Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1993-12-22
2001-11-06
Prouty, Rebecca E. (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S252300, C435S325000, C435S440000
Reexamination Certificate
active
06313279
ABSTRACT:
BACKGROUND OF THE INVENTION
In the mammalian central nervous system, L-glutamate serves as a major excitatory neurotransmitter. The interaction of glutamate with its membrane-bound receptors is believed to play a role in many important neuronal processes, including, for example, fast synaptic transmission, synaptic plasticity and long-term potentiation. These processes are fundamental to the maintenance of life and normal human abilities such as learning and memory. Monaghan D. T. et al., 8
Neuron
267 (1992).
Pharmacological characterization of receptors for L-glutamate has led to their classification into two families based on their biological function: the ionotropic receptors which are directly coupled to cation channels in the cell membrane, and the metabotropic receptors which function through coupling to G-proteins. A number of ionotropic receptors have been further characterized on the basis of the relatively specific agonists by which they can be activated. One major group comprises those receptors activated by N-methyl-D-aspartate (NMDA), which appears to have multiple allosteric modulatory sites. The other two groups consist of those receptors activated by kainate and/or amino-3-hydroxy-5-methyl-4-isoxozole propionate (AMPA). Collingridge G. L. et al., 40 Pharmacol. Rev. 143 (1989).
Molecular cloning studies of rodent ionotropic receptors have recently provided some information on the molecular structure of these proteins. The cDNAs for seven different subtypes of the kainate/AMPA group have been characterized. Heinemann S. et al., PCT publication, W091/06648 (1991), Keinanen K. et al., 249
Science
556 (1990), Sakimura K. et al., 272
FEBS Lett.
73 (1990), Werner P. et al., 341
Nature
742 (1991), Bettler B. et al., 8
Neuron
257 (1992). Splice variants, referred to as “flip” and “flop”, of some of these have been characterized as well. Sommer B. et al., 249
Science
1580 (1990). In addition, one member of the NMDA group has been cloned. Moriyoshi, K. et al., 354
Nature
31 (1991). An NNDA-related protein has also been reported. Kumar K. N. et al., 354
Nature
70 (1991). These proteins share varying degrees of homology with one another and are therefore believed to represent a gene superfamily. Based on analogy with other better characterized ion channel receptors, glutamate ionotropic receptors are expected to exist in vivo within the cell membrane as multisubunit assemblies of these subunits. Unwin N., 3
Neuron
665 (1989).
Moreover, at least two human glutamate receptors have been reported as cloned. The reported human receptors differ slightly from the present invention. Puckett C. et al., 88
Proc. Nat. Acad. Sci.
7557 (1991) and Sun W. et al., 89
Proc. Nat. Acad. Sci.
1443 (1992). The glutamate receptor cloned by Puckett et al. was named GluHI and was later identified to be the “flip” version of this particular receptor. The Sun W. et al. reference refers to the glutamate receptor they cloned as the HBGR1 receptor and explains that HBGR1 is presumed the “flop” version of GluHI. Sun et al. also discloses a partial clone of HBGR2, or GluH2.
In addition to its role in normal human physiology, interaction of L-glutamate with its receptors is believed to play a key role in many neurological disorders such as stroke, epilepsy and head trauma, as well as neurodegenerative processes such as Alzheimer's disease. Olney R. W., 17
Drug Dev. Resa.,
299 (1999). For this reason, understanding the molecular structure of human L-glutamate receptors will be important for understanding these disease processes as well as furthering the search for effective therapeutic agents. Up to the present, the search for therapeutic agents which will selectively bind and modulate the function of human glutamate receptors has been hampered by the unavailability of homogeneous sources of receptors to use for screens and tests of potential drug candidate compounds. The brain tissues commonly used by pharmacologists presently are derived from experimental animals (non-human) and furthermore contain mixtures of various types of glutamate receptors.
In searching for drugs for human therapy it is desirable to use receptors that are more analogous to those in the intact human brain than are the rodent receptors employed to date. The current invention provides a human receptor which can be used to search for drugs which modulate these receptors.
SUMMARY OF THE INVENTION
The present invention provides amino acid compounds which comprise the isolated amino acid sequence SEQ ID NO:1. In particular, the amino acid compound which is SEQ ID NO: 1 is preferred.
The invention also provides nucleic acid compounds which comprise an isolated nucleic acid sequence which encodes the amino acid compounds provided. Particularly, nucleic acid compounds which are DNA are preferred. Most preferred is the DNA compound SEQ ID NO:2. However, also preferred are those nucleic acid compounds which are sense mRNA.
Also provided by the present invention are recombinant nucleic acid vectors comprising the nucleic acids which encode SEQ ID NO:1. Preferred nucleic acid vectors are those which are DNA. Most preferred are recombinant DNA vectors which comprise SEQ ID NO:2. The recombinant DNA vector most preferred is plasmid pRS103.
Moreover, recombinant DNA vectors of the present invention preferably comprise a promoter positioned to drive expression of said isolated DNA sequence. A preferred recombinant DNA expression vector is one wherein the promoter functions in mammalian cells. A more preferred recombinant DNA expression vector is one wherein the promoter functions in COS-7 cells. Most preferred COS-7 cell DNA expression vectors further comprise SEQ ID NO:2.
Restriction fragments of the preferred vector are also provided. Particularly, the 4.2 kb (kilobase) EcoRI/Kpn1 and the 2.8 kb EcoRI/ClaI restriction fragment of pRS103 are provided.
The present invention also provides probes and primers useful for molecular biology techniques. Compounds which encode for SEQ ID NO:1 or a part thereof and which are at least 17 base pairs in length are provided. Preferably, the 17 base pair or more compound is DNA. Most preferred for this use are the DNA compounds which are SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
Further, this invention provides cells in which the nucleic acid compounds of the invention may be harbored. Oocytes wherein nucleic acid compounds of the invention express functional HSGluR1 receptor are provided. Moreover, oocytes wherein nucleic acids of the present invention express functional HSGluR1 receptor and wherein functional GluR2 receptor is also expressed is provided. Oocytes wherein nucleic acids of the present invention express functional HSGluR1 receptor and wherein functional GluR2 receptor is co-expressed, and wherein functional GluR3 receptor is additionally expressed is also provided. An oocyte wherein DNA expresses functional HSGluRl receptor is preferred. Most preferred is an oocyte wherein sense mRNA expresses functional HSGluR1 receptor.
Other host cells provided by the present invention include those which are transfected with a nucleic acid compound which encodes SEQ ID NO:1. Preferred cells include host cells transfected with a recombinant DNA vector. Preferred transfected host cells which encodes SEQ ID NO:1 are
E. coli
cells. The most preferred
E. coli
host cell is
E. coli
/pRS103.
Host cells which are transfected with a DNA vector which further comprise a promoter positioned to drive expression of functional HSGluR1 receptor are also provided. Preferably, the DNA vector comprises SEQ ID NO:2. Preferred host cells for expression of functional HSGluR1 are mammalian cells. Preferred mammalian cells for expression of functional HSGluR1 are COS-7 cells. Specifically, COS-7 cells which have been transfected with a DNA expression vector which expresses a functional HSGluR1 receptor and which further comprise a DNA vector which encodes a functional GluR2 receptor are provided. COS-7 cells which have been transfected with an DNA expression vector which expresses a functional HSGluR
Burnett J. Paul
Mayne Nancy G.
Sharp Robert L.
Snyder Yvonne M.
Eli Lilly and Company
Gaylo Paul J.
Prouty Rebecca E.
Wilson Alexander
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