG TGTCATCATC1140 - AGCATCACCA ACTGTGCCAC CAGCGTCTAT GCTGGCTTCG TCATCTTCTC CATCCTCGGC1200 - TTCATGGCCA ATCACCTGGG CGTGGATGTG TCCCGTGTGG CAGACCACGG CCCTGGCCTG1260 - GCCTTCGTGG CTTACCCCGA GGCCCTCACA CTACTTCCCA TCTCCCCGCT GTGGTCTCTG1320 - CTCTTCTTCT TCATGCTTAT CCTGCTGGGG CTGGGCACTC AGTTCTGCCT CCTGGAGACG1380 - CTGGTCACAG CCATTGTGGA TGAGGTGGGG AATGAGTGGA TCCTGCAGAA AAAGACCTAT1440 - GTGACCTTGG GCGTGGCTGT GGCTGGCTTC CTGCTGGGCA TCCCCCTCAC CAGCCAGGCA1500 - GGCATCTATT GGCTGCTGCT GATGGACAAC TATGCGGCCA GCTTCTCCTT GGTGGTCATC1560 - TCCTGCATCA TGTGTGTGGC CATCATGTAC ATCTACGGGC ACCGGAACTA CTTCCAGGAC1620 - ATCCAGATGA TGCTGGGATT CCCACCACCC CTCTTCTTTC AGATCTGCTG GCGCTTCGTC1680 - TCTCCCGCCA TCATCTTCTT TATTCTAGTT TTCACTGTGA TCCAGTACCA GCCGATCACC1740 - TACAACCACT ACCAGTACCC AGGCTGGGCC GTGGCCATTG GCTTCCTCAT GGCTCTGTCC1800 - TCCGTCCTCT GCATCCCCCT CTACGCCATG TTCCGGCTCT GCCGCACAGA CGGGGACACC1860 - CTCCTCCAGC GTTTGAAAAA TGCCACAAAG CCAAGCAGAG ACTGGGGCCC TGCCCTCCTG1920 - GAGCACCGGA CAGGGCGCTA CGCCCCCACC ATAGCCCCCT CTCCTGAGGA CGGCTTCGAG1980 - GTCCAGTCAC TGCACCCGGA CAAGGCGCAG ATCCCCATTG TGGGCAGTAA TGGCTCCAGC2040 - CGCCTCCAGG ACTCCCGGAT A2061. 7. A vector comprising a nucleic acid sequence encoding a glycine transporter protein, wherein the encoded glycine transporter protein has a protein sequence of SEQ ID NO: 2 or a protein sequence having four or fewer amino acid changes relative to SEQ ID NO: 2. 8. The vector of claim 7, wherein the vector further comprises an inducible promoter for inducibly expressing the glycine transporter protein. 9. A process of producing a recombinant cell that expresses a recombinant glycine receptor comprising transfecting a cell with the vector of claim 7. 10. An isolated host cell transfected with a nucleic acid encoding a glycine transporter and expressing the glycine transporter, wherein the encoded glycine transporter protein has a protein sequence of SEQ ID NO: 2 or a protein sequence having six or fewer amino acid changes relative to SEQ ID NO: 2. 11. An isolated host cell transfected with a nucleic acid encoding a glycine transporter and expressing the glycine transporter, wherein the encoded protein has a protein sequence of SEQ ID NO: 2 or a protein sequence having four or fewer amino acid changes relative to SEQ ID NO: 2. 12. The host cell of claim 11, wherein the encoded protein has a protein sequence of SEQ ID NO: 2 or a protein sequence having two or fewer amino acid changes relative to SEQ ID NO: 2. 13. The host cell of claim 11, expressing a recombinant glycine transporter encoded by the nucleic acid at its cell surface. 14. A process of producing a glycine transporter comprising expressing the protein in the host cell of claim 11. 15. The process of claim 14, further comprising at least one of (a) isolating membranes from said cells, which membranes comprise the glycine transporter or (b) extracting a protein fraction from the host cells which fraction comprises the glycine transporter. 16. The process of claim 14, wherein the nucleic acid further comprises an inducible promoter for inducibly expressing the glycine transporter protein and the process further comprises: growing the cell in a medium; and inducing the expression of the glycine transporter by adding an inducing agent into the medium. 17. An isolated vector comprising the nucleic acid segment of claim 5. 18. An isolated host cell transfected with the vector of claim 17 to contain nucleic acid encoding the glycine transporter isoform. 19. The isolated host cell of claim 18, wherein the glycine transporter isoform is expressed at the surface of the cell. 20. A process of producing a glycine transporter isoform comprising incubating the transfected host cell of claim 18 under conditions to express the glycine transporter isoform. 21. A process of producing a glycine transporter comprising expressing the protein in the host cell of claim 10. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
The present invention relates to nucleic acid encoding a "GlyT1d" member of the family of glycine transporters, to the isolated protein encoded by the nucleic acid, and to the field of drug discovery. Synaptic transmission is a complex form of intercellular communication that involves a considerable array of specialized structures in both the pre- and post-synaptic neuron. High-affinity neurotransmitter transporters are one such component, located on the pre-synaptic terminal and surrounding glial cells (Kanner and Schuldiner, CRC Critical Reviews in Biochemistry 22: 1032, 1987). Transporters sequester neurotransmitter from the synapse, thereby regulating the concentration of neurotransmitter in the