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
1998-11-13
2001-06-05
Ulm, John (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...
C435S252300, C435S320100, C536S023500
Reexamination Certificate
active
06242216
ABSTRACT:
TECHNICAL FIELD
The invention relates generally to receptor proteins and to DNA and RNA molecules encoding therefor. In particular, the invention relates to a nucleic acid sequence that encodes a human receptor P2X
2
and P2X
4
. The invention also relates to methods of using the receptors encoded thereby to identify compounds that interact with them. This invention further relates to compounds which act as antagonists and agonists to compounds which have reactivity with the various P2X receptor and methods utilized in determining said reactivity. The invention also involves therapeutic uses involving aspects of these receptors.
BACKGROUND OF THE INVENTION
P2 receptors have been generally categorized as either metabotropic nucleotide receptors or ionotropic receptors for extracellular nucleotides. Metabotropic nucleotide receptors (usually designated P2Y or P2Y
n
, where “n” is a subscript integer indicating subtype) are believed to differ from ionotropic receptors (usually designated P2X or P2X
n
) in that they are based on a different fundamental means of transmembrane signal transduction: P2Y receptors operate through a G protein-coupled system, while P2X receptors are ligand-gated ion channels. The ligand for these P2X receptors is ATP, and/or other natural nucleotides, for example, ADP, UTP, UDP, or synthetic nucleotides, for example 2-methylthioATP.
At least seven P2X receptors, and the cDNA sequences encoding them, have been identified to date. P2X
1
cDNA was cloned from the smooth muscle of the rat vas deferens (Valera et al (1994) Nature 371:516-519) and P2X
2
cDNA was cloned from PC12 cells (Brake et al. (1994) Nature 371:519-523). Five other P2X receptors have been found in cDNA libraries by virtue of their sequence similarity to P2X
1
and P2X
2
(P2X
3
: Lewis et al. (1995) Nature 377:432-435, Chen et al. (1995) Nature 377:428-431; P2X
4
: Buell et al. (1996) EMBO J. 15:55-62, Seguela et al. (1996) J. Neurosci. 16:448-455, Bo et al. (1995) FEBS Lett. 375:129-133, Soto et al. (1996) Proc. Natl. Acad. Sci. USA 93:3684-3688, Wang et al. (1996) Biochem. Biophys. Res. Commun.220:196-202; P2X
5
: Collo et al. (1996) J. Neurosci. 16:2495-2507, Garcia-Guzman et al. (1996) FEBS Lett. 388:123-127; P2X
6
: Collo et al. (1996), supra, Soto et al. (1996) Biochem. Biophys. Res. Commun. 223:456-460; P2X
7
: Surprenant et al. (1996) Science 272:735-738). For a comparison of the amino acid sequences of rat P2X receptors see Buell et al. (1996) Eur. J. Neurosci. 8:2221-2228.
Native P2X receptors form rapidly activated, nonselective cationic channels that are activated by ATP. Rat P2X
1
and rat P2X
2
have equal permeability to Na
+
and K
+
but significantly less to Cs
+
. The channels formed by the P2X receptors generally have high Ca
2+
permeability (P
Ca
/P
Na
≅4). The cloned rat P2X
1
, P2X
2
and P2X
4
receptors exhibit the same permeability for Ca
2+
observed with native receptors. However, the mechanism by which P2X receptors form an ionic pore or bind ATP is not known.
A variety of tissues and cell types, including epithelial, immune, muscle and neuronal, express at least one form of P2X receptor. The widespread distribution of P2X
4
receptors in the rat central nervous system suggests a role for P2X
4
-mediated events in the central nervous system. However, study of the role of individual P2X receptors is hampered by the lack of receptor subtype-specific agonists and antagonists. For example, one agonist useful for studying ATP-gated channels is &agr;,&bgr;-methylene-ATP (&agr;,&bgr;meATP). However, the P2X receptors display differential sensitivity to the agonist with P2X
1
and P2X
2
being &agr;,&bgr;meATP-sensitive and insensitive, respectively. Furthermore, binding of &agr;,&bgr;meATP to P2X receptors does not always result in channel opening. The predominant forms of P2X receptors in the rat brain, P2X
4
and P2X
6
receptors, cannot be blocked by suramin or PPADS. These two forms of the P2X receptor are also not activated by &agr;,&bgr;meATP and are, thus, intractable to study with currently available pharmacological tools.
