| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 31.12.02 |
| PATENT TITLE |
Human CNS receptors of the NMDA-R1 family |
| PATENT ABSTRACT |
Neurotransmission by excitatory amino acids (EAAS) such as glutamate is mediated via membrane-bound surface receptors. DNA coding for EAA receptors of one family of human NMDA-binding type receptors has now been isolated and receptor proteins characterized. Herein described are recombinant cell lines which produce the EAA receptor as a heterologous membrane-bound product. Also described are related aspects of the invention, which are of commercial significance. Included is use of the cell lines as a tool for discovery of compounds which modulate EAA receptor stimulation. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | December 10, 1993 |
| PATENT REFERENCES CITED |
P.N.A.S. 88:7557-7561, Sep. 1991, Puckett et al Molecular Cloning and Chromosomal Localization of One of the Human Glutamate Receptor Genes.* P.N.A.S. 86:9762-9766, Dec., 1989, Grandy et al Cloning of the cDNA and Gene for a Human D.sub.2 Dopamine Receptor.* Nature 347:76-79, Sep. 06, 1990, Zhou et al Cloning and Expression of Human and Rat D.sub.1 Dopamine Repecetors.* Barnett et al., "Rapid Generation of DNA Fragments by PCR Amplicaton of Crude, Synthetic, Oligonucleotides," Nucleic Acids Research, vol. 18, No. 10, p. 3094 (1990). Moriyoshi et al., "Molecular Cloning and Characterization of the Rat/NMDA Receptor," Nature, vol. 354, pp. 31-36 (Nov. 1991). Sakimura et al., "Primary Structure and Expression of the .sub.y 2 Subunit of the Glutamate Receptor Channel Selective for Kainate," Neuron, vol. 8, pp. 267-274 (Feb. 1992). Yamazaki et al., "Cloning, Expression and Modulation of a Mouse NMDA Receptor Subunit," FEBS Letters, vol. 300, No. 1, pp. 39-45 (Mar. 1992). Meguro et al., "Functional Characterization of a Heteromeric NMDA Receptor Channels Expressed from Cloned cDNAs," Nature, vol. 357, pp. 70-74 (May 1992). Monyer et al., "Heteromeric NMDA Receptors: Molecular and Functional Distinction of Subtypes," Science, vol. 256, pp. 1217 (May 1992). Anantharam et al., "Combinatorial RNA Splicing Alters the Surface Change on the NMDA Receptor," FEBS Letters, vol. 305, No. 1, pp. 27-30 (Jun. 1992). Sugihara et al., "Structures and Properties of Seven Isoforms of the NMDA Receptor Generated by Alternative Splicing," Biochemical and Biophysical Research Communications, vol. 185, No. 3, pp. 826-832 (Jun. 1992). Kutsuwada et al., "Molecular Diversity of the NMDA Receptor Channel," Nature, vol. 358, pp. 36-41 (Jul. 1992). Oksenberg et al., "A Single Amino-Acid Difference Confers Major Pharmacological Variation between Human and Rodent 5-HT.sub.18 Receptors," Nature, vol. 360, pp. 161-163 (Nov. 1992). Durand et al., "Cloning of an Apparent Splice Variant of the Rat N-methyl-D-aspartate Receptor NMDAR1 with Altered Sensitivity to Polyamines and Activators of Protein Kinase C," Proc. Natl. Acad. Sci. USA, vol. 89, pp. 9359-9363 (Oct. 1992). William Sun, et al., "Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors"; Proc. Natl. Acad. Sci. USA, vol. 89, pp. 1443-1447, Feb. 1992. Carmie Puckett, et al., "Molecular cloning and chromosomal localization of one of the human glutamate receptor genes"; Proc. Natl. Acad. Sci. USA, vol. 88, pp. 7557-7561, Sep. 1991. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. An isolated polynucleotide comprising a nucleotide sequence that codes for a human NMDAR1, wherein said NMDAR1 has the sequence of amino acids 1-867 of SEQ ID NO:2 with none to as many as 6 amino acid substitutions. 2. A recombinant DNA construct having incorporated therein a polynucleotide as defined in claim 1. 3. A cell that has been engineered genetically to produce a human NMDAR1, said cell having incorporated expressibly therein a heterologous polynucleotide as defined in claim 1. 4. A cell as defined in claim 3, which is a mammalian cell. 5. A membrane preparation derived from a cell as defined in claim 3. 6. A method of assaying a test ligand for binding with a human CNS receptor, which comprises the steps of incubating the test ligand under appropriate conditions with a human NMDAR1-producing cell as defined in claim 3, or with a membrane preparation derived therefrom, and then determining the extent of binding between the human NMDAR1 and the test ligand. 7. A method of assaying a test ligand for electrophysiological effect upon interaction with a human CNS receptor, which comprises the steps of incubating the test ligand under appropriate conditions with a human NMDAR1-producing cell as defined in claim 3 or with membrane preparation derived therefrom, and then determining ligand-induced electrical current across said cell or membrane. 