Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-10-23
2004-10-26
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C536S023100, C536S023500, C530S350000
Reexamination Certificate
active
06808893
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to protein-protein interactions and more specifically to Glutamate Transporter Associated Proteins involved in mediating glutamate transport, chloride transport and cytoskeletal stability and their association with glutamate transporter proteins.
BACKGROUND OF THE INVENTION
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system, acting on postsynaptic ionotropic glutamate receptors (particularly NMDA and AMPA receptors). In addition, glutamate stimulates a subset of metabotropic glutamate receptors (particularly the group I metabotropic glutamate receptors mGluR1a and mGluR5) concentrated in the postsynaptic membrane. The timely removal of glutamate from the synaptic cleft is critical to preventing desensitization resulting from continued exposure of the postsynaptic receptors to glutamate. Removal of glutamate from the synaptic cleft is mediated by a class of molecules known as glutamate transporter proteins located on surrounding astroglia and neurons. Five distinct, high-affinity, sodium-dependent glutamate transporters have been identified in animal and human central nervous system. Rat GLAST, GLT-1, EAAC1 (EAAT1, EAAT2 and EATT3, respectively, in human), EAAT4 and EAAT5 differ in structure, pharmacological properties and tissue distribution.
Glutamate transport is a sodium- and potassium-coupled process capable of concentrating intracellular glutamate up to 10,000-fold compared with the extracellular environment. The stoichiometry of the process has been studied and at several models exist proposing various ionic exchanges. In one model derived from salamander retinal glial cells, the transport process is coupled to the co-transport of two sodium ions and the counter-transport of one potassium ion and one hydroxyl ion. (Bouvier et al. (1992), Nature 360:471-474). Another model proposes that with EAAC1, one glutamate is co-transported with three sodium ions and one hydrogen ion, with the counter-transport of one potassium ion (Zerangue et al., Nature (1996) 383:634-637). Yet another study suggests that two sodium ions are co-transported with one glutamate molecule (Hart et al., Science (1998) 280:2112-2114).
The cloning of glutamate transporter subtypes and detailed electrophysiological studies of these proteins reveals that glutamate transporters also possess channel-like properties. The conduct chloride flux is not thermodynamically coupled to substrate transport, although at transportable substrate is required for the chloride conductance. The binding of glutamate to the transporter may change its conformational state to form the chloride channel.
In addition to their possible role in development and learning (due to their potential for modulating normal synaptic transmission), the regulation of synaptic glutamate transporters is likely to play an important role in acute and chronic neurological processes. They can be involved through the disruption of synaptic transmission as well as through glutamate mediated excitotoxicity. Several diseases are associated with disruptions in glutamate transport.
Loss of cerebellar Purkinje cell is the hall mark of several inherited neurodegenerative diseases, including the trinucleotide repeat diseases such as spinocerebellar ataxia type 1 (SCA1), and is commonly associated with neurotoxicity of chronic ethanol ingestion, and with certain paraneoplastic neurological disorders. Although the molecular event that initiates the disease is known—a trinucleotide repeat—the cellular mechanisms responsible for Purkinje cell degeneration is not known. The selective loss of glutamate transporters such as EAAT4 could make the protein an attractive candidate for a downstream event.
Similarly, dysregulation of glutamate transporter EAAC1 could also have pathological consequences. EAAC1 has the unusual localization to GABA pre-synaptic terminals. This transport could serve as a precursor transporter, supplying extracellular glutamate for GABA re-synthesis. GABA normally is synthesized, via glutamate amino decarboxylase, from glutamate. The source of this glutamate has been traditionally thought to be cellular glutamate. However, the unique localization of the glutamate transporter to GABA terminals suggests that these transporters supply precurser glutamate for GABA re-synthesis. Thus, EAAC1 could serve as an important step in GABAergic neurotransmission. Modulation of GABAergic metabolism is associated with a number of neurological disorders, including epilepsy, tremors, and spasticity. In addition, some theories of schizophrenia include disturbances of glutamate and GABA metabolism.
Accordingly, there is a need in the art for compounds that regulate glutamate transport and in particular, compounds and molecules that interact with glutamate transporter proteins.
SUMMARY OF THE INVENTION
The present invention provides a family of proteins that interact with glutamate transporter proteins. Through their interaction with glutamate transporter proteins, Glutamate Transporter Associated Proteins modulate glutamate transport, and also effect cytoskeletal organization and stability as well as chloride flux.
In one embodiment of the invention, there is provided a substantially pure polypeptide characterized as modulating intracellular glutamate transport, interacting with a glutamate transporter protein, and having an expression pattern in the brain. In addition, the polypeptide can have at least one PDZ domain, at least one regulatory G-protein domain, at lest one pleckstrin homology domain, at least one proline-rich domain and at least one guanine exchange factor domain. The polypeptide can have at least one pleckstrin homology domain, at least one spectrin repeat and at least one &agr;-actinin domain.
In an additional embodiment of the invention, there is provided a substantially pure polypeptide characterized as modulating intracellular glutamate transport; interacting with a glutamate transporter protein; having an expression pattern in neural non-neuronal tissues; having at least one kinase C domains; having four transmembrane domains; and being hydrophobic.
In another embodiment of the invention, there is provided a substantially pure polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or conservative variants thereof.
In still another embodiment of the invention, there is provided an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2; (b) a polynucleotide of (a), wherein T can be U; (c) a polynucleotide complementary to (a) or (b); (d) a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1; (e) degenerate variants of (a), (b), (c) or (d); and (f) a fragment of (a), (b), (c), (d) or (e) having at least 15 base pairs and hybridizes to a polynucleotide encoding a polypeptide as set forth in SEQ ID NO:2.
In yet another embodiment of the invention, there is provided an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4; (b) a polynucleotide of (a), wherein T can be U; (c) a polynucleotide complementary to (a) or (b); (d) a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:3; (e) degenerate variants of (a), (b), (c) or (d); and (e) a fragment of (a), (b), (c), (d) or (e) having at least 15 base pairs and hybridizes to a polynucleotide encoding a polypeptide as set forth in SEQ ID NO:4.
In still another embodiment of the invention, there is provided an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:6; (b) a polynucleotide of (a), wherein T can be U; (c) a polynucleotide complementary to (a) or (b); (d) a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:5; (e) degenerate variants of (a), (b), (c) or (d); and (f) a fr
Jackson Mandy
Law Robert
Lin Glen
Orlov Irina
Rothstein Jeffrey D.
Gray Cary Ware & Freidenrich LLP
Kunz Gary
The Johns Hopkins University School of Medicine
Wegert Sandra
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