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
2000-10-24
2003-03-18
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S252300, C435S320100, C435S325000, C536S023500, C530S350000
Reexamination Certificate
active
06534287
ABSTRACT:
2. FIELD OF THE INVENTION
The present invention relates to nucleic acid sequences coding for a newly identified splice variant of human metabotropic glutamate receptor 5 (mGluR5). The novel human receptor may be expressed in host cells which may be used to screen for agonist, antagonist, and modulatory molecules that act on the novel human mGluR5. These molecules acting on the novel human mGluR can be used to modulate the activity of the novel human receptor for the treatment of neurological disorders and diseases.
The invention also relates to nucleic acids encoding such receptors, genetically modified cells containing such nucleic acids, methods of screening for compounds that bind to or modulate the activity of such receptors, and methods of use relating to all of the foregoing.
3. BACKGROUND OF THE INVENTION
The following description provides a summary of information related to the background of the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to that invention.
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Glutamate produces its effects on central neurons by binding to and thereby activating cell surface receptors. These receptors have been subdivided into two major classes, the ionotropic and metabotropic glutamate receptors, based on the structural features of the receptor proteins, the means by which the receptors transduce signals into the cell, and pharmacological profiles.
The ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that, upon binding glutamate, open to allow the selective influx of certain monovalent and divalent cations, thereby depolarizing the cell membrane. In addition, certain iGluRs with relatively high calcium permeability can activate a variety of calcium-dependent intracellular processes. These receptors are multisubunit protein complexes that may be homomeric or heteromeric in nature. The various iGluR subunits all share common structural motifs, including a relatively large amino-terminal extracellular domain (ECD), followed by two transmembrane domains (TMD), a second smaller extracellular domain, and a third TMD, before terminating with an intracellular carboxy-terminal domain. Historically the iGluRs were first subdivided pharmacologically into three classes based on preferential activation by the agonists &agr;-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate (KA), and N-methyl-D-aspartate (NMDA). Later, molecular cloning studies coupled with additional pharmacological studies revealed a greater diversity of iGluRs, in that multiple subtypes of AMPA, KA and NMDA receptors are expressed in the mammalian CNS Hollman & Heinemann (1994),
Ann. Rev. Neurosci
. 17:31.
The metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors capable of activating a variety of intracellular second messenger systems following the binding of glutamate.
Activation of mGluRs in intact mammalian neurons can elicit one or more of the following responses: activation of phospholipase C, increases in phosphoinositide (PI) hydrolysis, intracellular calcium release, activation of phospholipase D, activation or inhibition of adenylyl cyclase, increases or decreases in the formation of cyclic adenosine monophosphate (cAMP), activation of guanylyl cyclase, increases in the formation of cyclic guanosine monophosphate (cGMP), activation of phospholipase A
2
, increases in arachidonic acid release, and increases or decreases in the activity of ion channels (e.g., voltage- and ligand-gated ion channels). Schoepp & Conn (1993),
Trends Pharmacol. Sci
. 14:13; Schoepp (1994),
Neurochem. Int
. 24:439; Pin & Duvoisin (1995),
Neuropharmacology
34:1.
Thus far, eight distinct mGluR subtypes have been isolated via molecular cloning, and named mGluR1 to mGluR8 according to the order in which they were discovered. Nakanishi (1994),
Neuron
13:1031; Pin & Duvoisin (1995),
Neuropharmacology
34:1; Knopfel et al. (1995),
J Med. Chem
. 38:1417. Further diversity occurs through the expression of alternatively spliced forms of certain mGluR subtypes. Pin et al. (1992),
Proc. Natl. Acad. Sci
. USA 89:10331; Minakami et al. (1994),
BBRC
199:1136; Joly et al. (1995),
J Neurosci
. 15:3970. All of the mGluRs are structurally similar, in that they are single subunit membrane proteins possessing a large amino-terminal ECD, followed by seven putative TMDs, and an intracellular carboxy-terminal domain of variable length.
