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锂电池百篇论文点评(-)
reviews of selected 100 recent papers for lithium batteries (aug.1, 2013 to sept.30, 2013).pdf 12页
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锂电池百篇论文点评(-)
reviews of selected 100 recent papers for lithium batteries (aug.1, 2013 to sept.30, 2013)
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第2卷第6期
储能科学与技术
V01．2No．6
2013年11月
Scienceand
EnergyStorage
Technology
锂电池百篇论文点评(—)
勇，徐凯琪，林明翔，唐代春，董金平，孙 洋，陈 彬，王 昊，贲留斌，黄学杰
(中国科学院物理研究所，北京100190)
摘要：该文是一篇近两个月的锂电池文献评述，我们以“lithium”和“battery～’为关键词检索了Web
氧化物正极材料的研究包括充放电循环过程中的反应机理、结构衍变以及制备条件对材料性能的影响等，高电
压的尖晶石结构LiNio，Ml。04材料的结构分析、改性以及与电解液的匹配受到人们较多的关注。高容量的Si基
负极材料一直是研究的热点，本期碳材料与锡。电解液添加剂、锂硫电池和锂空气电池均有多篇研究论文，理
论模拟X-作扩展到固体电解质、电解液添加剂作用机理、锂空气电池电极过程等。除了这些以材料为主的研究
之外，针对电池的原位分析、电池模型的研究论文也大量出现。
关键词：锂电池；正极材料：负极材料；电解质；电池技术
doi：10．39694．issn．．2
中图分类号：TM
文献标志码：A
Reviewsofselected100recent forlithiumbatteries
(Aug．1，2013Sept．30，2013)
Daichun，DONG
Yong．XUKaiqi，LINMingxiang，TANG Jinping，SUNYang，
CHENBin，WANGHao，BENLiubin，HUANG
(Institute
100190，China)
ofPhysics，ChineseAcademyofSciences，Bering
lithiumbatteries．W色
Abstract：Thisreview
bimonthlypaperhighlights publishedpapers
searchedtheWebofSciencebasedonthe
keywords“lithium'’and‘'battery。”，andPapers
ofthemwereselectedtobe
Aug．1，2013Sept．30，
highlighted．Layeredhigh
materialsarestillunderextensiv
正在加载中，请稍后...Methods of screening for compounds that modulate hormone receptor activity
United States Patent Application
The present invention provides methods for identifying an effective agent that modulates a biological activity of a nuclear hormone receptor. In a method of the invention, an isolated receptor-containing complex is assayed for an altered modification state as compared to a control modification state. The presence of an altered modification state serves to identify an effective agent that modulates a biological activity of the nuclear hormone receptor.
Inventors:
Zhao, Yi (Irvine, CA, US)
Thacher, Scott M. (Costa Mesa, CA, US)
Xiao, Jia-hao (Irvine, CA, US)
Kusari, Jyotirmoy (Irvine, CA, US)
Chandraratna, Roshantha A. (Laguna Hills, CA, US)
Application Number:
Publication Date:
04/24/2003
Filing Date:
08/02/2001
Export Citation:
THACHER SCOTT M.
XIAO JIA-HAO
KUSARI JYOTIRMOY
CHANDRARATNA ROSHANTHA A.
Primary Class:
International Classes:
G01N33/68; G01N33/74; G01N33/78; (IPC1-7): G01N33/53; G01N33/567
View Patent Images:
&&&&&&PDF help
Related US Applications:
August, 2008Rosenberg et al.November, 2005WangFebruary, 2009Akira et al.June, 2005Meier et al.November, 2009Boswell et al.February, 2004ThonnardDecember, 2006ChenJune, 2006Sih et al.December, 2008Peng et al.June, 2004Cotte et al.July, 2009Hanya et al.
Attorney, Agent or Firm:
CAMPBELL & FLORES LLP (4370 LA JOLLA VILLAGE DRIVE, SAN DIEGO, CA, 92122, US)
1. A method for identifying an effective agent that modulates a biological activity of a nuclear hormone receptor, comprising the steps of: (a) contacting said nuclear hormone receptor with one or more agents and a eukaryotic cell sample to form a test sample under conditions suitable to form a receptor- (b) isolating said receptor-containing complex f (c) providing to said isolated receptor-containing complex conditions suitable for modification of said receptor- and (d) assaying said isolated receptor-containing complex for an altered modification state occurring in said isolated receptor-containing complex as compared to a control modification state, wherein the presence of said altered modification state indicates that at least one of said one or more agents is an effective agent that modulates a biological activity of the nuclear hormone receptor.
2. A method for identifying an effective agent that modulates a biological activity of a nuclear hormone receptor, comprising the steps of: (a) contacting said nuclear hormone receptor with one or more agents and a eukaryotic cell sample to form a test sample under conditions suitable to form a receptor- (b) isolating said receptor-containing complex f (c) providing to said isolated receptor-containing complex conditions suitable for phosphorylation of said receptor- and (d) assaying said isolated receptor-containing complex for an altered phosphorylation state occurring in said isolated receptor-containing complex as compared to a control phosphorylation state, wherein the presence of said altered phosphorylation state indicates that at least one of said one or more agents is an effective agent that modulates a biological activity of the nuclear hormone receptor.
3. The method of claim 2, wherein said altered phosphorylation state is an increased phosphorylation state.
4. The method of claim 2, wherein said altered phosphorylation state is a decreased phosphorylation state.
5. A method for identifying an effective agent that modulates a biological activity of a nuclear hormone receptor, comprising the steps of: (a) contacting said nuclear hormone receptor with one or more agents and a eukaryotic cell sample to form a test sample under conditions suitable to form a receptor- (b) isolating said receptor-containing complex f (c) providing to said isolated receptor-containing complex conditions suitable for phosphorylation of said receptor- and (d) assaying said isolated receptor-containing complex for an altered phosphorylation state occurring in said isolated receptor-containing complex as compared to a control phosphorylation state, wherein said altered phosphorylation state is an altered phosphorylation state of said nuclear hormone receptor or an altered phosphorylation state of a 160 kDa protein, and wherein the presence of said altered phosphorylation state indicates that at least one of said one or more agents is an effective agent that modulates a biological activity of the nuclear hormone receptor.
6. The method of claim 5, wherein said altered phosphorylation state is an altered phosphorylation state of said nuclear hormone receptor.
7. The method of claim 5, wherein said altered phosphorylation state is an altered phosphorylation state of a 160 kDa protein.
8. The method of claim 1, wherein said one or more agents are present during step (b).
9. The method of claim 2, where said conditions suitable for modification of said receptor-containing complex are a magnesium concentration of 1 to 25 mM.
10. The method of claim 1, wherein said nuclear hormone receptor is selected from the group consisting of a retinoid X receptor (RXR), hepatocyte nuclear factor 4 (HNF4), testicular receptor, tailless gene homolog (TLX), Chicken ovalbumin upstream promoter transcription factor (COUP-TF), thyroid receptor (TR), retinoic acid receptor (RAR), peroxisome proliferator activated receptor (PPAR), reverse Erb (revErb), RAR-related orphan receptor (ROR), steroidogenic factor-1 (SF-1), liver receptor homolog-1 (LRH-1), liver X receptor (LXR), farnesoid X receptor (FXR), vitamin D receptor (VDR), ecdysone receptor (EcR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), neuron-derived activated receptor (NOR1), nuclear receptor related 1 (NURR1), estrogen receptor (ER), estrogen-related receptor (ERR), glucocorticoid receptor (GR), androgen receptor (AR), progesterone receptor (PR) and mineralocorticoid receptor (MR).
