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
1999-10-25
2003-04-29
Kemmerer, Elizabeth (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...
C435S366000, C435S320100, C435S252300, C435S325000, C536S023500, C536S023100
Reexamination Certificate
active
06555341
ABSTRACT:
This specification claims the benefit of PCT/GB/01206, filed Apr. 24, 1998, and GB Application 9708479.2, filed Apr. 25, 1997.
This invention concerns the cloning of a novel cDNA sequence encoding a particular subunit of the human GABA
A
receptor. In addition, the invention relates to a stable cell line capable of expressing said cDNA and to the use of the cell line in a screening technique for the design and development of subtype-specific medicaments.
Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. It mediates fast synaptic inhibition by opening the chloride channel intrinsic to the GABA
A
receptor. This receptor comprises a multimeric protein of molecular size 230-270 kDa with specific binding sites for a variety of drugs including benzodiazepines, barbiturates and &bgr;-carbolines, in addition to sites for the agonist ligand GABA (for reviews see MacDonald and Olsen,
Ann. Rev. Neurosci
., 1994, 17, 569; and Whiting et al,
Int. Rev. Neurobiol
., 1995, 38, 95).
Molecular biological studies demonstrate that the receptor is composed of several distinct types of subunit, which are divided into four classes (&agr;, &bgr;, &ggr; and &dgr;) based on their sequence similarities. To date, in mammals, six types of &agr; (Schofield et al.,
Nature
(London), 1987, 328, 221; Levitan et al.,
Nature
(London), 1988, 335, 76; Ymer et al.,
EMBO J
., 1989, 8, 1665; Pritchett & Seeberg,
J. Neurochem
., 1990, 54, 802; Luddens et al.,
Nature
(London), 1990, 346, 648; and Khrestchatisky et al.,
Neuron
, 1989, 3, 745), three types of &bgr; (Ymer et al.,
EMBO J
., 1989, 8, 1665), three types of &ggr; (Ymer et al.,
EMBO J
., 1990, 9, 3261; Shivers et al.,
Neuron
, 1989, 3, 327: and Knoflach et al,
FEBS Lett
., 1991, 293, 191) and one &dgr; subunit (Shivers et al.,
Neuron
, 1989, 3, 327) have been identified. More recently, a further member of the GABA receptor gene family, &egr;, has been identified (Davies et al,
Nature
, 1997, 385, 820). The polypeptide is 506 amino acids in length and exhibits greatest amino acid sequence identity with the GABA
A
receptor &ggr;
3
subunit (47%), although this degree of homology is not sufficient for it to be classified as a fourth &ggr; subunit.
The differential distribution of many of the subunits has been characterised by in situ hybridisation (Shivers et al.,
Neuron
, 1989, 3, 327; Wisden et al,
J. Neurosci
., 1992, 12, 1040; and Laurie et al,
J. Neurosci
, 1992, 12, 1063) and this has permitted it to be speculated which subunits, by their co-localisation, could theoretically exist in the same receptor complex.
Various combinations of subunits have been co-transfected into cells to identify synthetic combinations of subunits whose pharmacology parallels that of bona fide GABA
A
receptors in vivo (Pritchett et al.,
Science
, 1989, 245, 1389; Pritchett and Seeberg,
J. Neurochem
., 1990, 54, 1802; Luddens et al.,
Nature
(London), 1990, 346, 648; Hadingham et al,
Mol. Pharmacol
., 1993, 43, 970; and Hadingham et al.,
Mol. Pharmacol
., 1993, 44, 1211). This approach has revealed that, in addition to an &agr; and &bgr; subunit, either &ggr;
1
or &ggr;
2
(Pritchett et al.,
Nature
(London), 1989, 338, 582; Ymer et al.,
EMBO J
., 1990, 9, 3261; and Wafford et al.,
Mol. Pharmacol
., 1993, 44, 437) or &ggr;
3
(Herb et al.,
Proc. Natl. Acad. Sci. USA
, 1992, 89, 1433; Knoflach et al.,
FEBS Lett
., 1991, 293, 191; and Wilson-Shaw et al.,
FEBS Lett
., 1991, 284, 211) is also generally required to confer benzodiazepine sensitivity, and that the benzodiazepine pharmacology of the expressed receptor is largely dependent on the identity of the &agr; and &ggr; subunits present. Receptors containing a &dgr; subunit (i.e. &agr;&ggr;&dgr;) do not appear to bind benzodiazepines (Shivers et al.,
Neuron
, 1989, 3, 327; and Quirk et al.,
J. Biol. Chem