The eight mGluRs have been subdivided into three groups based on amino acid sequence homologies, the second messenger systems they utilize, and pharmacological characteristics. Nakanishi (1994),
Neuron
13:1031; Pin & Duvoisin (1995),
Neuropharmacology
34:1; Knopfel et al. (1995),
J Med. Chem
. 38:1417. The amino acid homology between mGluRs within a given group is approximately 70%, but drops to about 40% between mGluRs in different groups. For mGluRs in the same group, this relatedness is roughly paralleled by similarities in signal transduction mechanisms and pharmacological characteristics.
The Group I mGluRs comprise mGluR1, mGluR5, and their alternatively spliced variants. The binding of agonists to these receptors results in the activation of phospholipase C and the subsequent mobilization of intracellular calcium. For example, Xenopus oocytes expressing recombinant mGluR1 receptors have been utilized to demonstrate this effect indirectly by electrophysiological means. Masu et al. (1991),
Nature
349:760; Pin et al. (1992),
Proc. Natl. Acad. Sci. USA
89:10331. Similar results were achieved with oocytes expressing recombinant mGluR5 receptors. Abe et al. (1992),
J Biol. Chem
. 267:13361; Minakami et al. (1994),
BBRC
199:1136; Joly et al. (1995),
J Neurosci
. 15:3970. Alternatively, agonist activation of recombinant mGluR1 receptors expressed in Chinese hamster ovary (CHO) cells stimulated PI hydrolysis, cAMP formation, and arachidonic acid release as measured by standard biochemical assays. Aramori & Nakanishi (1992),
Neuron
8:757. In comparison, activation of mGluR5 receptors expressed in CHO cells stimulated PI hydrolysis and subsequent intracellular calcium transients, but no stimulation of cAMP formation or arachidonic acid release was observed. Abe et al. (1992),
J Biol. Chem
. 267:13361. However, activation of mGluR5 receptors expressed in LLC-PK1 cells does result in increased cAMP formation as well as PI hydrolysis. Joly et al. (1995),
J Neurosci
. 15:3970. The agonist potency profile for Group I mGluRs is quisqualate>glutamate=ibotenate>(2S,1′S,2′S)-2-carboxycyclopropyl)glycine (L-CCG-I)>(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). Quisqualate is relatively selective for Group I receptors, as compared to Group II and Group III mGluRs, but it also potently activates ionotropic AMPA receptors. Pin & Duvoisin (1995),
Neuropharmacology
34:1; Knopfel et al. (1995),
J Med. Chem
. 38:1417.
The Group II mGluRs include mGluR2 and mGluR3. Activation of these receptors as expressed in CHO cells inhibits adenlyl cyclase activity via the inhibitory G protein, G
i
, in a pertussis toxin-sensitive fashion. Tanabe et al. (1992),
Neuron
8:169; Tanabe et al. (1993),
J. Neurosci
. 13:1372. The agonist potency profile for Group II receptors is L-CCG-1>glutamate>A CPD>ibotenate>quisqualate. Preliminary studies suggest that L-CCG-I and (2S,1′R,2′R, 3′R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) are both relatively selective agonists for the Group II receptors versus other mGluRs (Knopfel et al. (1995),
J. Med. Chem
. 38:1417), but DCG-IV does exhibit agonist activity at iGluRs as well (Ishida et al. (1993),
Br. J. Pharmacol
. 109:1169).
The Group III mGluRs include mGluR4, mGluR6, mGluR7 and mGluR8. Like the Group II receptors, these mGluRs are negatively coupled to adenylyl cyclase to inhibit intracellular cAM
Hammerland Lance G.
Krapcho Karen J.
Levinthal Cynthia
Stormann Thomas M.
Jiang Dong
Madson & Metcalf
NPS Pharmaceuticals Inc.
Spector Lorraine
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