11. A method for identifying an effective agent that modulates a biological activity of a nuclear hormone receptor, comprising the steps of: (a) contacting said nuclear hormone receptor with one or more agents and a eukaryotic cell sample to form a test sample under conditions suitable to form a receptor-containing complex, wherein said nuclear hormone receptor is selected from the group consisting of a retinoid X receptor, retinoic acid receptor, progesterone receptor, estrogen receptor, androgen receptor and vitamin D (b) isolating said receptor-containing complex f (c) providing to said isolated receptor-containing complex conditions suitable for modification of said receptor- and (d) assaying said isolated receptor-containing complex for an altered modification state occurring in said isolated receptor-containing complex as compared to a control modification state, wherein the presence of said altered modification state indicates that at least one of said one or more agents is an effective agent that modulates a biological activity of the nuclear hormone receptor.
12. The method of claim 11, wherein said nuclear hormone receptor is selected from the group consisting of RARα, RARβ, RARγ, RXRα, RXRβ and RXRγ.
13. The method of claim 11, wherein said nuclear hormone receptor is a retinoid X receptor (RXR) selected from the group consisting of RXRα, RXRβ and RXRγ.
14. A method for identifying an effective agent that modulates a biological activity of a retinoid X receptor, comprising the steps of: (a) contacting said retinoid X receptor with one or more agents and a eukaryotic cell sample to form a test sample under conditions suitable to form a retinoid X receptor- (b) isolating said receptor-containing complex f (c) providing to said isolated receptor-containing complex conditions suitable for modification of said receptor- and (d) assaying said isolated receptor-containing complex for an altered phosphorylation state occurring in said isolated receptor-containing complex as compared to a control phosphorylation state, wherein the presence of said altered modification state indicates that at least one of said one or more agents is an effective agent that modulates a biological activity of said retinoid X receptor.
15. The method of claim 1, wherein said nuclear hormone receptor is exogenous to said cell.
16. The method of claim 1, wherein said nuclear hormone receptor is truncated.
17. The method of claim 16, wherein said truncated nuclear hormone receptor lacks the native DNA-binding domain.
18. The method of claim 16, wherein said truncated nuclear hormone receptor consists essentially of the ligand-binding domain.
19. The method of claim 1, wherein said nuclear hormone receptor is a variant with an increased ratio of cytoplasmic to nuclear localization as compared to wild type nuclear hormone receptor.
20. The method of claim 1, wherein said nuclear hormone receptor is a variant that lacks a functional DNA-binding domain.
21. The method of claim 1, wherein said nuclear hormone receptor is a fusion protein comprising a heterologous membrane-anchoring domain.
22. The method of claim 1, wherein said nuclear hormone receptor is a fusion protein comprising a heterologous epitope tag.
23. The method of claim 1, wherein said nuclear hormone receptor is a fusion protein comprising a heterologous protein kinase recognition sequence.
24. The method of claim 1, wherein step (b) comprises specific binding to said receptor-containing complex.
25. The method of claim 24, wherein step (b) comprises immunoprecipitation of said receptor-containing complex.
26. The method of claim 25, wherein said immunoprecipitation is performed using antibody immunoreactive with said nuclear hormone receptor or a heterologous epitope fused thereto.
27. The method of claim 26, wherein said immunoprecipitation is performed using antibody immunoreactive with said nuclear hormone receptor.
28. The method of claim 2, wherein step (c) comprises incubating said receptor-containing complex with ATP.
29. The method of claim 1, wherein said test sample comprises an exogenous heterodimeric partner of said nuclear hormone receptor.
30. The method of claim 1, wherein said test sample comprises an exogenous kinase that enhances detection of said altered modification state.
31. The method of claim 1, wherein said eukaryotic cell sample comprises viable cells.
32. The method of claim 1, wherein said eukaryotic cell sample is a whole cell lysate.
33. The method of claim 1, wherein said eukaryotic cell sample is a fractionated cell lysate.
34. The method of claim 1, wherein said receptor-containing complex comprises a serine/threonine kinase.
35. A method for identifying an improved effective agent that modulates a biological activity of a nuclear hormone receptor, comprising the steps of: (a) contacting said nuclear hormone receptor with one or more agents and a eukaryotic cell sample to form a test sample under conditions suitable to form a receptor- (b) isolating said receptor-containing complex f (c) providing to said isolated receptor-containing complex conditions suitable for modification of said receptor-containing complex, (d) assaying said isolated receptor-containing complex for an altered modification state occurring in said isolated receptor-containing complex as compared to a contro and (e) assaying for direct transcriptional activity of said nuclear hormone receptor contacted with said one or more agents, wherein the presence of said altered modification state combined with the absence of said direct transcriptional activity indicates that at least one of said one or more agents is an improved effective agent that modulates a biological activity of the nuclear hormone receptor.
36. A method for identifying an effective agent that modulates protein kinase A activity associated with a nuclear hormone receptor, comprising the steps of: (a) contacting said nuclear hormone receptor with one or more agents and a cell sample to form a test sample under conditions suitable to form a receptor- (b) isolating said receptor-containing complex f (c) contacting said isolated receptor-containing complex with a protein kinase A substrate under conditions suitable for phosphorylati and (d) assaying said substrate for an altered phosphorylation state as compared to a control phosphorylation state, wherein the presence of said altered phosphorylation state indicates that at least one of said one or more agents is an effective agent that modulates protein kinase A activity associated with said nuclear hormone receptor.
37. The method of claim 36, wherein said altered phosphorylation state is an increased phosphorylation state.
38. The method of claim 36, wherein said altered phosphorylation state is a decreased phosphorylation state.
39. The method of claim 36, wherein said substrate is a purified substrate.
40. A method for identifying an effective agent that modulates protein kinase A activity associated with a nuclear hormone receptor, comprising the steps of: (a) contacting said nuclear hormone receptor with one or more agents and a cell sample to form a test sample under conditions suitable to form a receptor- (b) isolating said receptor-containing complex f (c) contacting said isolated receptor-containing complex with a purified protein kinase A peptide substrate under conditions suitable for phosphorylati and (d) assaying said peptide substrate for an altered phosphorylation state as compared to a control phosphorylation state, wherein the presence of said altered phosphorylation state indicates that at least one of said one or more agents is an effective agent that modulates protein kinase A activity associated with said nuclear hormone receptor.
41. The method of claim 40, wherein said peptide substrate comprises a sequence selected from the group consisting of Arg-X-Ser, Arg-Arg-X-Ser, Arg-X-X-Ser, Lys-Arg-X-X-Ser and Arg-X-Lys-Arg-X-X-Ser-X (SEQ ID NO: 113), where X is independently any amino acid.
42. The method of claim 41, wherein said peptide substrate comprises the amino acid sequence Arg-X-Ser, where X is independently any amino acid.
43. The method of claim 42, wherein said peptide substrate comprises the amino acid sequence Arg-Arg-X-Ser, where X is independently any amino acid.
44. The method of claim 41, wherein said peptide substrate comprises the amino acid sequence Lys-Arg-X-X-Ser, where X is independently any amino acid.
