Abstract:
Disclosed herein are compositions and methods used for detecting and measuring ligands for nuclear receptors and intracellular lipid binding proteins in both in vitro and in vivo samples.

Description:
RELATED APPLICATIONS 
     The present application claims priority to and the benefit of U.S. Provisional Application 60/634,764, filed Dec. 9, 2004. 
    
    
     GOVERNMENTAL SUPPORT 
     The present invention was supported in part by a grant from the National Institutes of Health, RO1 CA68150. Therefore, the government has certain rights to this invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to nuclear hormone receptors, intracellular lipid binding proteins (iLBP), and their intracellular binding ligands. In particular, the invention pertains to detecting and measuring the concentration of ligand that binds to their cognate nuclear receptors and iLBPs. 
     BACKGROUND OF THE INVENTION 
     One method for cells receiving signals from the external environment is through ligand-receptor interactions. In one scenario, the receptor is integral to the cell and is embedded within the plasma membrane bilayer of the cell. The receptor may traverse the entire bilayer or reside within the layer having a portion of it exposed on the surface of the cell. Typical ligands that interact with these receptors are hydrophilic molecules. Through this interaction, secondary events are triggered leading to changes within the cytosol of the cell such as protein phosphorylation. 
     In contrast to hydrophilic ligands, hydrophobic molecules, such as steroid and thyroid hormones, pass through the plasma membrane of a cell and interact with specific receptor proteins residing within the cytoplasm or nucleus. Other hydrophobic molecules, such as retinoic acid, are metabolically synthesized in the cell itself. Similarly to externally derived hydrophobic molecules, hydrophobic compounds that are synthesized intracellularly interact with intracellular receptor proteins to exert their biological effects. 
     For example, steroid hormones (testosterone, estrogen, etc.) dissociate from plasma-binding proteins and cross the plasma membrane and enter target cells. Steroid hormone receptors are tissue-specific binding proteins found in low concentrations in the cytoplasm of the cell. When steroid receptors are occupied by ligand they change conformation and become activated with enhanced affinity for nuclear chromatin. The activated hormone-receptor complex accumulates in the nucleus bound to chromosomal DNA containing acceptor sites for the complex. The high affinity interaction of the steroid-hormone receptor complex with nuclear chromatin results in activation of DNA transcription and in the synthesis of specific mRNAs. 
     Hydrophobic ligands other than steroids similarly bind to and activate different nuclear receptors. For example, the receptors for thyroid hormones are found in the nucleus even in the absence of their ligand. Thyroid hormones enter cells and travel to the nucleus. Specific genes are under thyroid hormone control, and they are transcribed to particular mRNA in response to this ligand. In turn, translation of the mRNA results in the synthesis of specific cell proteins. 
     In addition to nuclear receptors, hydrophobic ligands bind in cells to proteins that are members of a family of homologous proteins termed intracellular lipid binding proteins (iLBPs). These proteins reside in the cytosol of cells. Some iLBPs move to the nucleus when they bind their cognate ligands. For example, the iLBP called adipocyte fatty acid binding protein (adipocyte FABP) moves from the cytosol to the nucleus following binding of its cognate ligands. Intracellular lipid binding proteins often share ligands with particular nuclear receptors. For example, the anti-diabetic drug troglitazone binds to the nuclear receptor termed peroxisome proliferator activated receptor γ (PPARγ) and also associates with adipocyte FABP. 
     Another example of nuclear receptor ligands are the vitamin A metabolites retinoic acids (“RA”) and their synthetic derivatives, collectively known as retinoids, which can be used in the treatment of a variety of pathologies ranging from dermatological disorders to cancer. 
     The retinoid members of the nuclear hormone receptor superfamily are responsive to compounds referred to as retinoids, which include retinoic acid and a series of natural and synthetic derivatives which have been found to exert profound effects on development and differentiation in a wide variety of systems. 
