Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-11-17
2002-10-08
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Reexamination Certificate
active
06462178
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention concerns novel G protein chimeras, nucleotide sequences encoding same, host cells transformed or transfected with same, methods of determining GPCR response to a molecule, and kits for same.
The guanine nucleotide-binding proteins (G proteins) are responsible for the efficient transmission of signals from agonist-bound cell surface receptors to different intracellular effectors. Approximately 5000 G protein-coupled receptors (GPCRs) are encoded by the human genome, and most fall into the categories of being G
q
-, G
i
-, or G
s
-coupled. Some G proteins are more promiscuous than others by possessing the ability to interact with a large panel of GPCRs. The most notable examples of promiscuous G proteins are the human G
16
and its murine homolog, G
15
. Both G
15
and G
16
link a variety of G
q
-, G
i
-, and G
s
-coupled receptors to stimulate phospholipase C (PLC); (Offermanns, S. and Simon, M., 1995, J. Biol. Chem., 270; 15175-15180; Lee, J. W. M. et al., 1998, J Neurochem., 70: 2203-2211).
G proteins are membrane-associated proteins that transduce signals from GPCRs to various intracellular effectors. G proteins within this class are heterotrimers, consisting of an &agr; subunit responsible for binding guanine nucleotides, a &bgr; subunit, and a &ggr; subunit. In mammalians, at least 16 distinct genes encode G protein &agr; subunits. Furthermore, 5 distinct &bgr; subunit genes as well as 12 &ggr; subunit genes have been identified (Clapham, D. E. and Neer, E. J., 1997, Annu. Rev. Pharmacol. Toxicol. 37, 167-203; Hildebrandt, J. D., 1997, Biochem. Pharmacol., 51, 325-339). In vivo, the &bgr; and &ggr; subunits form high-affinity non-dissociating complexes, thus, a large number of &bgr;&ggr; combinations are possible. Over the past decade, both the &agr; subunit and &bgr;&ggr; complexes have been shown to possess the ability to regulate effector systems (Clapham, D. E. and Neer, E. J., 1997, Annu. Rev. Pharmacol. Toxicol. 37, 167-203; Hildebrandt, J. D., 1997, Biochem. Pharmacol., 51, 325-339).
The G protein &agr; subunit family is divided into four subgroups based on their amino acid sequence homology and functional diversity. The G
s
family, including G&agr;
sL
, G&agr;
sS
and G&agr;
olf
, is routinely classified as G protein &agr; subunits able to mediate stimulatory regulation of adenylyl cyclase isoforms. The G
q
family of &agr; subunits, including G&agr;
q
, G&agr;
11
, G&agr;
14
and G&agr;
16
, promotes the activation of &bgr;-isoforms of PLC. G&agr;
12
and G&agr;
13
are recently identified as the regulators of Na
+
-H
+
exchangers and small molecular weight-G protein signaling cascades through the interaction with at least two guanine nucleotide exchange factors of Rho. The G
i
family, which contains 10 members—G&agr;
i1
, G&agr;
i2
, G&agr;
i3
, G&agr;
o1
, G&agr;
o2
, G&agr;
o3
, G&agr;
t1
, G&agr;
t2
, G&agr;
gust
(G&agr;
t3
), and G&agr;
z
—was originally defined as the G protein &agr; subunits closely related to those able to mediate inhibition of adenylyl cyclase (which is true of all G&agr;
i
subtypes, as well as G&agr;
z
). G&agr;
o
subtypes mainly regulate calcium ion channels, while G&agr;
t
, subtypes activate cGMP phosphodiesterases.
Because of their promiscuity, G
15
and G
16
have to date been recognised as being ideal candidates for linking “orphan” receptors (cloned receptors without a known ligand) to PLC and its downstream effectors. Hence, G
16
has received considerable attention as a potential tool for drug discovery (Milligan, G. et al., 1996, Trends in Pharmacol. Sci., 17: 235-237). Although G
15
and G
16
are more promiscuous than other G proteins, they are not true universal adapters for GPCRs. For example, the CCR2a chemokine receptor (Kuang, Y. et al., 1996, J. Biol. Chem., 271: 3975-3978), the &agr;
1A
- and &agr;
1C
-adrenoceptors (Wu, D. et al., 1992, J. Biol. Chem., 267: 25798-25802) are unable to recognize G
16
. Indeed, of thirty-three different GPCR examined to date (Offermanns, S. and Simon, M., 1995, supra; Lee, J. W. M. et al., 1998, supra; Kuang, Y. et al., 1996, supra; Wu, D. et al., 1992, supra; Wu, D. et al., 1993, Science, 261: 101-103; Zhu, X. and Birnbaumer, L., 1996, PNAS USA, 93: 2827-2831; Parmentier, M. L. et al., 1998, Mol. Pharmacol., 53: 778-786), at least six receptors are incapable of activating G
16
.
