Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-03-26
2003-02-04
Romeo, David S. (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S395000, C424S001110, C424S450000
Reexamination Certificate
active
06515107
ABSTRACT:
The present invention relates to human metabotropic glutamate receptor (hmGluR) proteins, isolated nucleic acids coding therefor, host cells producing the proteins of the invention, methods for the preparation of such proteins, nucleic acids and host cells, and uses thereof. Furthermore, the invention provides antibodies directed against the hmGluR proteins of the invention.
Metabotropic glutamate receptors (hmGluR) belong to the class of G-protein (guanine nucleotide binding protein) coupled receptors which upon binding of a glutamatergic ligand may transduce an extracellular signal via an intracellular second messenger system such as calcium ions, a cyclic nucleotide, diacylglycerol and inositol 1,4,5-triphosphate into a physiological response. Possessing seven putative transmembrane spanning segments, preceded by a large extracellular amino-terminal domain and followed by a large carboxy-terminal domain metabotropic glutamate receptors are characterized by a common structure. Based on the degree of sequence identity at the amino acid level the class of mGluR can be divided into different subfamilies comprising individual receptor subtypes (Nakanishi, Science 258, 597-603 (1992)). Each mGluR subtype is encoded by a unique gene. Regarding the homology of an individual mGluR subtype to another subtype of a different subfamily, the amino acid sequences are less than about 50% identical. Within a subfamily the degree of sequence identity is generally less than about 70%. Thus a particular subtype may be characterized by its amino acid sequence homology to another mGluR subtype, especially a subtype of the same mammalian species. Furthermore, a particular subtype may be characterized by its region and tissue distribution, its cellular and subcellular expression pattern or by its distinct physiological profile, e.g. by its electrophysiological and pharmacological properties.
The amino acid L-glutamate being the major excitatory neurotransmitter, glutamatergic systems are presumed to play an important role in numerous neuronal processes including fast excitatory synaptic transmission, regulation of neurotransmitter releases, long-term potentation, learning and memory, developmental synaptic plasticity, hypoxic-ischemic damage and neuronal cell death, epileptiform seizures, as well as the pathogenesis of several neurodegenerative disorders. Up to today, no information is available on human metabotropic glutamate receptor (hmGluR) subtypes, e.g. on their amino acid sequence or tissue distribution. This lack of knowledge particularly hampers the search for human therapeutic agents capable of specifically influencing any disorder attributable to a defect in the glutamatergic system. In view of the potential physiological and pathological significance of metabotropic glutamate receptors, there is a need for human receptor subtypes and cells producing such subtypes in amounts sufficient for elucidating the electrophysiological and pharmacological properties of these proteins. For example, drug screening assays require purified human receptor proteins in an active form, which have not yet been attainable.
It is an object of the present invention to fulfill this need, namely to provide distinct hmGluR subtypes, nucleic acids coding therefor and host cells producing such subtypes. In particular, the present invention discloses the hmGluR subfamily comprising the subtype designated hmGluR4, and the individual proteins of said subfamily. In the following, said subfamily will be referred to as the hmGluR4 subfamily. Contrary to other hmGluR subtypes the members of this subfamily are potently activated by L-2-amino-4-phosphobutyric acid (AP4) and, when expressed e.g. in Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells, negatively coupled to adenylate cyclase via G protein. Using a system comprising a recombinant hmGluR subtype of the invention in screening for hmGluR reactive drugs offers (among others) the possibilities of attaining a greater number of receptors per cell giving greater yield of reagent and a higher signal to noise ratio in assays as well as increased receptor subtype specificity (potentially resulting in greater biological and disease specificity).
More specifically, the present invention relates to a hmGluR subtype characterized in that its amino acid sequence is more than about 65% identical to the sequence of the hmGluR4 subtype having the amino acid sequence depicted in SEQ ID NO:2.
According to the invention the expression “hmGluR subtype” refers to a purified protein which belongs to the class of G protein-coupled receptors and which upon binding of a glutamatergic ligand transduces an extracellular signal via an intracellular second messenger system. In such case, a subtype of the invention is characterized in that it modifies the level of a cyclic nucleotide (cAMP, cGMP). Alternatively, signal transduction may occur via direct interaction of the G protein coupled to a receptor subtype of the invention with another membrane protein, such as an ion channel or another receptor. A receptor subtype of the invention is believed to be encoded by a distinct gene which does not encode another metabotropic glutamate receptor subtype. A particular subtype of the invention may be characterized by its distinct physiological profile, preferably by its signal transduction and pharmacological properties. Pharmacological properties are e.g. the selectivity for agonists and antagonist responses.
As defined herein, a glutamatergic ligand is e.g. L-glutamate or another compound interacting with, and particularly binding to, a hmGluR subtype in a glutamate like manner, such as ACPD (1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid), an ACPD-like ligand, e.g. QUIS (quisqualate), AP4, and the like. Other ligands, e.g. (R,S)-&agr;-methylcarboxyphenylglycine (MCPG) or &agr;-methyl-L-AP4, may interact with a receptor of the invention in such a way that binding of glutamatergic ligand is prevented.
As used hereinbefore or hereinafter, the terms “purified” or “isolated” are intended to refer to a molecule of the invention in an enriched or pure form obtainable from a natural source or by means of genetic engineering. The purified proteins, DNAs and RNAs of the invention may be useful in ways that the proteins, DNAs and RNAs as they naturally occur are not, such as identification of compounds selectively modulating the expression or the activity of a hmGluR of the invention.
Purified hmGluR of the invention means a member of the hmGluR4 subfamily which has been identified and is free of one or more components of its natural environment Purified hmGluR includes purified hmGluR of the invention in recombinant cell culture. The enriched form of a subtype of the invention refers to a preparation containing said subtype in a concentration higher than natural, e.g. a cellular membrane fraction comprising said subtype. If said subtype is in a pure form it is substantially free from other macromolecules, particularly from naturally occurring proteinaceous contaminations. If desired, the subtype of the invention may be solubilized. A preferred purified hmGluR subtype of the invention is a recombinant protein. Preferably, the subtype of the invention is in an active state meaning that it has both ligand binding and signal transduction activity. Receptor activity is measured according to methods known in the art, e.g. using a binding assay or a functional assay, e.g. an assay as described below.
Preferred hmGluR subtypes of the hmGluR4 subfamily are subtypes hmGluR4, hmGluR7 and hmGluR6. A particularly preferred hmGluR4 subtype is the protein having the amino acid sequence set forth in SEQ ID NO:2. A hmGluR7-type protein may comprise a polypeptide selected from the group consisting of the polypeptides having the amino acid sequences depicted in SEQ ID NOs: 4, 6, 8 and 10, respectively. Such hmGluR7 subtype is preferred. Particularly preferred are the hmGluR7 subtypes having the amino acid sequences set forth in SEQ ID NOs: 12 and 14, respectively. A preferred hmGluR6-type protein comprise
Flor Peter Josef
Knöpfel Thomas
Kuhn Rainer
Lindauer Kristin
Püttner Irene
Murphy Joseph F.
Novartis Corporation
Romeo David S.
Wildman David E.
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