45. The method of claim 44, wherein said peptide substrate comprises the amino acid sequence Arg-X-Lys-Arg-X-X-Ser-X (SEQ ID NO: 113), where X is independently any amino acid.
46. The method of claim 43, wherein said peptide substrate comprises an amino acid sequence selected from the group consisting of LRRASLG (SEQ ID NO: 59) and GRTGRRNSI (SEQ ID NO: 60).
47. The method of claim 40, 41, 42, 43, 44, 45 or 46, wherein said peptide substrate has at most ten residues.
48. The method of claim 39, wherein said purified substrate is myelin basic protein.
49. The method of claim 39, wherein said purified substrate has a Km of less than 20 μM for protein kinase A.
50. A method for identifying an effective agent that modulates protein kinase A activity associated with a nuclear hormone receptor, comprising the steps of: (a) contacting said nuclear hormone receptor with one or more agents and a cell sample to form a test sample under conditions suitable to form a receptor-containing complex, wherein said nuclear hormone receptor is selected from the group consisting of a retinoid X receptor (RXR), retinoic acid receptor (RAR) and a peroxisome proliferator activated receptor (PPAR); (b) isolating said receptor-containing complex f (c) contacting said isolated receptor-containing complex with a protein kinase A substrate under conditions suitable for phosphorylati and (d) assaying said substrate for an altered phosphorylation state as compared to a control phosphorylation state, wherein the presence of said altered phosphorylation state indicates that at least one of said one or more agents is an effective agent that modulates protein kinase A activity associated with said nuclear hormone receptor.
51. The method of claim 50, wherein said nuclear hormone receptor is a retinoid X receptor (RXR).
52. The method of claim 51, wherein said retinoid X receptor is RXRα.
53. The method of claim 36, 51 or 52, wherein said nuclear hormone receptor is a variant with an increased ratio of cytoplasmic to nuclear localization as compared to wild type nuclear hormone receptor.
54. The method of claim 36, 51 or 52, wherein said nuclear hormone receptor is a fusion protein comprising a heterologous membrane-anchoring domain.
55. The method of claim 36, 51 or 52, wherein said nuclear hormone receptor is a fusion protein comprising a pleckstrin homology domain.
56. The method of claim 36, 51 or 52, wherein said nuclear hormone receptor is a fusion protein comprising a heterologous epitope tag.
57. The method of claim 36, 51 or 52, wherein said cell sample comprises an exogenous nucleic acid molecule encoding said nuclear hormone receptor.
58. The method of claim 36, 51 or 52, wherein said nuclear hormone receptor is endogenous to said cell sample.
59. The method of claim 36, 51 or 52, wherein said cell sample comprises an exogenous nucleic acid molecule encoding a heterodimeric partner of said nuclear hormone receptor.
60. The method of claim 36, 51 or 52, wherein said cell sample comprises an exogenous nucleic acid molecule encoding a catalytic subunit of protein kinase A.
61. The method of claim 36, wherein said cell sample comprises viable eukaryotic cells.
62. The method of claim 36, wherein said cell sample is a eukaryotic whole cell lysate.
63. The method of claim 36, wherein said one or more agents are present during step (b).
64. The method of claim 36, wherein step (b) comprises specific binding to said receptor-containing complex.
65. A method for identifying an effective agent that modulates protein kinase A activity associated with a nuclear hormone receptor, comprising the steps of: (a) contacting said nuclear hormone receptor with one or more agents and a cell sample to form a test sample under conditions suitable to form a receptor- (b) immunoprecipitating said receptor-containing complex from said test sample, thereby isolating said receptor- (c) contacting said isolated receptor-containing complex with a protein kinase A substrate under conditions suitable for phosphorylati and (d) assaying said substrate for an altered phosphorylation state as compared to a control phosphorylation state, wherein the presence of said altered phosphorylation state indicates that at least one of said one or more agents is an effective agent that modulates protein kinase A activity associated with said nuclear hormone receptor.
66. The method of claim 65, wherein said immunoprecipitation is performed using antibody immunoreactive with said nuclear hormone receptor.
67. The method of claim 65, wherein said nuclear hormone receptor is a fusion protein comprising a heterologous epitope tag and said immunoprecipitation is performed using antibody immunoreactive with said epitope tag.
68. The method of claim 65, wherein step (d) comprises detecting radiolabeled substrate.
Description:
[0001] This application is based on, and claims the benefit of, U.S. Provisional Application No. 60/284,797, filed Apr. 18, 2001, and entitled NOVEL METHODS OF SCREENING FOR COMPOUNDS THAT MODULATE HORMONE RECEPTOR ACTIVITY, and which is incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates generally to the fields of biochemistry and molecular medicine and, in particular, to drugs that regulate post-translational modifications of nuclear hormone receptors or associated proteins. [0004] 2. Background Information [0005] Nuclear hormone receptors are a large family of gene regulatory, DNA-binding proteins that bind hormonally and nutritionally derived lipophilic ligands. Over 300 nuclear hormone receptors have been identified to date, including, for example, the retinoid X receptor, retinoic acid receptor, progesterone receptor, estrogen receptor, androgen receptor and vitamin D receptor (Whitfield et al., J. Cell. Biochem. Suppl. 32/33:110-122 (1999); Laudet et al., Cell 97:161-163 (1999); and Sluder et al., Genome Res. 9:103-120 (1999)). Nuclear hormone receptors have been conserved throughout evolution and play a role in cell growth and proliferation, development and homeostasis. Not surprisingly, nuclear hormone receptors have been implicated in disease. Retinoic acid receptors can play a role in, for example, acute promyelocyt thyroid hormone receptor is involved in thyroid hormone resistance and
vitamin D receptors play a role in type 2D-dependent rick peroxisome proliferator activated receptor (PPAR) contributes to obesity and Type II and the estrogen receptor plays a role in some forms of breast cancer (Lazar, J. Invest. Medicine 47:364-368 (1999)). Progress has been made in understanding the role of nuclear hormone receptors and their ligands in disease, and in identifying hormone receptor ligands with therapeutic activity. [0006] In the case of the retinoid receptors, retinoid ligands have been developed as therapeutics for a variety of disorders. Current retinoid therapies include differentiation of acute promyelocytic leukemia (APL); treatment of nodulocystic acne, a severe form
tr prevention of secondary h topical ther and reversal of UV-mediated photodamage (Thacher et al., Current Pharm. Design 6:25-58 (2000)). Unfortunately, the dosage of these retinoid ligands is limited by significant side effects, including irritation and inflammation of skin and mucous membranes, elevation of serum triglycerides, dysregulation of bone formation and resorption, headaches, hypothyroidism, and fetal malformation. Thus, there is a need for a new generation of retinoid and other hormone-based therapeutics which can have, for example, greater selectivity and fewer side effects. [0007] Nuclear hormone receptors have long been known to be DNA-binding proteins that can activate or repress transcription of target genes. In most cases, transcriptional activity of the hormone receptor is controlled in a ligand-dependent manner. Current assays for identifying therapeutic ligands are based on the transcriptional activity of the nuclear hormone receptor of interest. However, compounds identified using these assays often are characterized by significant side effects. [0008] Thus, there is a need for novel assays which can be used to identify therapeutic hormone receptor ligands but which do not rely on the transcriptional activity of the nuclear hormone receptor. The present invention satisfies this need and provides related advantages as well. SUMMARY OF THE INVENTION [0009] The present invention provides a method for identifying an effective agent that modulates a biological activity of a nuclear hormone receptor. The method includes the steps of contacting the nuclear hormone receptor with one or more agents and a eukaryotic cell sample to form a test sample under conditions suitable to form a receptor- isolating the receptor-containing complex