synapse, as well as its duration in the synapse therein, which together influence the magnitude of synaptic transmission. Further, by preventing the spread of transmitter to neighboring synapses, transporters maintain the fidelity of synaptic transmission. Last, by sequestering released transmitter into the presynaptic terminal, transporters allow for transmitter reutilization. Neurotransmitter transport is dependent on extracellular sodium and the voltage difference across the membrane; under conditions of intense neuronal firing, as for example during a seizure, transporters can function in reverse, releasing neurotransmitter in a calcium-independent non-exocytotic manner (Attwell et al., Neuron 11: 401-407, 1993). Pharmacologic modulation of neurotransmitter transporters thus provides a means for modifying synaptic activity, which provides useful therapy for the treatment of neurological and psychiatric disturbances. The amino acid glycine is a major neurotransmitter in the mammalian nervous system, functioning at both inhibitory and excitatory synapses. By nervous system, both the central and peripheral portions of the nervous system are intended. These distinct functions of glycine are mediated by two different types of receptor, each of which is associated with a different class of glycine transporter. The inhibitory actions of glycine are mediated by glycine receptors that are sensitive to the convulsant alkaloid, strychnine, and are thus referred to as "strychnine-sensitive." Such receptors contain an intrinsic chloride channel that is opened upon binding of glycine to the receptor, by increasing chloride conductance, the threshold for firing of an action potential is increased. Strychnine-sensitive glycine receptors are found predominantly in the spinal cord and brainstem, and pharmacological agents that enhance the activation of such receptors will thus increase inhibitory neurotransmission in these regions. Glycine functions in excitatory transmission by modulating the actions of glutamate, the major excitatory neurotransmitter in the central nervous system. See Johnson and Ascher, Nature 325: 529-531, 1987; Fletcher et al., Glycine Transmission, (Otterson and Storm-Mathisen, eds., 1990), pp. 193-219. Specifically, glycine is an obligatory co-agonist at the class of glutamate receptor termed N-methyl-D-aspartate (NMDA) receptor. Activation of NMDA receptors increases sodium and calcium conductance, which depolarizes the neuron, thereby increasing the likelihood that the neuron will fire an action potential. NMDA receptors are widely distributed throughout the brain, with a particularly high density in the cerebral cortex and hippocampal formation. Molecular cloning has revealed the existence in mammalian brains of two classes of glycine transporters, termed GlyT1 and GlyT2. GlyT1 is found predominantly in the forebrain, and its distribution corresponds to that of glutamatergic pathways and NMDA receptors (Smith, et al., Neuron 8: 927-935, 1992). Molecular cloning has further revealed the existence of three variants of GlyT-1, termed GlyT-1a, GlyT-1b and GlyT-1c (Kim et al., Molecular Pharmacology 45: 608-617, 1994), each of which displays a unique distribution in the brain and peripheral tissues. GlyT2, in contrast, is found predominantly in the brain stem and spinal cord, and its distribution corresponds closely to that of strychnine-sensitive glycine receptors (Liu et al., J. Biol. Chem. 268: 22802-22808, 1993Jursky and Nelson, J. Neurochem. 64: 1026-1033, 1995. These observations are consistent with the view that, by regulating the synaptic levels of glycine, GlyT1d and GlyT2 selectively influence the activity of NMDA receptors and strychnine-sensitive glycine receptors, respectively. Sequence comparisons of GlyT1 and GlyT2 have revealed that these glycine transporters are members of a broader family of sodium-dependent neurotransmitter transporters, including, for example, transporters specific for gamma-amino-n-butyric acid (GABA) and others. Uhl, Trends in Neuroscience 15: 265-268, 1992: Clark and Amara, BioEssays 15: 323-332, 1993. Overall, each of these transporters includes 12 putative transmembrane domains that predominantly contain hydrophobic amino acids. Compounds that inhibit or activate glycine transporters would be expected to alter receptor function, and provide therapeutic benefits in a variety of disease states. For example, inhibition of GlyT2 can be used to diminish the activity of neurons having strychnine-sensitive glycine receptors via increasing synaptic levels of glycine, thus diminishing the transmission of pain-related (i.e., nociceptive) information in the spinal cord, which has been shown to be mediated by these receptors. Yaksh, Pain, 111-123 (1989). Additionally, enhancing inhibitory glycinergic transmission through strychnine-sensitive glycine receptors in the spinal cord can be used to decrease muscle hyperactivity, which is useful in treating diseases or conditions associated with increased muscle contraction, such as spasticity, myoclonus, and epilepsy (Truong et al., Movement Disorders, 3, 77-87 (1988); Becker, FASEB J., 4, 2767-2774 (1990)). Spasticity that can be treated via modulation of glycine receptors is associated with epilepsy, stroke, head trauma, multiple sclerosis, spinal cord injury, dystonia, and other conditions of illness and injury of the nervous system. NMDA receptors are critically involved in memory and learning (Rison and Stanton, Neurosci. Biobehav. Rev., 19, 533-552 (1995); Danysz et al., Behavioral Pharmacol., 6, 455-474 (1995)); and, furthermore, decreased function of NMDA-mediated neurotransmission appears to underlie, or contribute to, the symptoms of schizophrenia (Olney and Farber, Archives General Psychiatry 52: 998-1007 (1996)). Thus, agents that inhibit GlyT-1 and thereby increase glycine activation of NMDA receptors can be used as novel antipsychotics and anti-dementia agents, and to treat other diseases in which cognitive processes are impaired, such as attention deficit disorders and organic brain syndromes. Conversely, over-activation of NMDA receptors has been implicated in a number of disease states, in particular the neuronal death associated with stroke and possibly neurodegenerative diseases, such as Alzheimer's disease, multi-infarct dementia, AIDS dementia, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis or other conditions in which neuronal cell death occurs, such as stroke and head trauma. Coyle & Puttfarcken, Science 262: 689-695, 1993; Lipton and Rosenberg, New Engl. J. of Medicine, 330: 613-622, 1993; Choi, Neuron 1: 623-634, 1988. Thus, pharmacological agents that increase the activity of GlyT-1 will result in decreased glycine-activation of NMDA receptors, which activity can be used to treat these, and related, disease states. Similarly, drugs that directly block the glycine site on the NMDA receptors can be used to treat these and related disease states. SUMMARY OF THE INVENTION The invention provides an isolated nucleic acid encoding a glycine transporter protein comprising a nucleic acid wherein: (a) the encoded protein has a protein sequence of SEQ ID 2 or a protein sequence having at least about 99% sequence identity with SEQ ID 2; or (b) a glycine transporter protein-encoding portion of the nucleic acid has at least about 95% sequence identity with SEQ ID 1. The invention further provides a cell comprising a nucleic acid encoding a glycine transporter, wherein the nucleic acid is functionally associated with a promoter, wherein: (a) the encoded protein has a protein sequence of SEQ ID 2 or a protein sequence having at least about 99% sequence identity with SEQ ID 2; or (b) a protein-encoding portion of the nucleic acid has at least about 95% sequence identity with SEQ ID 1. A glycine transporter can be isolated from such a cell. The invention further provides a method for characterizing a bioactive agent for treatment of a nervous system disorder or condition or for identifying bioactive agents for treatment of a nervous system disorder or condition, the method comprising (a) providing a first assay composition comprising (i) a cell according to the invention or (ii) an isolated glycine transporter protein comprising the amino acid sequence encoded by the nucleic acid, or the amino acid sequence resulting from cellular processing of the amino acid sequence encoded by the nucleic acid of the vector, (b) contacting the first assay composition with the bioactive agent or a prospective bioactive agent, and (c) measuring the amount of glycine transport exhibited by the assay composition. Additionally, the invention provides a nucleic acid comprising an amplification primer or nuclease protection probe effective to identify GlyT1d and to distinguish GlyT1d from GlyT1a, GlyT1b, GlyT1c and GlyT2, which can be a vector comprising of the nucleic acid. The invention further provides an isolated glycine transporter protein having a sequence of SEQ ID 2 or a sequence having at least about 99% sequence identity with SEQ ID 2, which can comprise isolated membranes in which the glycine transporter protein is an integral protein. |
| PATENT PHOTOCOPY | Available on request |
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