     Retinoic acid-dependent transcription factors, referred to as RARs (retinoic acid receptors), have been identified. Currently, three different RAR subtypes (alpha, beta and gamma) and several isoforms of each are known to exist in mammals. RARs share sequence homology with other members of the superfamily of nuclear hormone receptors. This family of proteins encompasses ligand-dependent transcription factors that regulate the expression of particular target genes upon binding of specific ligands. Different RAR subtypes are expressed in distinct patterns throughout development and in the mature organism. 
     Additional members of the nuclear hormone superfamily of receptors that respond to retinoids have been identified. These are termed retinoid X receptors (RXRs): RXR-α (see Mangelsdorf et al., in Nature 345: 224-229 (1990)), RXR-β (see Hamada et al., Proc. Natl. Acad. Sci. USA 86: 8289-8293 (1989)), and RXR-γ (see Mangelsdorf et al., Genes and Development 6:329-344 (1992)). 
     Although both RARs and RXRs respond to retinoic acids, these receptors differ in several important aspects. First, RAR and RXR are significantly divergent in primary structure. These sequence differences are reflected in differential responsiveness of RAR and RXR to various vitamin A metabolites and synthetic retinoids. In addition, distinctly different patterns of tissue distribution are seen for RAR and RXR. Furthermore, while RXR can activate transcription as a homodimer, i.e. on its own, the transcriptional activity of RAR is mediated through RAR-RXR heterodimers. Finally, RXR homodimers bind to response elements that are distinct from the DNA sequences that are recognized by RAR-RXR heterodimers, and thus RXR-RXR and RXR-RAR complexes regulate the expression of different genes. 
     Retinoid therapy is complicated by the toxicity of these compounds at pharmacological doses. Existing methods for retinoid detection and quantification consist of organic solvent extractions and HPLC analyses, procedures that are too time-consuming and expensive to be used in the hospital/clinic setting. Consequently, as currently practiced, retinoid treatment is not individualized for particular patients but is administered by ‘standard’ dosing. This is so despite the high toxicity of these compounds and the large patient-to-patient variability in resulting plasma concentrations of RA. 
     Certain diseases affect or are affected by processes that alter physiological events that are associated with specific ligand-receptor interactions. Clearly, the detection and quantitation of ligands that bind to nuclear receptors is important diagnostically as well as for monitoring physiological effects during a treatment regime. 
     SUMMARY OF THE INVENTION 
     The present invention relates to nuclear receptors, intracellular lipid binding proteins, and their cognate intracellular binding ligands. Embodiments of the present invention are directed toward detecting and quantitating ligands of nuclear receptors and intracellular lipid binding proteins. In particular, the present invention pertains to methods designed to measure ligands whose cognate receptor resides intracellularly, either in the cytoplasm or in the nucleus of a cell. In one particular aspect, the receptors are nuclear hormone receptors. In another aspect, the receptors are intracellular lipid binding proteins. The methods of the present invention are directed to detecting and measuring ligands from various different source materials. 
     One embodiment of the present invention is directed to the detection and measurement of a ligand from a sample. In one aspect of this embodiment, the sample preparation comprises a homogenous ligand preparation. In another aspect, the sample comprises a heterogeneous composition of ligands, wherein the population of ligands differ in their affinity for a particular receptor protein (also referred to as a sensor protein). In this embodiment, a titration curve is established using known quantities of a standard ligand that interacts with a known receptor protein. For example, the ligand can be retinoic acid and the receptor can be a retinoid nuclear receptor or a cellular retinoic acid binding protein. Typically, the receptor protein is labeled prior to incubation with a ligand. Following the establishment of a titration curve, the sample containing a putative ligand can be admixed with the labeled receptor protein preparation. The signal generated following this incubation can be compared to the titration curve in order to ascertain the concentration of the sample ligand. The receptor used for establishing the standard curve is typically the same receptor used in the assay of the sample ligand. 