providing to the isolated receptor-containing complex conditions suitable for modification of the receptor- and assaying the isolated receptor-containing complex for an altered modification state occurring in the isolated receptor-containing complex as compared to a control modification state, where the presence of the altered modification state indicates that at least one of the one or more agents is an effective agent that modulates a biological activity of the nuclear hormone receptor. In a method of the invention, the altered modification state can be, for example, an increased or decreased phosphorylation state. In particular embodiments, a method of the invention is practiced by assaying for an altered phosphorylation state of a nuclear hormone receptor or an altered phosphorylation state of a 160 kDa protein. [0010] In a method of the invention, a nuclear hormone receptor is contacted with one or more agents and a eukaryotic cell sample. In one embodiment, the one or more agents with which the nuclear hormone receptor is contacted are present during isolation of the receptor-containing complex from the test sample. [0011] In a method of the invention, the isolated receptor-containing complex is provided with conditions suitable for modification of the receptor-containing complex, for example, for phosphorylation of the receptor-containing complex. In one embodiment, the conditions are a magnesium concentration of 1 to 25 mM. [0012] The methods of the invention rely on a nuclear hormone receptor. In particular embodiments, the nuclear hormone receptor is a retinoid X receptor (RXR), hepatocyte nuclear factor 4 (HNF4), testicular receptor, tailless gene homolog (TLX), chicken ovalbumin upstream promoter transcription factor (COUP-TF), thyroid receptor (TR), retinoic acid receptor (RAR), peroxisome proliferator activated receptor (PPAR), reverse Erb (revErb), RAR-related orphan receptor (ROR), steroidogenic factor-1 (SF-1), liver receptor homolog-1 (LRH-1), liver X receptor (LXR), farnesoid X receptor (FXR), vitamin D receptor (VDR), ecdysone receptor (EcR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), neuron-derived activated receptor (NOR1), nuclear receptor related 1 (NURR1), estrogen receptor (ER), estrogen-related receptor (ERR), glucocorticoid receptor (GR), androgen receptor (AR), progesterone receptor (PR) or mineralocorticoid receptor (MR). In one embodiment, the nuclear hormone receptor is a retinoid X receptor, retinoic acid receptor, progesterone receptor, estrogen receptor, androgen receptor or vitamin D receptor. In another embodiment, the nuclear hormone receptor is a retinoid X receptor such as RXRα, RXRβ or RXRγ. [0013] A nuclear hormone receptor useful in a screening method of the invention also can be a truncated nuclear hormone receptor. Such a truncated receptor can be, for example, a truncated hormone receptor lacking a functional DNA-binding domain or a truncated nuclear hormone receptor containing at least the ligand-binding domain of the receptor. In other embodiments, a nuclear hormone receptor useful in the invention is a fusion protein that contains a heterologous sequence from a different nuclear hormone receptor or from a protein that is not a nuclear hormone receptor. A nuclear hormone receptor useful in the invention can be a fusion protein that contains, for example, a heterologous membrane-anchoring domain, heterologous epitope tag or heterologous protein kinase recognition sequence, or any combination of these heterologous sequences. A nuclear hormone receptor useful in the invention also can be a variant with an increased ratio of cytoplasmic to nuclear localization as compared to wild type nuclear hormone receptor, or a variant that lacks a functional DNA-binding domain. [0014] In a method of the invention, isolation of the receptor-containing complex can be achieved using a variety of means including specific binding to the receptor-containing complex, for example, immunoprecipitation of the receptor-containing complex. Immunoprecipitation can be performed, for example, using antibody immunoreactive with the nuclear hormone receptor. [0015] A method of the invention can be practiced with a variety of eukaryotic cell samples, including viable cells, which can be, for example, transiently o or a fractionated cell lysate. The test sample containing the nuclear hormone receptor and one or more agents to be assayed also can include, if desired, an exogenous heterodimeric partner of the nuclear hormone receptor, or an exogenous kinase that enhances detection of an altered modification state. In one embodiment, the isolated receptor-containing complex includes a serine/threonine kinase. [0016] The present invention also provides a method for identifying an effective agent that modulates a biological activity of a retinoid X receptor. The method includes the steps of contacting the retinoid X receptor and a eukaryotic cell sample with one or more agents t and assaying a protein in the test sample for an altered modification state as compared to a control modification state, where the presence of the altered modification state indicates that at least one of the one or more agents is an effective agent that modulates a biological activity of the retinoid X receptor. [0017] Further provided by the invention is a method for identifying an improved effective agent that modulates a biological activity of a nuclear hormone receptor. The method includes the steps of contacting the nuclear hormone receptor with one or more agents and a eukaryotic cell sample to form a test sample under conditions suitable to form a receptor- isolating the receptor-containing complex
providing to the isolated receptor-containing complex conditions suitable for modification of the receptor- assaying the isolated receptor-containing complex, or a component thereof, for an altered modification state occurring in the isolated receptor-containing complex as compared to a contro and assaying for direct transcriptional activity of the nuclear hormone receptor contacted with the one or more agents, where the presence of an altered modification state combined with the absence of direct transcriptional activity indicates that at least one of the one or more agents is an improved effective agent that modulates a biological activity of the nuclear hormone receptor. [0018] The present invention also provides a method for identifying an effective agent that modulates protein kinase A activity associated with a nuclear hormone receptor. A method of the invention is practiced by contacting nuclear hormone receptor with one or more agents and a cell sample to form a test sample under conditions suitable to form a receptor- isolating the receptor-containing complex
