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
2000-12-08
2004-02-10
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...
C435S320100, C435S325000, C435S252300, C435S006120, C530S350000, C536S023500
Reexamination Certificate
active
06689585
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to isolated polynucleotide molecules which encode a novel transmembrane G-protein coupled receptor designated GABA-B2. The novel receptor appears to be activated by the neurotransmitter y-amino butyric acid (GABA).
BACKGROUND OF THE INVENTION
&ggr;-amino butyric acid (GABA) is the principal inhibitory neurotransmitter in the brain, whose action is mediated by two types of receptors, GABA-A and GABA-B. GABAergic inhibitory neurons typically form short pathways (e.g. from striatum to substantia nigra and from cerebellar cortex to deep cerebellar nuclei), although at least one long pathway projecting from the posterior hypothalamus to the cerebral cortex has also been recognized. This long pathway is believed to provide a direct pathway by which limbic, emotional and visceral information may be transferred to the cortex (Vincent et al.,
Science
220: 1309-1311, 1993).
GABA-B receptors, such as the GABA-B1
a
and GABA-B1
b
receptors, are predominantly present in the brain where they are believed to play a major role in learning and memory. In view of these functions and the known benefit of GABA-B agonists (e.g. baclofen) in the treatment of spasticity, anxiety and depression, there is considerable interest in isolating genes encoding GABA-B receptor subtypes so as to, enable the recombinant production of GABA-B receptors for the development of novel therapeutics.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides an isolated polyniucleotide molecule encoding a GABA-B2 receptor or a functionally equivalent fragment thereof.
Preferably, the encoded GABA-B2 receptor is characterised by the N-terminal amino acid sequence: MASPRSSGQP (SEQ ID NO: 1)
More preferably, the isolated polynucleotide molecule encodes a human GABA-B2 receptor of about 941 amino acids.
Most preferably, the isolated polynucleotide molecule encodes a GABA-B2 receptor having an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 2.
The polynucleotide molecule of the first aspect may comprise a nucleotide sequence substantially corresponding to, or showing at least 90% (more preferably, at least 95%) homology to that shown at nucleotides 1 to 3256 or nucleotides 140 to 2962 of SEQ ID NO: 3 or any portion thereof encoding a functionally equivalent GABA-B2 receptor fragment.
The isolated polynucleotide molecule may be incorporated into plasmids or expression vectors (including viral vectors), which may then be introduced into suitable bacterial, yeast, insect and mammalian host cells. Such host cells may be used to express the GABA-B2 receptor encoded by the isolated polynucleotide molecule.
Accordingly, in a second aspect, the present invention provides a mammalian, insect, yeast or bacterial host cell transformed with the polynucleotide molecule of the first aspect.
In a third aspect, the present invention provides a method of producing GABA-B2 receptors or functionally equivalent fragments thereof, comprising culturing the host cell of the second aspect under conditions enabling the expression of GABA-B2 receptors or functionally equivalent fragments thereof.
Preferably, the host cell is mammalian or of insect origin. Where the cell is mammalian, it is presently preferred that it be a Chinese hamster ovary (CHO) cell, monkey kidney (COS) cell or human embryonic kidney 293 cell. Where the cell is of insect origin, it is presently preferred that it be an insect Sf9 cell.
In a preferred embodiment, the GABA-B2 receptors or fragments thereof are expressed onto the surface of the host cell.
By using the polynucleotide molecule of the present invention it is possible to obtain GABA-B2 receptor protein or fragments thereof in a substantially pure form.
Accordingly, in a fourth aspect, the present invention provides a GABA-B2 receptor or a functionally equivalent fragment of said receptor, in a substantially pure form.
In a fifth aspect, the present invention provides an antibody capable of specifically binding to the GABA-B2 receptor of the fourth aspect. Such antibodies may be produced by any of the methods routine to the art.
In a sixth aspect, the present invention provides a non-human animal transformed with a polyniucleotide molecule according to the first aspect of the present invention.
In a seventh aspect, the present invention provides a method for detecting agonist or antagonist agents of a GABA-B2 receptor, comprising contacting a GABA-B2 receptor, functionally equivalent fragment thereof or a cell transfected with and expressing the polynucleotide molecule of the first aspect, with a test agent under conditions enabling the activation of a GABA-B2 receptor, and detecting an increase or decrease in activity of the GABA-B2 receptor or functionally equivalent fragment thereof.
An increase or decrease in activity of the receptor or functionally equivalent fragment thereof may be detected by measuring changes in cAMP production, Ca
2+
levels or IP3 turnover after activating the receptor molecule with specific agonists or antagonists.
In a further aspect, the present invention provides an oligonucleotide or polynucleotide probe comprising a nucleotide sequence of 10 or more nucleotides, the probe comprising a nucleotide sequence such that the probe specifically hybridises to the polynucleotide molecule of the first aspect under high stringency conditions (Samibrook et al.,
Molecular cloning: a laboratory manual
, Second Edition, Cold Spring Harbor Laboratory Press).
In a still further aspect, the present invention provides an antisense oligonucleotide or polynucleotide molecule comprising a nucleotide sequence capable of specifically hybridising to an mRNA molecule which encodes a GABA-B2 receptor so as to prevent translation of the mRNA molecule.
Such antisense oligonucleotide or polynucleotide molecules may include a ribozyme region to catalytically inactivate mRNA to which it is hybridised.
The polynucleotide molecule of the first aspect of the invention may be a dominant negative mutant which encodes a gene product causing an altered phenotype by, for example, reducing or eliminating the activity of endogenous GABA-B2 receptors.
The term “substantially corresponding” as used herein in relation to amino acid sequences is intended to encompass minor variations in the amino acid sequences which do not result in a decrease in biological activity of the GABA-B2 receptor. These variations may include conservative amino acid substitutions. The substitutions envisaged are:
G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, N&agr;-alkalamiino acids.
The term “substantially corresponding” as used herein in relation to nucleotide sequences is intended to encompass minor variations in the nucleotide sequences which due to degeneracy in the DNA code do not result in a change in the encoded protein. Further, this term is intended to encompass other minor variations in the sequence which may be required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded protein.
The term “functionally equivalent fragment/s” as used herein is intended to refer to fragments of the GABA-B2 receptor that exhibit binding specificity and activity that is substantially equivalent to the GABA-B2 receptor from which it/they is/are derived.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Reference to percent homology made in this specification have been calculated using the BLAST program blastn as described by Altschul, S. F. et al., “Capped BLAST and PSI-BLAST: a new generation of protein database search programs”,
Nucleic Acids Research
, Vol. 25, No. 17, pp. 3389-3402 (1997).
REFERENCES:
Nature 396: 679-682 (1998) by White JH e
Clark & Brody
Jiang Dong
Spector Lorraine
The Garvan Institute of Medical Research
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