     Another embodiment of the present invention is directed to the screening of ligands that bind to nuclear receptors and to measurements of the binding affinities of such ligands. In one aspect of this embodiment, synthetic compounds that potentially serve as therapeutic agents, acting either as activators or inhibitors of particular receptors, are tested for their capacity for receptor binding. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       For a better understanding of the present invention, reference in the detailed description is made to the accompanying figures, wherein: 
         FIG. 1  illustrates the position of L29 residue of the CRABP-I protein in the (B) absence (apo) and (A) presence (holo) retinoic acid; there is a shift in position of L29 upon binding of retinoic acid; 
         FIG. 2  A shows the amino acid sequences of mutants of intracellular lipid binding proteins to be used as sensor proteins to detect and measure different cognate ligands; * indicates mutation point; B is a Table of names and accession numbers iLBPs and exact nature of point mutations; 
         FIG. 3  is a titration plot of fluorescein-labeled mutant (L29C) CRABP-I with all trans-retinoic acid; 
         FIG. 4  is a titration plot of fluorescein-labeled mutant RXR with 9-cis-RA; ligand binding was followed by monitoring changes in the fluorescence of the labeled protein (λex—490 nm; λem.—517 nm); 
         FIG. 5  is a calibration curve for use in measurements of retinoic acid in cell extracts using fluorescein-labeled CRABP-I-L29C; it shows a linear relationship between initial slopes of titrations of mutant BMF-CRABP-I and concentration of retinoic acid in standard solutions; and 
         FIG. 6  is an example for using the protocol to measure the degradation of retinoic acid in cancer cells, a time course for disappearance of retinoic acid from mammary carcinoma MCF-7 cells is shown; MCF-7 cells were treated with 1 μM retinoic acid for 1 hour; Retinoic acid was removed from the media, cells were lysed in ethanol at the indicated times, and retinoic acid concentrations were monitored using the BMF-CRABP-I-L29C assay; Inset shows the same data plotted on a log scale to extract the half-life of the compound in the cells. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to nuclear receptors, intracellular lipid-binding proteins (iLBP), and their intracellular binding ligands. Embodiments of the present invention are directed toward detecting and quantitating ligands of nuclear receptors and iLBPs. In particular, the present invention pertains to methods designed to measure ligands whose cognate receptor resides within a cell. The methods of the present invention are directed to detecting and measuring ligands which can originate from a variety of different source materials. Another embodiment of the present invention is directed towards identification and screening of ligands that potentially bind to nuclear receptors and to measurements of their receptor binding affinities. In particular, the methods serve to test whether natural or synthetic compounds can bind to a particular receptor and enable ready screening of multiple compounds. 
     There is significant homology between the various known nuclear hormone receptors. The super-family of nuclear receptors is comprised of hormone binding proteins that operate as ligand-dependent transcription factors. The family contains several branches including steroid receptors, receptors that belong to the retinoid/thyroid class, and receptor for which specific ligands have not yet been identified. These hormone binding proteins have the intrinsic ability to bind to specific DNA sequences. Following binding, the transcriptional activity of target gene (i.e., a gene associated with the specific DNA sequence) is modulated as a function of the ligand bound to the receptor. 
     The DNA-binding domains of all of these nuclear receptors are related, consisting of 66-68 amino acid residues, and possessing about 20 invariant amino acid residues, including nine cysteines. See, U.S. Pat. No. 6,576,676, the entire teaching of which is incorporated herein by reference. 