., 1994, 269, 16020). Combinations of subunits have been identified which exhibit the pharmacological profile of a BZ
1
type receptor (&agr;
1
&bgr;
1
&ggr;
2
) and a BZ
2
type receptor (&agr;
2
&bgr;
1
&ggr;
2
or &agr;
3
&bgr;
1
&ggr;
2
, Pritchett et al.,
Nature
(London), 1989, 338, 582), as well as GABA
A
receptors with a novel pharmacology, &agr;
5
&bgr;
2
&ggr;
2
(Pritchett and Seeberg,
J. Neurochem
., 1990, 54, 1802), &agr;
4
&bgr;
2
&ggr;
2
(Wisden et al,
FEBS Lett
., 1991, 289, 227) and &agr;
6
&bgr;
2
&ggr;
2
(Luddens et al.,
Nature
(London), 1990, 346, 648). The pharmacology of these expressed receptors appears similar to that of those identified in brain tissue by radioligand binding, and biochemical expperiments have begun to determine the subunit composition of native GABA receptors (McKernan & Whiting,
Tr. Neurosci
., 1996, 19, 139). The exact structure of receptors in vivo has yet to be definitively elucidated.
The present invention relates to a new class of GABA receptor subunit, hereinafter referred to as the theta subunit (&thgr; subunit).
The nucleotide sequence for the theta subunit, together with its deduced amino acid sequence corresponding thereto, is depicted in
FIG. 1
of the accompanying drawings.
The present invention accordingly provides, in a first aspect, a DNA molecule encoding the theta subunit of the human GABA receptor comprising all or a portion of the sequence depicted in
FIG. 1
, or a modified human sequence.
In an alternative aspect, the present invention provides a DNA molecule encoding the theta subunit of the human GABA receptor comprising all or a portion of the sequence depicted in
FIG. 2
, or a modified human sequence.
The term “modified human sequence” as used herein referes to a variant of the DNA sequences depicted in FIG.
1
and FIG.
2
. Such variants may be naturally occuring allelic variants or non-naturally occuring or “engineered” variants. Allelic variation is well known in the art in which the nucleotide sequence may have a substitution, deletion or addition of one or more nucleotides without substantial alteration of the function of the encoded polypeptide. Particularly preferred allelic variants arise from nucleotide substitution based on the degeneracy of the genetic code.
The sequencing of the novel cDNA molecules in accordance with the invention can conveniently be carried out by the standard procedure described in accompanying Example 1; or may be accomplished by alternative molecular cloning techniques which are well known in the art, such as those described by Maniatis et al. in
Molecular Cloning, A Laboratory Manual
, Cold Spring Harbor Press, New York, 2nd edition, 1989.
In a further aspect, the present invention also relates to polynucleotides (for example, cDNA, genomic DNA or synthetic DNA) which hybridize under stringent conditions to the DNA molecules depicted in FIG.
1
and FIG.
2
. As used herein, the term “stringent conditions” will be understood to require at least 95% and preferably at least 97% identity between the hybridized sequences. Polynucleotides which hybridize under stringent conditions to the DNA molecules depicted in FIG.
1
and
FIG. 2
preferably encode polypeptides which exhibit substantially the same biological activity or function as the polypeptides depicted in FIG.
1
and
FIG. 2
, respectively.
The present invention further relates to a GABA theta subunit polypeptide which has the deduced amino acid sequence of
FIG. 1
or
FIG. 2
, as well as fragments, analogs and derivatives thereof.
The terms “fragment”, “derivative” and “analog” when referring to the polypeptide of
FIG. 1
or
FIG. 2
, means a polypeptide which retains essentially the same biological activity or function as the polypeptide depicted in
FIG. 1
or FIG.
2
. Thus, an analog may be, for example, a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polypeptide of
FIG. 1
or
FIG. 2
may be one in w
Bonnert Timothy Peter
Whiting Paul John
Kemmerer Elizabeth
Merck Sharp & Dohme Ltd.
Tribble Jack L.
Wegert Sandra
Yablonsky Michael D.
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