Receptor

Details Receptor Receptor

1999-09-03

2003-02-11

Eyler, Yvonne (Department: 1646)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C435S006120, C435S007210, C435S069100, C435S252300, C435S320100, C435S325000, C514S002600, C536S023500, C436S501000

Reexamination Certificate

active

06518399

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the novel GABA
B
receptor subtypes GABA
B
-R1c and GABA
B
-R2 as well as to a novel, functional GABA
B
receptor which comprises a heterodimer of GABA
B
-R1 and GABA
B
-R2 receptor subunits. The present invention also relates to variants of the receptors, nucleotide sequences encoding the receptors and variants thereof and novel vectors, stable cell lines, antibodies, screening methods, methods of treatment and methods of receptor production.
BACKGROUND OF THE INVENTION
GABA (&ggr;-amino-butyric acid) is the main inhibitory neurotransmitter in the central nervous system (CNS) activating two distinct families of receptors; the ionotropic GABA
A
and GABA
C
receptors for fast synaptic transmissions, and the metabotropic GABA
B
receptors governing a slower synaptic transmission. GABA
B
receptors are members of the superfamily of 7-transmembrane G protein-coupled receptors. Activation results in signal transduction through a variety of pathways mediated principally via members of the G
i
/G
o
family of pertussis toxin-sensitive G proteins. GABA
B
receptors have been shown to inhibit N, P/Q and T-type Ca
2+
channels in a pertussis toxin-sensitive manner (Kobrinsky et al., 1993; Menon-Johansson et al., 1993; Harayama et al., 1998) and indeed there is also some evidence for direct interactions between GABA
B
receptors and Ca
2+
channels since Ca
2+
channel ligands can modify the binding of GABA
B
agonists (Ohmori et al., 1990). GABA
B
receptor-mediated Ca
2+
channel inhibition is the principle mechanism for presynaptic inhibition of neurotransmitter release. Post-synaptically the major effect of GABA
B
receptor activation is to open potassium channels, to generate post-synaptic inhibitory potentials. Autoradiographic studies show that GABA
B
receptors are abundant and heterogeneously distributed throughout the CNS, with particularly high levels in the molecular layer of the cerebellum, interpeduncular nucleus, frontal cortex, olfactory nuclei and thalamic nuclei. GABA
B
receptors are also widespread in the globus pallidus, temporal cortex, raphe magnus and spinal cord (Bowery et al., 1987). GABA
B
receptors are an important therapeutic target in the CNS for conditions such as spasticity, epilepsy, Alzheimer's disease, pain, affective disorders and feeding. GABA
B
receptors are also present in the peripheral nervous system, both on sensory nerves and on parasympathetic nerves. Their ability to modulate these nerves gives them potential as targets in disorders of the lung, GI tract and bladder (Kerr and Ong, 1995; 1996; Malcangio and Bowery, 1995).
Despite the widespread abundance of GABA
B
receptors, considerable evidence from neurochemical, electrophysiological and behavioural studies suggests that multiple subtypes of GABA
B
receptors exist. This heterogeneity of GABA
B
receptors may allow the development of selective ligands, able to target specific aspects of GABA
B
receptor function. This would lead to the development of drugs with improved selectivity profiles relative to current compounds (such as baclofen) which are relatively non-selective and show a variety of undesirable behavioural actions such as sedation and respiratory depression. Multiple receptor subtypes are best classified by the differing profiles of agonist and antagonist ligands.
To date screening for GABA
B
ligands and subsequent structure/activity determinations has relied on radioligand binding assays to rat brain membranes. Further analysis of such ligands in animal models has indicated differences in their behavioural profile. However, due to the absence of cloned GABA
B
receptors the molecular basis for such differences has not been defined, and therefore it has not been possible to optimise GABA
B
ligands for therapeutic use.
GABA
B
receptors were first described nearly 20 years ago (Hill and Bowery, 1981), but despite extensive efforts using conventional expression cloning strategies, for example in Xenopus oocytes, or cloning based on sequence homology, the molecular nature of the GABA
B
receptor remained elusive. The development of a high affinity antagonist for the receptor finally allowed Kaupmann et al, (1997) to expression clone the receptor from a rat cerebral cortex cDNA using a radioligand binding assay. Two splice variants of the receptor were identified, GABA
B
-R1a encoding a 960 amino acid protein and GABA
B
-R1b, encoding an 844 amino acid protein, differing only in the lengths of their N-termini. These two splice variants have distinct spatial distributions within the brain, but both reside within neuronal rather than glial cells. Pharmacologically, the two splice variants are similar, showing binding affinities for a range of antagonists, but about 10 fold lower than those of native receptors, as well as agonist displacement constants which are about 100-150 a fold lower than those of native receptors. These observations have led to speculation that the cloned receptor was a low affinity receptor and an additional high affinity, pharmacologically distinct GABA
B
receptor subtype could exist in the brain. Alternatively, it was argued that G-protein coupling was inefficient or the receptor was desensitising in the recombinant systems used.
A number of groups working in the area have, however, found that the cloned receptor fails to behave as a functional GABA
B
receptor either in mammalian cells or in Xenopus oocytes. The present invention describes the cloning of a novel human GABA
B
receptor subtype, GABA
B
-R2, the identification of a novel splice variant GABA
B
-R1c, and the surprising observation that GABA
B
-R1 and GABA
B
-R2 strongly interact via their C-termini to form heterodimers. Co-expression of GABA
B
-R1 and GABA
B
-R2 allows trafficking of GABA
B
-R1 to the cell surface and results in a high affinity functional GABA
B
receptor in both mammalian cells and Xenopus oocytes.
These surpising findings provide a unique opportunity to define GABA
B
subtypes at the molecular level, which in turn will lead to the identification of novel subtype-specific drugs.
SUMMARY OF THE INVENTION
According to one embodiment of the present invention there is provided an isolated GABA
B
-R2 receptor protein or a variant thereof.
According to another embodiment of the invention there is provided an isolated GABA
B
-R2 receptor protein having amino acid sequence provided in
FIG. 1B
, or a variant thereof.
According to a further embodiment of the invention there is provided a nucleotide sequence encoding a GABA
B
-R2 receptor or a variant thereof, or a nucleotide sequence which is complementary thereto.
According to a further embodiment of the invention there is provided a nucleotide sequence encoding a GABA
B
-R2 receptor, as shown in
FIG. 1A
, or a variant thereof, or a nucleotide sequence which is complementary thereto.
According to a further embodiment of the invention there is provided an expression vector comprising a nucleotide sequence as referred to above which is capable of expressing a GABA
B
-R2 receptor protein or a variant thereof.
According to a still further embodiment of the invention there is provided a stable cell line comprising a vector as referred to above.
According to another embodiment of the invention there is provided an antibody specific for a GABA
B
-R2 receptor protein or a variant thereof.
According to another embodiment of the invention there is provided an isolated GABA
B
-R1c receptor protein or a variant thereof.
According to another embodiment of the invention there is provided an isolated GABA
B
-R1c receptor protein having amino acid sequence provided in
FIG. 2
, or a variant thereof.
According to another embodiment of the invention there is provided a nucleotide sequence encoding a GABA
B
-R1c receptor protein or a variant thereof, or a nucleotide sequence which is complementary thereto.
According to another embodiment of the invention there is provided an expression vector comprising a nucleotide sequence as referred to above, which is capable of ex

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