contacting the isolated receptor-containing complex with a protein kinase A substrate under conditions suitable for phosphorylat and assaying the substrate for an altered phosphorylation state as compared to a control phosphorylation state, where the presence of the altered phosphorylation state indicates that at least one of the one or more agents is an effective agent that modulates protein kinase A activity associated with the nuclear hormone receptor. In a method of the invention for identifying an effective agent that modulates protein kinase A activity associated with a nuclear hormone receptor, the altered phosphorylation state can be an increased or decreased phosphorylation state. [0019] A substrate useful in a method of the invention can be, for example, a purified substrate and, in one embodiment, has a Km of less than 20 μM for protein kinase A. Peptide substrates, including purified peptide substrates, are useful in the methods of the invention and, in one embodiment, the peptide substrates have at most ten residues. A peptide substrate useful in the invention can include, for example, the amino acid sequence Arg-X-Ser, Arg-Arg-X-Ser, Arg-X-X-Ser, Lys-Arg-X-X-Ser or Arg-X-Lys-Arg-X-X-Ser-X (SEQ ID NO: 113), where X is independently any amino acid. A peptide substrate useful in the methods of the invention can include, for example, the sequence Arg-Arg-X-Ser and, in particular embodiments, can contain the sequence LRRASLG (SEQ ID NO: 59) or GRTGRRNSI (SEQ ID NO: 60). In particular embodiments, such a peptide substrate has a length of at most ten residues. A method of the invention also can be practiced with purified protein substrates, for example, myelin basic protein. [0020] The invention also provides a method for identifying an effective agent that modulates protein kinase A activity associated with a nuclear hormone receptor by contacting the nuclear hormone receptor with one or more agents and a cell sample to form a test sample under conditions suitable to form a receptor-containing complex, where the receptor is retinoid X receptor (RXR), retinoic acid receptor (RAR) or peroxisome proliferator activated receptor (PPAR); isolating the receptor-containing complex
contacting the isolated receptor-containing complex with a protein kinase A substrate under conditions suitable for phosphorylat and assaying the substrate for an altered phosphorylation state as compared to a control phosphorylation state, where the presence of the altered phosphorylation state indicates that at least one of the one or more agents is an effective agent that modulates protein kinase A activity associated with the nuclear hormone receptor. In one embodiment, the nuclear hormone receptor is a retinoid X receptor (RXR) and can be, for example RXRα. [0021] A method of the invention also can be practiced with a nuclear hormone receptor, which is a variant or fusion protein rather than wild type receptor. In one embodiment, the nuclear hormone receptor is a variant with an increased ratio of cytoplasmic to nuclear localization as compared to wild type nuclear hormone receptor. In another embodiment, the nuclear hormone receptor is a fusion protein that contains a heterologous membrane-anchoring domain. In a further embodiment, the nuclear hormone receptor is a fusion protein containing a pleckstrin homology domain. In yet another embodiment, the nuclear hormone receptor is a fusion protein containing a heterologous epitope tag. [0022] A method of the invention for identifying an effective agent that modulates protein kinase A activity associated with a nuclear hormone receptor can be practiced with endogenous or exogenous receptor. In one embodiment, the invention is practiced with a cell sample containing an exogenous nucleic acid molecule encoding the nuclear hormone receptor. In another embodiment, the nuclear hormone receptor is endogenous to the cell sample. A cell sample to be used in a screening methods of the invention can contain an exogenous nucleic acid molecule encoding a heterodimeric partner of the nuclear hormone receptor or an exogenous nucleic acid molecule encoding a catalytic subunit of protein kinase A. In such a cell sample, the nuclear hormone receptor can be endogenous or exogenous to the cell. [0023] A method of the invention can be practiced with a variety of types of cell samples. In one embodiment, the cell sample contains viable eukaryotic cells, and, in another embodiment, the cell sample is a eukaryotic whole cell sample. In one embodiment, isolation of the substrate is performed in the presence of the one or more agents. In a method of the invention, isolation of the receptor-containing complex can be performed by a variety of means, for example, by specific binding to the receptor-containing complex. [0024] Further provided by the invention is a method for identifying an effective agent that modulates protein kinase A activity associated with a nuclear hormone receptor by contacting the nuclear hormone receptor with one or more agents and a cell sample to form a test sample under conditions suitable to form a receptor- immunoprecipitating the receptor-containing complex from the test sample to isolate the receptor- contacting the isolated receptor-containing complex with a protein kinase A substrate under conditions suitable for phosphorylat and assaying the substrate for an altered phosphorylation state as compared to a control phosphorylation state, where the presence of the altered phosphorylation state indicates that at least one of the one or more agents is an effective agent that modulates protein kinase A activity associated with the nuclear hormone receptor. Immunoprecipitation can be performed, for example, using antibody immunoreactive with the nuclear hormone receptor. Where the nuclear hormone receptor is a fusion protein containing a heterologous epitope tag, the immunoprecipitation can be performed, for example, using antibody immunoreactive with the epitope tag. A variety of means can be used to assay the protein kinase A substrate for an altered phosphorylation state including, for example, detecting radiolabeled substrate.BRIEF DESCRIPTION OF THE DRAWINGS [0025] FIG. 1 shows the amino acid sequences of human RXRα (SEQ ID NO: 1), human RXRβ (SEQ ID NO: 2) and human RXRγ (SEQ ID NO: 3). [0026] FIG. 2 shows a schematic view of nuclear receptor functional domains. Modular diagrams in the top panel are drawn to scale and aligned at the conserved E1 domain. The DNA-binding region consists of two (Cys)4-type zinc-finger motifs (C4 Zn fingers), followed by a C-terminal extension (CTE) of varying length. Dimerization and ligand-binding contacts determined by X-ray crystallography also are shown in the top panel. The center panel shows a selected portion of the DNA-binding domain for several receptors, with solid circles indicating DNA contacts as determined by X-ray crystallography for human RXRα, human TRβ, human ERα and rat GR. Jellyfish RXR is shown for comparison. The lower panel details three subregions of the ligand-binding domain in several nuclear hormone receptors, including the conserved E1 domain that supports dimerization and participate h9, which particip and the AF2 region, which contains ligand contacts and effects transactivation. Residues highly conserved among all nuclear hormone receptors are present in the E1 subregion and highlighted. SEQ ID NOS: are shown in parenthesis. [0027] FIG. 3 shows activation of MAP kinases by RXR-specific agonist AGN194204. (A) Activation of MAP kinases (MAPKs) by AGN194204 in 3T3-L1 fibroblasts. 3T3-L1 fibroblasts were starved in DMEM with 0.1% calf bovine serum for overnight and then stimulated with vehicle (DMSO, lane 0), 30 nM insulin (lane 1), 100 nM AGN194204 (lane 2), 30 nM insulin plus 100 nM AGN194204 (lane 3) for 5 minutes, or 100 nM AGN194204 for 30 minutes followed by the additional of 30 nM insulin for 5 minutes (lane 4). Cells were lysed directly with 1×SDS sample buffer and the cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-MAPK. (B) Activation of MAP kinase by AGN194204 in 3T3 differentiated adipocytes. Differentiated 3T3 adipocytes were starved, stimulated, and manipulated as described above. (C) Dose and time dependent activation of MAP kinases by AGN194204 in 3T3-L1 fibroblasts. Cells were starved as described above, then stimulated by different doses of AGN194204 (as indicated in left panel) for 5 minutes and for variable time (as indicated in right panel) at 10-10 M concentration. Detection of MAP kinase phosphorylation was performed as described above with anti-phospho-MAPK from Promega. [0028] FIG. 4 shows the effect of MAPK pathway on epitope-tagged RXR in transfected HEK 293 cells. The top panel shows Western blotting with anti-RXR antibody (D20). The middle panel shows Western blotting with anti-Flag antibody (M2). The bottom panel shows Western blotting