8. A process for obtaining a human EAA receptor in substantially homogeneous form, which comprises the steps of culturing cells having incorporated expressibly therein a polynucleotide as defined in claim 1, and then recovering the cultured cells. 9. A process according to claim 8, comprising the subsequent step of obtaining a membrane preparation from the cultured cells. 10. A human NMDAR1, in a form essentially free from other proteins of human origin, said NMDAR1 being encoded by a polynucleotide as defined in claim 1. 11. An isolated polynucleotide which codes for a receptor selected from the group consisting of: (a) NMDAR1-1 having the sequence of amino acids 1-867 of SEQ ID NO: 2; (b) NMDAR1-2 having the sequence of SEQ ID NO: 25; (c) NMDAR1-3A having the sequence of SEQ ID NO: 26; (d) NMDAR1-3C having the sequence of SEQ ID NO: 27; (e) NMDAR1-3B having the amino acid sequence of NMDAR1-3C except that residue 470 is a lysine residue; (f) NMDAR1-4 having the amino acid sequence of SEQ ID NO: 28; (g) NMDAR1-5 having the amino acid sequence of SEQ ID NO: 29; (h) NMDAR1-6 having the amino acid sequence of SEQ ID NO: 30; (i) NMDAR1-7 having the amino acid sequence of SEQ ID NO: 31; (j) NMDAR1-8 having the amino acid sequence of SEQ ID NO: 32. 12. An isolated polynucleotide encoding a human NMDAR1-1 having at least 99.6% amino acid identity with the 1-845 amino acid region of SEQ ID NO:2 of the NMDAR1-1. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention is concerned with applications of recombinant DNA technology in the field of neurobiology. More particularly, the invention relates to the cloning and expression of DNA coding for excitatory amino acid (EAA) receptors, especially human EAA receptors. BACKGROUND TO THE INVENTION In the mammalian central nervous system (CNS), the transmission of nerve impulses is controlled by the interaction between a neurotransmitter substance released by the "sending" neuron which then binds to a surface receptor on the "receiving" neuron, to cause excitation thereof. L-glutamate is the most abundant neurotransmitter in the CNS, and mediates the major excitatory pathway in vertebrates. Glutamate is therefore referred to as an excitatory amino acid (EAA) and the receptors which respond to it are variously referred to as glutamate receptors, or more commonly as EAA receptors. Using tissues isolated from mammalian brain, and various synthetic EAA receptor agonists, knowledge of EAA receptor pharmacology has been refined somewhat. Members of the EAA receptor family can be grouped into three main types based on differential binding to such agonists. One type of EAA receptor, which in addition to glutamate also binds the agonist NMDA (N-methyl-D-aspartate), is referred to as the NMDA type of EAA receptor. Two other glutamate-binding types of EAA receptor, which do not bind NMDA, are named according to their preference for binding with two other EAA receptor agonists, namely AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate), and kainate (2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetate). Accordingly, receptors which bind glutamate but not NMDA, and which bind with greater affinity to kainate than to AMPA, are referred to as kainate type EAA receptors. Similarly, those EAA receptors which bind glutamate but not NMDA, and which bind AMPA with greater affinity than kainate are referred to as AMPA type EAA receptors. The glutamate-binding EAA receptor family is of great physiological and medical importance. Glutamate is involved in many aspects of long-term potentiation (learning and memory), in the development of synaptic plasticity, in epileptic seizures, in neuronal damage caused by ischemia following stroke or other hypoxic events, as well as in other forms of neurodegenerative processes. The development of therapeutics which modulate these processes has been very difficult, due to the lack of any homogeneous source of receptor material with which to discover selectively binding drug molecules, which interact specifically at the interface of the EAA receptor. The brain derived tissues currently used to screen candidate drugs are heterogeneous receptor sources, possessing on their surface many receptor types which interfere with studies of the EAA receptor/ligand interface of interest. The search for human therapeutics is further complicated by the limited availability of brain tissue of human origin. It would therefore be desirable to obtain cells that are genetically engineered to produce only the receptor of interest. With cell lines expressing cloned receptor cDNA, a substrate which is homogeneous for the desired receptor is provided, for drug screening programs. Non-human cDNAs which appear to encode the NMDA-type of EAA receptor have recently been identified and isolated. A cDNA encoding a subunit polypeptide of an NMDA receptor in rat, designated NR1, has been isolated as described by Moriyoshi et al. in Nature 354: 31, 1991. This work has been extended to demonstrate six isoforms of NR1, presumably generated by combinations of alternative RNA splicing in the amino- and carboxy-terminal regions of NR1 (Anantharam et al. FEBS Lett. 305: 27, 1992; Durand et al. Proc. Natl. Acad. Sci. USA 89: 9359, 1992; Nakanishi et al. Proc. Natl. Acad. Sci. USA 89: 8552, 1992; Sugihara et al. Biochem. Biophys. Res. Commun. 