Nucleic acids encoding a functional human purinoreceptor...

Details Nucleic acids encoding a functional human purinoreceptor... Nucleic acids encoding a functional human purinoreceptor...

1998-11-13

2001-04-10

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, C530S350000, C536S023500

Reexamination Certificate

active

06214581

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 P
2
X
3
and P
2
X
6
. 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 P
2
X receptor and methods utilized in determining said reactivity. The invention also involves therapeutic uses involving aspects of these receptors. The invention relates generally to receptor ligand screening methods. In particular, the invention relates to a method of using stably transfected cell lines that express human purinoreceptors to identify purinoreceptor ligands.
BACKGROUND OF THE INVENTION
P
2
receptors have been generally categorized as either metabotropic nucleotide receptors or ionotropic receptors for extracellular nucleotides. Metabotropic nucleotide receptors (usually designated P
2
Y or P
2
Y
n
, where “n” is a subscript integer indicating subtype) are believed to differ from ionotropic receptors (usually designated P
2
X or P
2
X
n
) in that they are based on a different fundamental means of transmembrane signal transduction: P
2
Y receptors operate through a G protein-coupled system, while P
2
X receptors are ligand-gated ion channels. The ligand for these P
2
X receptors is ATP, and/or other natural nucleotides, for example, ADP, UTP, UDP, or synthetic nucleotides, for example 2-methylthioATP.
At least seven P
2
X receptors, and the cDNA sequences encoding them, have been identified to date. P
2
X
1
cDNA was cloned from the smooth muscle of the rat vas deferens (Valera et al. (1994) Nature 371:516-519) and P
2
X
2
cDNA was cloned from PC
12
cells (Brake et al. (1994) Nature 371:519-523). Five other P
2
X receptors have been found in cDNA libraries by virtue of their sequence similarity to P
2
X
1
and P
2
X
2
(P
2
X
3
: Lewis et al. (1995) Nature 377:432-435, Chen et al. (1995) Nature 377:428-431; P
2
X
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; P
2
X
5
: Collo et al. (1996) J. Neurosci. 16:2495-2507, Garcia-Guzman et al. (1996) FEBS Lett. 388:123-127; P
2
X
6
: Collo et al. (1996), supra, Soto et al. (1996) Biochem. Biophys. Res. Commun. 223:456-460; P
2
X
7
: Surprenant et al. (1996) Science 272:735-738). For a comparison of the amino acid sequences of rat P
2
X receptors see Buell et al. (1996) Eur. J. Neurosci. 8:2221-2228.
Native P
2
X receptors form rapidly activated, nonselective cationic channels that are activated by ATP. Rat P
2
X
1
and rat P
2
X
2
have equal permeability to Na
+
and K
+
but significantly less to Cs
+
. The channels formed by the P
2
X receptors generally have high Ca
2+
permeability (P
Ca
/P
Na
≈4). The cloned rat P
2
X
1
, P
2
X
2
and P
2
X
4
receptors exhibit the same permeability for Ca
2+
observed with native receptors. However, the mechanism by which P
2
X 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 P
2
X receptor. The widespread distribution of P
2
X
4
receptors in the rat central nervous system suggests a role for P
2
X
4
-mediated events in the central nervous system. However, study of the role of individual P
2
X 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 P
2
X receptors display differential sensitivity to the agonist with P
2
X
1
and P
2
X
2
being &agr;,&bgr;meATP-sensitive and insensitive, respectively. Furthermore, binding of &agr;,&bgr;meATP to P
2
X receptors does not always result in channel opening. The predominant forms of P
2
X receptors in the rat brain, P
2
X
4
and P
2
X
6
receptors, cannot be blocked by suramin or PPADS. These two forms of the P
2
X receptor are also not activated by &agr;,&bgr;meATP and are, thus, intractable to study with currently available pharmacological tools.
A therapeutic role for P
2
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 P
2
X receptor subtype and, in particular, agents that will be effective in vivo, as well as for methods for identifying P
2
X receptor-specific agonist and antagonist compounds. P
2
purinoreceptors have been generally categorized as either metabotropic nucleotide receptors or ionotropic receptors for extracellular nucleotides. Metabotropic nucleotide receptors, designated P
2
Y
n
, and the ionotropic receptors, designated P
2
X
n
, are distinguished on the basis of their respective transmembrane signal transduction mechanisms as well as structural differences; the P
2
Y
n
receptors operate through a G protein-coupled system, while the P
2
X
n
receptors are ligand-gated ion channels. The ligand for these receptors may be ATP and/or another natural nucleotide such as ADP, UTP and UDP, or a synthetic nucleotides such as 2-methylthioATP.
At least seven P
2
X receptors, and the cDNA sequences therefore, have been identified to date. P
2
X
1
cDNA has been cloned from the smooth muscle of the rat vas deferens (Valera et al. (1994)
Nature
371:516-519) and P
2
X
2
cDNA was cloned from PC
12
cells (Brake et al. (1994)
Nature
371:519-523). Five other P
2
X receptors have been found in rat neuronal cDNA libraries by virtue of their sequence similarity to P
2
X
1
and P
2
X
2
(P
2
X
3
: Lewis et al. (1995)
Nature
377:432-435, Chen et al. (1995)
Nature
377:428-431; P
2
X
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; P
2
X
5
: Collo et al. (1996)
J. Neurosci.
16:2495-2507, Garcia-Guzman et al. (1996)
FEBS Lett.
388:123-127; P
2
X
4
: Collo et al. (1996), supra, Soto et al. (1996)
Biochem. Biophys. Res. Commun.
223:456-460; P
2
X
7
Surprenant et al. (1996)
Science
272:735-738. For a comparison of the amino acid sequences of rat P
2
X receptors see Buell et al. (1996)
Eur. J. Neurosci.
8:2221-2228. -38-
Native P
2
X receptors form rapidly-activated, nonselective cationic channels that are activated by ATP. P
2
X
1
and P
2
X
2
have equal permeability to Na
+
and K
+
but significantly less to Cs
+
. The channels formed by the P
2
X receptors generally have high Ca
2+
permeability (P
Ca
/P
Na
4
). The cloned rat P
2
X
1
, P
2
X
2
, and P
2
X
4
receptors exhibit the same permeability for Ca
2+
observed with native receptors. However, the mechanism

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