A therapeutic role for P2 receptors has been suggested, for example, for cystic fibrosis (Boucher et al. (1995) in: Belardinelli et al. (eds) Adenosine and Adenine Nucleotides: From Molecular Biology to Integrative Physiology (Kluwer Acad., Norwell Mass.) pp 525-532), diabetes (Loubatiéres-Mariani et al. (1995) in: Belardinelli et al. (eds), supra, pp 337-345), immune and inflammatory diseases (Di Virgilio et al. (1995) in: Belardinelli et al. (eds), supra, pp 329-335), cancer (Rapaport (1993) Drug Dev. Res. 28:428-431), constipation and diarrhea (Milner et al. (1994) in: Kamm et al. (eds.) Constipation and Related Disorders: Pathophysiology and Management in Adults and Children (Wrightson Biomedical, Bristol) pp 41-49), behavioral disorders such as epilepsy, depression and aging-associated degenerative diseases (Williams (1993) Drug. Dev. Res. 28:438-444), contraception and sterility (Foresta et al. (1992) J. Biol. Chem. 257:19443-19447), and wound healing (Wang et al. (1990) Biochim. Biophys. Res. Commun. 166:251-258).
Accordingly, there is a need in the art for specific agonists and antagonists for each P2X receptor subtype and, in particular, agents that will be effective in vivo, as well as for methods for identifying P2X receptor-specific agonist and antagonist compounds.
SUMMARY OF THE INVENTION
The present invention relates to human P2X
2
and P2X
4
receptors.
In one embodiment, a DNA molecule or fragments thereof is provided, wherein the DNA molecule encodes aforementioned human P2X receptors, or subunits thereof.
In another embodiment, a recombinant vector comprising such DNA molecules, or fragments thereof, is provided.
In another embodiment, the subject invention is directed to a human P2X
2
and P2X
4
receptor polypeptides, either alone or in multimeric form.
In still other embodiments, the invention is directed to messenger RNA encoded by the DNA, recombinant host cells transformed or transfected with vectors comprising the DNA or fragments thereof, and methods of producing recombinant P2X polypeptides using such cells.
In yet another embodiment, the invention is directed to a method of expressing the above human P2X receptors, or a subunit thereof, in a cell to produce the resultant P2X-containing receptors.
In a further embodiment, the invention is directed to a method of using such cells to identify potentially therapeutic compounds that modulate or otherwise interact with the above P2X-containing receptors.
In another embodiment, therapeutic uses involving P2X modulators, such as an ATP agonist or antagonist are contemplated.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.
REFERENCES:
patent: 5607836 (1997-03-01), Boucher et al.
patent: 5733916 (1998-03-01), Neely
patent: 9533048 (1995-07-01), None
patent: 9533048 (1995-12-01), None
patent: 9741222 (1997-06-01), None
patent: 9818916 (1998-07-01), None
patent: 9842835 (1998-10-01), None
Brändle, U., et al., “Desensitization of the P2X2receptor controlled by alternative splicing”, FEBS Letters, 404:294-298 (1997).
Cook, S. P., et al., “Distinct ATP receptors on pain-sensing and stretch-sensing neurons”,letters to nature, 387:505-508 (1997).
Garcia-Guzman, M., et al., “Molecular characterization and pharmacological properties of the human P2X3purinoceptor”,Molecular Brain Research, 47:59-66 (1997).
Garcia-Guzman, M., et al., “Characterization of Recombinant Human P2X4 Receptor Reveals Pharmacological Differences to the Rat Homologue”,The American Soc of Pharm. And Exp. Therapeutics, 51:109-118 (1997).
Koshimizu, T-a., et al., “Functional Role of Alternative Splicing in Pituitary P2X2Receptor-Channel Activation and Desensitization”,Molecular Endocrinology, 12(7):901-913 (19998).
Urano, T., et al., “Cloning of P2XM, a Novel Human P2X Receptor Gene Regulated by p53”,Cancer Research, 57:3281-3287 (1997).
McMahon R. A.: “H. sapiens P2X2A receptor” Ac
Burgard Edward C.
Lynch Kevin J.
Metzger Randy E.
Niforatos Wende
Touma Edward B.
Abbott Laboratories
Becker Cheryl L.
Goller Mimi C.
Ulm John
LandOfFree
Nucleic acids encoding a functional human purinoreceptor... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleic acids encoding a functional human purinoreceptor..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acids encoding a functional human purinoreceptor... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2437166