with anti-phospho-MAPK antibody. [0029] FIG. 5 shows that RXRα associates with a protein kinase in the presence of RXR-specific ligand. (A) AGN194204-dependent protein kinase activity in Flag-RXR immunocomplexes, which were immunoprecipitated from transfected cells using antibody against the “Flag” epitope tag fused to the RXR receptor. HEK 293 cells were transiently transfected with Flag-RXR expression vector and stimulated without (-) or with (+) AGN-7 M) for 10 minutes. Total cell lysates were immunoprecipitated with anti-Flag antibody (M2). Aliquots of the immunocomplexes were used for the in vitro kinase reaction with [γ-32P]ATP and separated on SDS-PAGE. Phosphorylated proteins were detected by autoradiography (top panel). Additional aliquots of the immunocomplexes were subjected to immunoblotting with polyclonal anti-RXR antisera (D20; bottom panel). (B) Phosphoamino acid analysis. Flag-RXR and the 160-kDa protein were phosphorylated in vitro and separated on SDS-PAGE as described above, then transferred onto PVDF membrane, detected by autoradiography, and cut out for hydrolysis with acid (6 N HCl). The phosphorylated amino acids and standards (P-Ser, P-Thr, and P-Tyr) were separated by thin-layer chromatography. Positions of the standards P-Ser, P-Thr, and P-Tyr were visualized by ninhydrin staining and are indicated by arrows. (C) In vivo metabolic labeling of transfected HEK 293 cells. HEK 293 cells were transfected with Flag-RXR expression vector. Cells were washed with phosphate-free medium and starved for 90 minutes before incubation with [32P] orthophosphate for another 4 hours. Before harvesting, cells were stimulated with vehicle, RXR-specific ligand AGN192404, RXR-specific antagonist AGN195393, or RAR-specific agonist TTNPB for 10 minutes. Total cell lysates were immunoprecipitated with anti-Flag antibody (M2) and subjected to separation on SDS-PAGE. Phosphorylated proteins were detected by autoradiography. [0030] FIG. 6 shows the dose and RXR ligand-dependence of in vitro kinase activity in Flag-RXR immunocomplexes. (A) Dose of AGN194204 dependent kinase activity was determined in HEK 293 cells transfected with Flag-RXR expression vector and stimulated with the indicated dose of RXR-specific ligand AGN194204 for 10 minutes. Cell lysates were immunoprecipitated with anti-Flag antibody (M2) in the absence (-) or presence (+) of AGN194204 in the immunoprecipitation buffer. In vitro kinase reaction with [γ-32P]ATP, separation on SDS-PAGE, and detection of the phosphorylated proteins were performed as described above. (B) RXR-specific ligand dependent kinase activity. HEK 293 cells were transfected with vector alone or Flag-RXR expression vector, and stimulated with vehicle, RXR-specific ligand (AGN194204, AGN195029, AGN192620, AGN195203, and AGN195184), or RXR-specific antagonist AGN195393 for 15 minutes. Total cell lysates were subjected to immunoprecipitation, and the in vitro kinase reaction performed as described above. [0031] FIG. 7 shows a schematic representation of the structure of RXRα deletion mutants. [0032] FIG. 8 shows identification of RXRα regions required for association with a protein kinase. HEK 293 cells transiently transfected with Flag-RXRα or mutant expression vectors were treated with RXR-specific ligand AGN194204 for 10 minutes. Cell lysis, immunoprecipitation with anti-Flag (M2), in vitro kinase assay, and detection of the phosphorylated proteins in the RXRα immunoprecipitated complexes were performed as described above. [0033] FIG. 9 shows identification of proteins interacting with Flag-RXR in transfected HEK 293 cells by two-dimensional gel electrophoresis. HEK 293 cells were transfected with Flag-RXR and lysed 48 hours after transfection. Immunocomplexes were prepared with anti-Flag antibody (M2), solubilized in IEF-sample buffer, and applied to separation on two-dimensional gel electrophoresis. Proteins were visualized by silver staining. [0034] FIG. 10 shows the influence of heat shock protein 90 (HSP90) inhibition on RXR-associated kinase activity. HEK 293 cells were transfected with Flag-RXR expression vector and treated with 1 μM of the HSP90 inhibitor geldanamycin for 30 minutes or overnight as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody (M2) prior to in vitro kinase reaction with [γ-32P]ATP, separation on SDS-PAGE, and detection of the phosphorylated proteins as described above. [0035] FIG. 11 shows the influence of overexpressed MEK1 mutants and protein tyrosine kinase JAK1 on RXRα-associated kinase activity in transiently transfected HEK 293 cells. (A) Influence of the MAPK pathway on the kinase in the FLAG-RXR immunocomplexes. HEK 293 cells were cotransfected with Flag-RXR expression vector, together with vector alone, wild-type MEK1, constitutively active MEK1 mutant (CA), or dominant negative MEK1 mutant (DN). Transfected cells were lysed 48 hours after transfection with or without prior stimulation with AGN194204 for 10 minutes. Preparation of immunocomplexes and in vitro kinase reactions were carried out as described above. (B) Influence of overexpressed JAK1 on kinase activity in Flag-RXR immunocomplexes. HEK 293 cells were transfected with vector alone (lane 1) or Flag-RXR (lanes 2-8) plus varying concentrations of JAK1 expression vector (lane 2: 0 lane 3: 8 lane 4: 24 lane 5: 74 lane 6: 220 lane 7: 670 lane 8: 2 μg). Transfected cells were lysed following incubation with AGN194204 for 10 in vitro kinase reactions were then performed as described above. [0036] FIG. 12 shows pharmacokinetic analysis of RXR compounds in induction of kinase activity using a gel-based kinase assay. (A) Phosphorylation of 160 kDa protein. (B) Phosphorylation of RXRα. Stable RXRα-expressing 293 cells were treated with compounds at indicated doses for 10 minutes. Cells were then harvested and analyzed by gel-based kinase assays using γ-32P-ATP as described in FIG. 5A. Phosphorylated proteins were quantified by exposing gels to a PhosphorImager (Molecular Dynamics). [0037] FIG. 13 shows a high throughput kinase assay for screening nuclear hormone receptor compounds capable of activating kinase activity. (A) This panel illustrates the principle of a multiple-well plate-based kinase assay using RXR receptor. Extracts are prepared from Flag-RXR-overexpressing cells and added to a multiple-well plate in which the wells are coated with scintillation materials and protein A. In the presence of RXR compounds, RXR forms complexes with kinases and p160 protein. Addition of anti-Flag antibodies enables the complexes to be brought to the proximity of the scintillation materials on the well wall and specifically attached to the plate. Non-specific proteins are washed away by buffer. The kinase reaction is initiated by adding kinase buffer containing γ-33P-ATP. After the reaction, phosphorylated complexes remain attached to the plate while free γ-33P-ATP is washed away. Radioactivity generated from the phosphorylated proteins is quantified using a plate-format scintillation counter, which represents the compound activity. (B) An exemplary high throughput assay. 293 cells were transfected with Flag-RXRα or Flag-PH-RXRα and treated with or without AGN194204. Cell lysis was performed as described in FIG. 5A. Recombinant Flag-PH-RXRα contained the plekstrin homology domain (PH) from IRS-1 between Flag and RXRα. Cell lysate (200 μl) was mixed with the anti-Flag antibody, added to each well of a 96-well Flashplate (NEN-Dupont) coated with protein A, and then incubated at 4° C. for 4 hours with gentle shaking. Approximately 200 μg total cell lysate and 0.5 μg antibody were added per well. After the incubation, the plate was washed four times with the lysis buffer with or without AGN194204. Kinase buffer (50 μl), which contains 40 mM Tris-HCL at pH 7.5, 10 mM MgCl2, and 25 μCi γ-33P-ATP (Amersham), were added to each well and incubated at room temperature for 20 minutes. After the reaction, the plate was washed 4 times with 200 μl of kinase buffer without the γ-33P-ATP and then counted in the Microbeta counter (Wallac). [0038] FIG. 14 shows determination of protein kinase A activity in cells transfected with RXRα or PH-RXRα. FIG. 14A shows the effect of several protein kinase inhibitors on PKA activity using kemptide (LRRASLG; SEQ ID NO: 59) as a substrate. 