     A member of the superfamily can be identified as a protein which contains the above-mentioned invariant amino acid residues, which are part of the DNA-binding domain of steroid receptors such as the human glucocorticoid receptor (amino acids 421-486), the estrogen receptor (amino acids 185-250), and the mineralocorticoid receptor (amino acids 603-668), and retinoid/thyroid receptor-like proteins such as the human retinoic acid receptor (amino acids 88-153). The highly conserved amino acids of the DNA-binding domain of members of the superfamily are as follows: 
     Cys-X-X-Cys-X-X-Asp*-X-Ala*-X-Gly*-X-Tyr*-X-X-X-X-Cys-X-X-Cys-Lys*-X-Phe-Phe-X-Arg*-X-X-X-X-X-X-X-X-X-(X-X-) Cys-X-X-X-X-X-(X-X-X-) Cys-X-X-X-Lys-X-X-Arg-X-X-Cys-X-X-Cys-Arg*-X-X-Lys*-Cys-X-X-X-Gly*-Met (SEQ ID No 1), 
     wherein X designates non-conserved amino acids within the DNA-binding domain; the amino acid residues denoted with an asterisk are residues that are almost universally conserved, but for which variations have been found in some identified hormone receptors; and the residues enclosed in parenthesis are optional residues (thus, the DNA-binding domain is a minimum of 66 amino acids in length, but can contain several additional residues). 
     Members of the nuclear hormone superfamily of receptors include steroid receptors such as glucocorticoid receptor, mineralocorticoid receptor, progesterone receptor, androgen receptor, and the like; plus thyroid/retinoid-like receptors such as retinoid receptors (RAR-α, RAR-β, RAR-γ receptors, and the like), plus RXR-α, RXR-β, RXR-γ receptors, and the like; thyroid receptors, such as TR-α, TR-β, and the like; the vitamin D receptor as well as other gene products which, by their structure and properties, are considered to be members of the superfamily. For a comprehensive list of nuclear receptor family members see: The Nuclear Receptor FactsBook, Laudet, V. and Gronemeyer, H. 2002, Academic Press, London and San Diego, the entire teaching of which is incorporated herein by reference. 
     In addition to binding to nuclear receptors, hydrophobic ligands associate in cells with members of the family of homologous proteins known as Intracellular Lipid Binding Proteins (iLBP). Members of this family are small soluble proteins of a molecular weight of about 15 kDa. The iLBPs can be recognized by their highly conserved three dimensional structure. These proteins are comprised of a structure termed beta-clam, in which two 5-stranded beta sheets are arranged orthogonally to form a ligand binding pocket. In iLBPs, a single helix-loop-helix “lid” is situated over the entrance to the ligand binding pocket and appears to limit access to the pocket. Although similar in structure, iLBPs bind different hydrophobic molecules with distinct selectivities. For example, this family includes cellular retinol binding proteins (CRBPs) that bind retinol and retinaldehyde, cellular retinoic acid binding proteins (CRABPs) that associate with retinoic acid, and multiple forms of fatty acid binding proteins (FABPs) that bind a variety of fatty acids, metabolic derivatives of fatty acids such as prostaglandins, and other hydrophobic ligands. 
     One embodiment of the present invention is directed to the detection and measurement of a ligand from a sample. In one aspect of this embodiment, the sample preparation comprises a homogenous ligand preparation. In another aspect, the sample comprises a heterogeneous composition of ligands, wherein the population of ligands differ in their affinity for a particular receptor protein. In this embodiment, a titration curve is established using known quantities of a standard ligand that interacts with a known nuclear receptor protein. For example, the ligand can be retinoic acid and the receptor can be a retinoid receptor. Typically, the receptor protein is labeled by methods well known to those skilled in the art prior to incubation with a ligand. Following the establishment of a titration curve, the sample containing a putative ligand can be admixed with the labeled receptor protein preparation. The signal generated following this incubation can be compared to the titration curve in order to determine the concentration of the sample ligand. The receptor used for establishing the standard curve is typically the same receptor used in the assay of the sample ligand. 
     A standard titration curve must first be established. To accomplish this goal, the receptor protein must be labeled with, e.g., with a fluorescent label, using methods well known to those skilled in the art. One example of a suitable label is fluorescein. Other forms of labeling well known to those skilled in the art can also be employed, e.g., use of radioactive labels. The receptor protein serves as a sensor and can be referred to as a sensor protein. A known quantity of labeled sensor protein can be aliquoted into several vials. The sensor protein should be in a suitable buffer such that its affinity to its cognate ligand will be preserved. An example of such a buffer comprises about 20 mM Hepes, ˜pH 8.0, about 100 mM KCl, about 1 mM EDTA, and about 1 mM dithiothreitol (DTT). The buffer may vary depending upon the sensor protein used. 
