Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Rodent cell – per se
Reexamination Certificate
1996-09-09
2001-04-10
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Rodent cell, per se
C435S069100, C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06214615
ABSTRACT:
This invention relates to the molecular cloning and expression of neurotransmitter receptors. In particular, it relates to transformed or transfected cells and cell lines expressing genes for a class of human dopamine D2 receptors, the proteins comprising the receptors, the DNA encoding such proteins, vectors for transforming or transfecting cells with such DNA, methods of isolating the DNA and preparing the cell lines, and methods of using the cells and cell lines.
BACKGROUND OF THE INVENTION
Neurons communicate with various target cells by the release of neurotransmitters. Neurotransmitters exert their effects by binding to sites called receptors that are located on the extracellular surface of their respective target cells. Numerous neurotransmitters have been identified, one of which is dopamine. Schizophrenia and Parkinson's disease are believed to be related at least in part to disturbances in the operation of dopamine and its receptors.
A major function of a neurotransmitter receptor is to transmit information into the interior of a target cell, causing various rapid responses of the cell. One of the major classes of receptors is known as G protein-coupled receptors. They act by coupling to a family of signal-transducing proteins located on the cytoplasmic surface of the plasma membrane. These proteins are called G proteins because they bind guanine nucleotides when activated by neurotransmitter receptors. After activation, G proteins are able to regulate a variety of cellular events, including the activity of ion channels and enzymes.
Several of the genes that code for mammalian G protein-coupled receptors have been cloned. These include the alpha-2 and beta-2 adrenergic receptors, five subtypes of muscarinic acetylcholine receptors, three subtypes of serotonin receptors, the substance K receptor, and a rat dopamine D2 receptor. All of the G protein-coupled neurotransmitter receptors cloned to date have a predicted structure having seven transmembrane domains and several regions of strong homology, suggesting that they may have arisen from a common ancestral gene.
The cloning of subtypes of some of these neurotransmitter receptors indicates a molecular basis for pharmacological heterogeneity. For example, several subtypes of muscarinic, serotonergic, and adrenergic receptors have now been cloned. See Bonner et al.,
Science,
237: 527-532 (1987); Bonner et al.,
Neuron,
1: 403-410 (1988); Peralta et al.,
EMBO J.,
6: 3923-3929 (1987); Kubo et al.,
Nature,
323: 411-416 (1986); Julius et al.,
Science,
241: 558-564 (1988); Fargin et al.,
Nature,
335: 358-360 (1988); Kobilka et al.,
Science,
228: 650-656 (1987); and Dixon et al.,
Nature,
321: 75-79 (1986), all of which are incorporated herein by reference. These receptor subtypes are expressed in distinct regions of the brain and body.
The cloning of the muscarinic acetylcholine receptors provides an example of how the general technique of homology screening was applied to the identification of a family of genes encoding a G protein-coupled receptor. See Bonner et al.,
Science,
237: 527-532 (1987) and Bonner et al.,
Neuron,
1: 403-410 (1988), both of which are incorporated herein by reference.
In the work reported in
Science,
the authors made a probe from the highly conserved transmembrane region near the 5′ region of the porcine brain muscarinic receptor gene with certain modifications. The probe was used to screen a rat cerebral cortex cDNA library of clones in the pCD mammalian expression vector. The clones were characterized before isolation by using Southern blots of portions of the library. Dilutions were done to obtain a single band by Southern blotting. The cDNA inserts were transfected into mouse fibroblast (A9) or COS-7 cells. The transfected cells were assayed for the ability of the cell membranes to bind a muscarinic antagonist. The authors found four different muscarinic receptor genes.
The
Neuron
article reports the cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes in an approach similar to that in the
Science
article. A human genomic library was screened using the rat m1 receptor gene as a probe, which identified the human m5 receptor gene. The homologous rat gene was isolated from a rat genomic library, using a portion of the human gene as a probe. The coding portion of the human gene was inserted into the plasmid pCD-PS, a mammalian expression vector that utilizes an SV40 promoter, and expressed in CHO cells.