Most of the GPCRs that fail to activate G
16
belong to the G
i
-coupled receptor subfamily. The term G
i
-coupled receptors stands for a group of seven transmembrane receptors that can interact with all three subtypes of G&agr;
i
(G&agr;
i1-3
)as well as G&agr;
z
. Binding of a proper agonist to the receptor triggers the activation of the associated &agr; subunits by promoting the release of GDP and the uptake of GTP. These receptors are widely distributed in different receptor categories, including aminergic, hormonal, peptidergic, purinergic, and chemokine. The G
i
-coupled receptors constitute an exceedingly large GPCR subfamily which encompass many newly discovered receptors such as those for chemokines, however, approximately 15% of the G
i
-coupled receptors examined to date cannot activate G
16
. As previously mentioned, this poses a serious concern for using G
16
as an adapter of orphan receptors in drug screening protocols due to the large number of receptors which may not couple to or effectively couple to G&agr;
16
.
The underlying rationale for the intense interest in orphan GPCRs is based in their history of being excellent therapeutic targets. Over the past several decades, drug discovery programs world-wide have combined to produce greater than 200 novel drugs that possess activity or antagonizing properties towards GPCRs. As an example, it is estimated that the majority of drug discovery initiatives within the pharmaceutical industry are focused on this signalling pathway (Roush, W., 1996, Science, 271, 1056-1058). Likewise, the significance and complexity of GPCRs is readily apparent in the number of cases of genetic diseases that are known to be linked to various defects in these receptors (Dryja, T. P., et al., 1990, Nature, 343, 364-366; Sung, C-H. et al., 1991, Proc. Nal. Acad. Sci. U.S.A., 88, 6481-6485; Parma, J. et al., 1993, Nature, 365, 649-651; Shenker, A., et al., 1993, Nature, 365, 652-654; van den Oiweland, A. M., et al., 1992, Nat. Genet, 2, 99-102; Pan, Y., et al., 1992, Nat. Genet., 2, 103-106; Rosenthal, W. et al., 1993, J. Biol. Chem., 268, 13030-13033; Pollak, M. R., et al., 1993, Cell, 75, 1297-1303; Pollak, M. R., et al., 1994, Nat. Genet., 8, 303-307; Schipsni, E., et al., 1995, Science, 268, 98-100; Walston, J. et al., 1995, New Engl. J. Med., 333, 343-347; Widen, E., et al., 1995, New Engl. J. Med., 333, 348-351; Clement, K., et al., 1995, New Engl. J. Med., 333 352-354; Wajnrajch, M. P., et al., 1996, Nat. Genet., 12, 88-90; Clark, A. J. L., et al., 1993, Lancet, 341,461-462; Hager, J., et al., 1995, Nat Genet., 9, 299-304). As a result of the proven link of GPCRs to a wide variety of diseases and the historical success of drugs that target these receptors, characterisation of orphan GPCRs are among the most promising molecular targets for future drug discovery platforms. The ability to couple orphan GPCRs to down stream effectors via a small number of discriminating G proteins would greatly accelerate validation of GPCRs as potential drug targets and hence, further accelerate the discovery of novel therapeutics.
In light of the exceedingly large number of GPCRs, the characterisation of this class of proteins in toti is impractical, yet it remains necessary to identify ligands, particularly therapeutically effective ligands, for orphan receptors. Thus there is a need for an improved G protein with an increased promiscuity for binding to GPCRs (particularly G
i
-coupled receptors) in order to link them to PLC and other downstream effectors. In theory, this would provide the means to identify useful ligands that bind to orphan receptors and result in the activation of an engineere
Carlson Karen Cochrane
Dority & Manning PA
Yong Hou Wong
LandOfFree
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