Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-11-12
2004-09-14
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S069100, C435S320100, C435S325000, C536S023500, C530S350000
Reexamination Certificate
active
06790626
ABSTRACT:
OBJECT OF THE PRESENT INVENTION
The present invention concerns a new receptor having a preference for pyrimidine nucleotides preferably uridine triphopshate over purine nucleotides and the nucleic acid molecule encoding said receptor, vectors comprising said nucleic acid molecule, cells transformed by said vector, antibodies directed against said receptor, nucleic acid probes directed against said nucleic acid molecule, pharmaceutical compositions comprising said produces and non human transgenic animals expressing the receptor according to the invention or the nucleic acid molecule according to said receptor.
The invention further provides methods for determining ligand binding, detecting expression, screening for drugs, molecular binding specifically to said receptor and treatment involving the receptor according to the invention.
BACKGROUND OF THE INVENTION
The cloning of several receptors for ATP has been reported since 1993. In keeping with the latest nomenclature proposal, these P2 purinergic receptors can be subdivided into two classes: G protein-coupled receptors, or P2Y receptors, and receptors with intrinsic ion channel activity or P2X receptors (2). Two distinct rat P2X receptors have been cloned, respectively from the vas deferens (3) and phaechromocytoma PC12 cells (4): they have a characteristic topology, with two hydrophobic putatively membrane-spanning segments and an ion pore motif reminiscent of potassium channels. In the P2Y family, the sequences of two subtypes, both coupled to phospholipase C, have been published: chick (5), turkey (6), bovine (7), mouse and rat (8) P2Y1 receptors (formerly called P2Y); murine (9, 10), rat (11) and human (12) P2Y2 receptors (previously named P2U) on the other hand. In addition, a P2Y3 receptor, with a preference for ADP over ATP, has been cloned from chick brain, but its sequence is not yet published (13). Furthermore, the 6H1 orphan receptor, cloned from activated chicken T lymphocytes, exhibits a significant degree of homology to the P2Y1 and P2Y2 receptors, suggesting that it also belongs to the P2Y family, although its responsiveness to nucleotides has not yet been demonstrated (14).
SUMMARY OF THE INVENTION
This invention provides a receptor having a preference for pyrimidine nucleotides preferably uridine triphopshate over purine nucleotides. A receptor having a preference for pyrimidine nucleotides over purine nucleotides means a receptor for which pyrimidine nucleotides and purine nucleotides are not equally active and equipotent. This means that the receptor according to the invention in presence of these agonists presents a functional response (preferably the accumulation of Inositol triphosphate (IP3), diacylglycerol (DAG), or calcium ions) to lower concentration of pyrimidine nucleotides, preferably uridine triphopshate, than to purine nucleotides or a more important functional response to similar concentration of pyrimidine nucleotide than to purine nucleotide.
The inositol phosphate (IP3) accumulation after addition of said agonists is described in the specification thereafter.
Advantageously, the receptor according to the invention has at least a twofold, preferably a tenfold to one hundredfold preference for pyrimidine nucleotides over purine nucleotides.
A preferred embodiment of the receptor according to the invention is characterized by a preference for uridine triphosphate over adenine nucleotides.
The receptor according to the invention is a receptor, preferably a G protein-coupled receptor, which belongs structurally to the purinergic receptor family (P2Y family) but functionally is a pyrimidinergic receptor, preferably a UTP-specific receptor.
According to a preferred embodiment of the present invention, the receptor is a human receptor.
Said receptor has an amino acid sequence having more than 60% homology with the amino acid sequence shown in FIG.
1
. Preferably, the amino acid sequence of the receptor according to the invention has at least the amino acid sequence shown in
FIG. 1
or a portion thereof.
A portion of the amino acid sequence means a peptide or a protein having the same binding properties as the receptor according to the invention (i.e. peptide or a protein which is characterized by a preference for pyrimidine nucleotides, preferably UTP, over purine nucleotides).
The present invention is also related to a nucleic acid molecule, such as a DNA molecule or an RNA molecule, encoding the receptor according to the invention.
Preferably, said DNA molecule is a cDNA molecule or a genomic DNA molecule.
Preferably, said nucleic acid molecule has more than 60% homology to the DNA sequence shown in FIG.
1
.
Preferably, the nucleic acid molecule according to the invention is at least the DNA sequence shown in
FIG. 1
or portion thereof. “A portion of a nucleic acid sequence” means a nucleic acid sequence encoding at least a portion of amino acid sequence as described above.
The present invention is also related to a vector comprising the nucleic acid molecule according to the invention. Preferably, said vector is adapted for expression in a cell and comprises the regulatory elements necessary for expressing the amino acid molecule in said cell operatively linked to the nucleic acid sequence according to the invention as to permit expression thereof.
Preferably, said cell is chosen among the group consisting of bacterial cells, yeast cells, insect cells or mammalian cells. The vector according to the invention is a plasmid or a virus, preferably a baculovirus, an adenovirus or a semliki forest virus.
The plasmid may be the pcDNA3-P2Y4.
The present invention concerns also the cell (preferably a mammalian cell, such as a 1321N1 cell) transformed by the vector according to the invention. Advantageously, said cell is preferably non neuronal in origin and is chosen among the group consisting of a COS-7 cell, an LM(tk-) cell, an NIH-3T3 cell or a 1321N1 cell.
The present invention is also related to a nucleic acid probe comprising the nucleic acid molecule according to the invention, of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included in the sequence of the nucleic acid molecule encoding the receptor according to the invention. Said nucleic acid probe may be a DNA or an RNA molecule.
The invention concerns also an antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding the receptor according to the invention so as to prevent translation of said mRNA molecule or an antisense oligonucleotide having a sequence capable of specifically hybridizing to the cDNA molecule encoding the receptor according to the invention.
Said antisense oligonucleotide may comprise chemical analogs of nucleotide or substances which inactivate mRNA, or be included in an RNA molecule endowed with ribozyne activity.
Another aspect of the present invention concerns a ligand other than purine and pyrimidine nucleotides (preferably an antibody) capable of binding to a receptor according to the invention and an anti-ligand (preferably also an antibody) capable of competitively inhibiting the binding of said ligand to the receptor according to the invention.
Preferably, said antibody is a monoclonal antibody.
The present invention concerns also the monoclonal antibody directed to an epitope of the receptor according to the invention and present on the surface of a cell expressing said receptor.
The invention concerns also the pharmaceutical composition comprising an effective amount of oligonucleotide according to the invention, effective to decrease the activity of said receptor by passing through a cell membrane and binding specifically with mRNA encoding the receptor according to the invention in the cell so as to prevent its translation. The pharmaceutical composition comprises also a pharmaceutically acceptable carrier capable of passing through said cell membrane.
Preferably, in said pharmaceutical composition, the oligonucleotide is coupled to a substance, such as a ribozyme, which inactivates mRNA.
Preferably, the pharmaceutically
Boeynaems Jean-Marie
Communi Didier
Parmentier Marc
Pirotton Sabine
Euroscreen S.A.
Eyler Yvonne
Murphy Joseph F.
Palmer & Dodge LLP
Williams Kathleen M.
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