On the basis of functional and pharmacological data, central dopamine receptors have been divided into D1 and D2 subtypes. Dopamine D1 and D2 receptors stimulate and inhibit adenylate cyclase, respectively, by coupling to different G proteins. See Kebabian et al.,
Nature,
277: 93-96 (1979), incorporated herein by reference. D2 receptors are known to be abundant within various forebrain regions innervated by dopaminergic neurons projecting from the mesencephalon. See Bjorklund and Lindvall, “Dopamine Containing Systems in the CNS,” in
Handbook of Chemical Neuroanatomy Vol.
2:
Classical Transmitters in the CNS,
Bjorklund and Hokfelt, eds., (Elsevier, Amsterdam, 1984), pp. 55-122; Fuxe et al, “Dopaminergic Systems in the Brain and Pituitary,” in
Basic and Clinical Aspects of Neuroscience,
Fluckiger et al., eds., (Springer Verlag & Sandox, Heidelberg, 1985), pp. 11-25; Anden et al.,
Life Sci.,
3: 523-530 (1964); Anden et al.,
Life Sci.,
4: 1275-1279 (1965); and Dahlstrom et al.,
Acta. Physiol. Scand.,
62: 485-486 (1964), all of which are incorporated herein by reference. D2 receptors have also been shown to be presynaptically located on dopaminergic neurons, where they modulate dopamine release. See Roth,
Ann N.Y. Acad. Science,
430: 27-53 (1984) and White et al.,
J. Pharm. Exp. Ther.,
231: 275-280 (1986), both of which are incorporated herein by reference.
Abnormalities in central dopaminergic function have been implicated in the pathophysiology of schizophrenia and Parkinson's disease. See Stevens,
Arch. Gen. Psychiatry,
29: 177-189 (1973); Synder,
Amer. J. Psychiatry,
133: 197-202 (1976); and Hornykiewicz,
Wien. Klin. Wochenschr.,
75: 309-312, all of which are incorporated herein by reference. Schizophrenia is treated with antipsychotic drugs which block central D2 receptors, and Parkinson's disease is treated by drugs which lead to their stimulation. See Baldessarini, “Drugs and the Treatment of Psychiatric Disorders,” in
The Pharmacological Basis of Therapeutics,
Gilman et al., eds., (Macmillan, New York, 1985) pp. 387-445; Bianchine et al., “Drugs for Parkinson's Disease, Spasticity, and Acute Muscle Spasms,” in
The Pharmacoliogical Basis of Therapeutics,
Gilman et al., eds., (Macmillan, New York, 1985) pp. 473-490; Iversen et al.,
Science,
188: 1084-1089 (1975); and Seeman et al.,
Nature,
261: 717-719 (1976), all of which are incorporated herein by reference.
Prolonged treatment with antipsychotic agents is associated with extrapyramidal (motor) side effects such as tardive dyskinesia. Since the motor side effects are believed to be mediated by receptors located within the striatum, and since antipsychotic activity is believed to be mediated by sites within limbic and cortical regions, investigation of a potential heterogeneity of D2 receptors between these brain regions has become a focus for drug development. See Borison et al.,
Brain Res. Bull.,
11: 215-218 (1983); Leonard et al.,
Biochem. J.,
248: 595-602 (1987); and Baldessarini et al.,
Annu. Rev. Neurosci.,
3: 23-41 (1980), all of which are incorporated herein by reference.
Dopamine is the major catecholamine present in retina, where its synthesis and release is stimulated by light. Invone et al.,
Science,
202:901-902 (1978), incorporated herein by reference. As in the brain, retinal dopamine receptors have been divided into two subtypes, based on their pharmacological and functional properties. See Elena et al.,
Curr. Eye Res.,
8:75-83 (1989) and Qu et al.,
J. Pharm. Exp. Ther.,
248:621-625 (1989), both of which a
Brann Mark R.
Stormann Thomas M.
Fitch Even Tabin & Flannery
Gucker Stephen
Kunz Gary L.
The United States of America as represented by the Department of
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