293 cells were transfected with PH-RXRα. Sample 1: 0.1% DMSO (10 minutes). Sample 2: 10-7 M AGN minutes). Sample 3: 0.1% DMSO (30 minutes). Sample 4: 10-7 M AGN minutes). Sample 5: 10-7 M AGN minutes) with inhibitors of PKC and CaMDK. Sample 6: 10-7 M AGN minutes) with PKA inhibitor. Sample 7: No kemptide substrate or AGN194204. Sample 8: 10-7 M AGN194204 with no kemptide substrate. [0039] FIG. 14B shows determination of PKA activity using substrate GRTGRRNSI (SEQ ID NO: 60). Sample 1: 0.1% DMSO (10 minutes). Sample 2: 10-7 M AGN minutes). Sample 3: 0.1% DMSO (30 minutes). Sample 4: 10-7 M AGN minutes). Sample 5: No immunoprecipitate. [0040] FIG. 14C shows determination of PKA activity in 293 cells transfected with PH-RXRα or RXRα using kemptide (LRRASLG; SEQ ID NO: 59) as a substrate. 293 cells were transfected with PH-RXRα (samples 1 to 4) or RXRα (samples 5 and 6) and treated with DMSO or AGN194204 for 10 minutes. Sample 1: 0.1% DMSO. Sample 2: 10-7 M AGN194204. Sample 3: 0.1% DMSO with PKA inhibitor. Sample 4: 10-7 M AGN194204 with PKA inhibitor. Sample 5: 0.1% DMSO. Sample 6: 10-7 M AGN194204. [0041] FIG. 15A shows PKA activity in 3T3-L1 adipocytes in the absence and presence of AGN194204. Untransfected cells were incubated in the absence and presence of AGN194204 prior to immunoprecipitation with antibody against RXRα. Kinase activity was determined in the immunoprecipitate using kemptide (LRRASLG; SEQ ID NO: 59) as a substrate. Samples 1,3: 0.1% DMSO (10 minutes). Samples 2,4: 10-7 M AGN minutes). [0042] FIG. 15B shows PKA activity in cells stably overexpressing FLAG-RXRα and transfected with the catalytic subunit of PKA. Where indicated, cells were treated with AGN194204 for 10 minutes. Sample 1: N immunoprecipitation with beads only. Sample 2: 10-7 M AGN194204; immunoprecipitation with beads only. Sample 3: N immunoprecipitation with anti-FLAG antibody. Sample 4: 10-7 M AGN194204; immunoprecipitation with anti-FLAG antibody. Sample 5: N immunoprecipitation with beads only. Sample 6: 10-7 M AGN194204; immunoprecipitation with beads only. Sample 7: N immunoprecipitation with anti-PKA antibody. Sample 8: 10-7 M AGN194204; immunoprecipitation with anti-PKA antibody. [0043] FIG. 16 shows that the RXRα associated protein kinase phosphorylates exogenous protein substrates. HEK 293 cells were transiently transfected with vector alone or FLAG-RXR expression vector and stimulated without (-) or with (+) 10-7 M AGN194204 for 10 minutes. Total cell lysates were immunoprecipitated with anti-FLAG antibody (M2). Immunoprecipitated material was used for in vitro kinas reaction with γ-32P-ATP alone or with addition of exogenous mixed histones or myelin basic protein (MBP). The phosphorylated proteins were separated on SDS-PAGE and detected by autoradiography. [0044] FIG. 17 shows that RXRα is associated with and phosphorylated by protein kinase A. A. In vivo ligand independent association between RXRα and PKA. 293 cells were transfected with expression vectors encoding FLAG-RXRα and the catalytic subunit of PKA and treated with or without AGN194204 for 10 minutes. Left panel: Cells were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitates analyzed by SDS-PAGE and blotting with anti-PKA antibody. Right panel: Cells were immunoprecipitated with anti-PKA antibody, and the immunoprecipitates analyzed by SDS-PAGE and blotting with anti-FLAG antibody. B. Phosphorylation of RXRα by PKA. RXRα receptors immunoprecipitated from 293 cells treated with or without AGN194204 were incubated with purified PKA and γ-ATP. PKA I and PKC I indicate inhibitors of PKA and PKC, respectively. [0045] FIG. 18 shows exemplary pleckstrin homology (PH) domains. Amino acid identity in five or more PH domains is indi amino acid homology in five or more PH domains is indicated by grey shading. Plec-N, human pleckstrin, residues 1 to 105 (SEQ ID NO: 114); Plec-C, human pleckstrin, residues 239 to 350 (SEQ ID NO: 115); RasGAP, human Ras GTPase activating protein, residues 292 to 404 (SEQ ID NO: 116); Akt, human serine/threonine kinase AKT2, residues 1 to 118 (SEQ ID NO: 117); Spectrin, human β-spectrin, residues 2192 to 2311 (SEQ ID NO: 118); BARK, human β-adrenergic receptor kinase, residues 553 to 656 (SEQ ID NO: 119); Tiam1-PH1, mouse GDP-GTP exchanger Tiam-1, residues 440 to 546 (SEQ ID NO: 120); Grb7, mouse growth factor receptor bound protein 7, residues 224 to 345 (SEQ ID NO: 121); Dynamin, human GTPase dynamin 1, residues 515 to 629 (SEQ ID NO: 122); Irsl, rat insulin receptor substrate 1, residues 7 to 119 (SEQ ID NO: 123); RasGRFPH1, rat Ras guanine nucleotide release factor, residues 17 to 134 (SEQ ID NO: 124); RasGRFPH2, rat Ras guanine nucleotide release factor, residues 451 to 584 (SEQ ID NO: 125); &10& indicates omitted residues EKGKINKGRL (SEQ ID NO: 126); Dbl, human product of the dbl proto-oncogene, residues 704 to 813 (SEQ ID NO: 127); Vav, mouse product of the vav proto-oncogene, residues 397 to 508 (SEQ ID NO: 128); Sos, human son of sevenless protein, residues 438 to 548 (SEQ ID NO: 129); Bcr, human break point cluster region gene product, residues 728 to 870 (SEQ ID NO: 130); &19& indicates omitted residues GSKATERLKK KLSEQESLL (SEQ ID NO: 131); Btk, human Bruton's tyrosine kinase, residues 1 to 137 (SEQ ID NO: 132); &20& indicates omitted residues PPERQIPRRG EESSEMEQIS (SEQ ID NO: 133); PLC-delta, rat phospholipase Cδ1, residues 15 to 134 (SEQ ID NO: 134); PLC-gamma, human phospholipase Cy1, residues 22 to 146 (SEQ ID NO: 135); &7& indicates omitted rat residues DRYQEDP (SEQ ID NO: 136).DETAILED DESCRIPTION OF THE INVENTION [0046] The present invention is directed to the surprising discovery that a kinase can be directly associated with a nuclear hormone receptor such as a retinoid X receptor, and can phosphorylate this receptor as well as associated proteins in a ligand-dependent manner. Based on these findings, the invention provides novel assays for identifying nuclear hormone receptor ligands and regulators. These assays do not depend upon transcriptional activity of the nuclear hormone receptor, nor do they require that the nuclear hormone receptor have DNA-binding activity. [0047] As disclosed herein, in vivo labeling with [32P] ortho-phosphate demonstrated that retinoid X receptor a (RXRα) can be modified by phosphorylation and that this phosphorylation can be stimulated by the RXR-specific agonist AGN194204 but not by the RXR antagonist AGN195393, or the RAR-specific agonist TTNPB (see, also, Table 2). As further disclosed herein in Example IIA, HEK 293 cells were transfected with an epitope tagged Flag-RXR complexes immunoprecipitated with anti-Flag antibody were resuspended in kinase buffer and used for an in vitro kinase reaction with [γ-32P] ATP. Phosphorylation of RXR and another protein in the RXR immunocomplex of about 160 kDa was observed following treatment with RXR-specific ligand (see FIG. 5A). Phosphoamino acid analysis of [γ-32P]ATP-labeled RXR and 160 kDa protein demonstrated that these proteins are phosphorylated on serine and threonine but not tyrosine, indicating that a serine/threonine kinase is associated with RXR (Example IIB and FIG. 5B). As shown in Example IIC, phosphorylation was dependent on any of several RXR-specific agonists but was not stimulated by the RXR-specific antagonist AGN195393 or the RAR-specific agonist AGN195183 (TTNPB), indicating that kinase activity was associated with the RXR receptor in a ligand-dependent manner (FIG. 6B). As further disclosed herein, various RXR deletion mutants containing an intact E region, which contains the critical core of the ligand-binding domain, recruited kinase activity to the immunocomplex in the presence of RXR-specific agonist, demonstrating that the RXR ligand-binding domain is sufficient for RXR interaction with the associated kinase (see Example III). Additional results disclosed herein demonstrate that cotransfection of the tyrosine kinase JAK1 with RXR in HEK 293 cells augmented ligand-dependent phosphorylation of RXRα and p160 in RXR-containing immunocomplexes. [0048] Based on these discoveries, the present invention provides a method for identifying an effective agent that modulates a biological activity of a nuclear hormone receptor. The method includes the steps of contacting the nuclear hormone receptor with one or more agents and a eukaryotic cell sample to form a test sample under conditions suitable to form a receptor- isolating the receptor-containing complex