185: 826, 1992). DNA encoding NR1 and one of its isoforms have also been cloned from mouse brain by Yamazaki et al. as described in FEBS Lett. 300: 39, 1992. Other rat NMDA receptor subunits, designated NR2A, NR2B and NR2C, have also been identified (Monyer et al. Science 256: 1217, 1992), as well as mouse NMDA receptor subunits which have been designated .epsilon.1, .epsilon.2 and .epsilon.3 (Meguro et al. Nature 357: 70, 1992 and Kutsuwada et al. Nature 358: 36, 1992). There has emerged from these molecular cloning advances, a better understanding of the structural features of NMDA receptors and their subunits, as they exist in the non-human brain. According to the current model, each NMDA receptor is heteromeric, consisting of individual membrane-anchored subunits, each with four transmembrane regions, and extracellular domains that dictate ligand-binding properties and contribute to the ion-gating function served by the receptor complex. In the search for therapeutics useful to treat CNS disorders in humans, it is highly desirable to obtain knowledge of human NMDA-type EAA receptors. A specific understanding of these human receptors would provide a means to screen for compounds that react therewith, i.e. to stimulate or inhibit receptor activity, and thus providing a means to identify compounds having potential therapeutic utility in humans. Non-human mammalian models are not suitable for this purpose despite significant receptor sequence homology, as minute sequence discrepancies can cause dramatic pharmacological variation between species homologues of the same receptor (Oksenberg et al., Nature, 360:161, 1992). It is therefore particularly desirable to provide cloned cDNA encoding human EAA receptors, and cell lines expressing these receptors in a homogeneous fashion, in order to generate a screening method for compounds therapeutically useful in humans. These, accordingly, are objects of the present invention. Another object of the present invention is to provide in isolated form a DNA molecule which codes for a human EAA receptor. It is another object of the present invention to provide a cell that has been genetically engineered to produce an N-methyl-D-aspartate-type human EAA receptor. SUMMARY OF THE INVENTION Human cDNAs encoding a family of EAA receptors, which bind glutamate with an affinity typical of EAA receptors and exhibit ligand binding properties characteristic of NMDA-type EAA receptors, have been identified and characterized. A representative member of this human EAA receptor family is herein designated human NMDAR1-1. Sequence-related cDNAs encoding naturally occurring variants of the human NMDAR1-1 have also been identified, and constitute additional members of this receptor family as do fragments of NMDAR1 receptors, herein referred to as the human NMDAR1 receptor family. The present invention thus provides, in one of its aspects, an isolated polynucleotide, consisting either of DNA or of RNA, which codes for a human NMDAR1 or for fragments thereof characterized by at least one of MK-801-binding or glutamate-binding. In another aspect of the present invention, there is provided a cell that has been genetically engineered to produce a human EAA receptor belonging to the herein-defined NMDAR1 family. In related aspects of the present invention, there are provided recombinant DNA constructs and relevant methods useful to create such cells. In another aspect of the present invention, there is provided a method for evaluating interaction between a test ligand and a human EAA receptor, which comprises the steps of incubating the test ligand with a genetically engineered cell of the present invention, or with a membrane preparation derived therefrom, and then assessing said interaction by determining receptor/ligand binding. Other aspects of the present invention, which encompass various applications of the discoveries herein described, will become apparent from the following detailed description, and from the accompanying drawings in which: |
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| PATENT PHOTOCOPY | Available on request |
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