providing to the isolated receptor-containing complex conditions suitable for modification of the receptor- and assaying the isolated receptor-containing complex for an altered modification state occurring in the receptor-containing complex as compared to a control modification state, where the presence of the altered modification state indicates that at least one of the agents is an effective agent that modulates a biological activity of the nuclear hormone receptor. In a method of the invention, the altered modification state can be, for example, an increased or decreased phosphorylation state. [0049] In the methods of the invention, an effective agent that modulates a biological activity of a nuclear hormone receptor is identified by an altered modification state occurring in an isolated receptor-containing complex. Such an altered modification state can be, without limitation, an altered phosphorylation state of the nuclear hormone receptor such as increased phosphorylation of an RXR receptor or an altered phosphorylation state of an associated 160 kDa protein such as increased phosphorylation of a 160 kDa protein. In one embodiment, the altered modification state is any modification state other than a phosphorylation state of the cofactor TIF1α. In another embodiment, the altered modification state is any modification state other than a phosphorylation state of MAP kinase (MAPK). In a further embodiment, the altered modification is any modification state other than a phosphorylation state of thyroid hormone receptor. In yet a further embodiment, the altered modification state is any modification state other than a phosphorylation state of TIF1α, MAPK, or thyroid receptor, or any combination thereof. [0050] As used herein, the term “altered modification state” means a post-translational modification of one or more components of the isolated receptor-containing complex which is significantly increased, decreased or qualitatively distinct from a control modification state of the same one or more components. Such an altered modification state can result from an enzymatic modification or from a nonenzymatic, chemical modification, and can be reversible or irreversible. Exemplary modification states include phosphorylation states, for example, the extent of serine, threonine, tyrosine, histidine or ly adenylation and ADP- methylation states, for example, the extent of methylation at the α-amino group or on the side chains of Lys, Arg, and H
and conformational states. [0051] One skilled in the art understands that an “altered modification state” can represent a quantitative or qualitative difference as compared to a control modification state. For example, where the control modification state is a particular amount of serine phosphorylation on a component protein or proteins, an altered modification state can be, for example, an increased amount of serine phosphorylation on the same or a different residue of the same component protein or proteins, a decreased amount of serine phosphorylation on the same or a different residue of the same component protein or proteins, or an equal amount of tyrosine or threonine phosphorylation on the same component protein or proteins. [0052] It is understood that, where there is a quantitative difference between the altered and control modification states, the difference does not fall within the inherent variability of the assay. In general, where the altered modification state is increased or decreased as compared to the control modification state, the altered modification state is increased or decreased by 50% or more as compared to the control modification state. An altered modification state also can represent an increase or decrease of 100% or more, or an increase or decrease of 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold or more relative to the control modification state. [0053] An altered modification state can be determined for the receptor-containing complex as a whole, or can be determined for one or more individual components of the complex such as the nuclear hormone receptor, a heterodimeric partner of the nuclear hormone receptor or another associated protein such as a kinase or a 160 kDa protein. It is understood that an altered modification state of any one component of the receptor-containing complex serves to identify an “effective agent,” regardless of whether there is an altered modification state of any other components of the isolated receptor-containing complex. [0054] As used herein, the term “control modification state” means the same post-translational modification of the same one or more components occurring in an isolated receptor-containing complex, where the nuclear hormone receptor contained in the isolated complex has not been contacted with the one or more agents. [0055] A control modification state is determined using a cell sample such as a eukaryotic cell sample that is not contacted with the one or more agents to be assayed. For convenience, a eukaryotic cell sample that is not contacted with the one or more agents is designated herein the “control eu” a eukaryotic cell sample contacted with the one or more agents is referred to herein as a “test eukaryotic cell sample.” The control eukaryotic cell sample can be the same sample as the test eukaryotic cell sample, provided that the control modification state is determined prior to contacting the test eukaryotic cell sample with the one or more agents. The control eukaryotic cell sample also can be different from the test eu the control eukaryotic cell sample can be contacted, if desired, with vehicle or with reference agent having a known effect on the modification state to be assayed. [0056] To determine a control modification state in a control eukaryotic cell sample different from the test eukaryotic cell sample, one skilled in the art would use a corresponding cell or tissue type and would culture the cells or tissue under the same conditions as the cells or tissue from which the test eukaryotic cell sample is prepared. Such a corresponding cell or tissue type preferably has the same amount and type of nuclear hormone receptor and the same amount and type of heterodimeric partner, and, most preferably, is of the identical cell or tissue type used to prepare the test eukaryotic cell sample. Even more preferably, cells or tissue of the identical cell or tissue type are grown under the same conditions as the cells or tissue from which the eukaryotic cell sample is prepared. As disclosed herein, for example, HEK 293 cells were transiently transfected with FLAG-RXRα and contacted with known RXR- as a control, the same transiently transfected HEK 293 cells were treated with control vehicle (see Example II). One skilled in the art understands that transiently transfected FLAG-RXRα HEK 293 cells or another immortalized human kidney cell line expressing a similar level of RXRα can be used as a control eukaryotic cell sample, where the test eukaryotic cell sample is FLAG-RXRα transfected HEK 293 cells. One skilled in the art further understands that a control modification state can be determined empirically before, after, or simultaneously with an assay performed to determine an altered modification state or can be determined, if desired, by referencing a historical value. [0057] As disclosed herein, an altered modification state can occur rapidly upon treatment with one or more agents. As disclosed herein, RXRα is associated with a protein kinase in cells treated with RXR-specific ligand for only 10 minutes. Thus, in one embodiment of the invention, a nuc