     Next, using predetermined concentrations, a suitable ligand can be added, under conditions suitable for affinity binding, to the various vials containing the sensor protein. Each vial receiving a different ligand concentration. For example, the sensor protein may be present at concentrations ranging between 0.05 and 1 μM. Ligand can then be added at concentrations in that range between 1/10 to 2-fold of the protein concentration. In this range, a complete titration curve can be obtained. As ligand-protein association rates are rapid, measurements can be carried out immediately following mixing. The source of the ligand can be from a commercial source, alternatively, the ligand can be synthesized using an intact or extract cell system or an automated platform. 
     The labeled sensor protein has a particular emission signal absent any ligand. When the ligand interacts with and binds to the sensor, the signal changes. The interaction between the ligand and sensor induces a detectable signal change in, e.g., fluorescence. For example, as the concentration of ligand increases, the signal elaborated from the labeled sensor diminishes due to conformational changes in the sensor protein. Other patterns in signal elaboration are considered to be within the scope of the present invention. Regardless of the signal platform employed, it is important that a relationship exist between signal elaboration and changes in ligand concentration and that such relationship can be exploited in order to detect and measure ligand. 
     A standard titration curve can then be established. (See the Example below for a typical titration curve using the methods of the present invention.) It is this standard curve that can be used to ascertain the concentration of a ligand within a sample. 
     In order to ascertain if a particular sample comprises a ligand and, if so, the quantity of the ligand, an aliquot of sample can be added to a vial comprising the labeled sensor protein. The sensor protein in the reaction vial should be under the same or analogous conditions to those which were used to generate the standard curve. The presence of a ligand in the sample can be inferred from a change in spectral emission. Additionally, the quantity of the ligand can be computed using the titration curve previously established. 
     In the present embodiment, the sample includes, but is not limited to, tissue and cell extracts from animal and plant. The sample includes biological fluids such a sera, urine, aqueous humor, vitreous, bodily excretions, blood and alike. Tissues such as kidney, liver, lung, eye, muscle, and intestine can serve as sources for the biological sample. Mammals such as human, rodent, sheep, pig, cow and alike can serve as sources for the biological sample. Established cell lines, such as carcinoma cells and primary cells in culture, can serve as sources for the biological sample. 
     Suitable ligands for the present embodiment include, but are not limited to, ligands that bind to a nuclear receptor protein and ligands that bind to intracellular lipid binding proteins. These ligands can be natural or synthetically produced. Ligands of the present embodiment include modifications and derivatives of parent ligands. Modified ligands include, but are not limited to, chemically modified ligands. Derivatives include fragments of a parent ligand in which the fragment demonstrates affinity for the parent&#39;s cognate receptor. This principle holds true for any modified or derived ligand, i.e., there has to be a discriminating affinity between the ligand and the receptor. The affinity need not match that of the parents, however, it has to be sufficient enough so as to be useful in the present invention. Agonists are also within the scope of this invention. In some instances, agonists can be understood as derivatives or modifications of parent ligands. 
     Suitable receptors for the present embodiment include, but are limited to, receptors that reside within the interior of a cell. Nuclear receptors and iLBPs are included within this definition of suitable receptors. Examples of such receptors include, but are not limited to, estrogen receptors, glucocorticoid receptors, thyroid hormone receptors, vitamin D receptors, CRABPs, CRBPs, and the like. Receptors can be isolated from nature or can be recombinantly produced using techniques well known to those skilled in the art. Receptors of the present embodiment need not have the complete complement of amino acid residues as found in nature. In one aspect, the receptors can have a percent homology ranging from about 95% to about 100%, in still another aspect, the percent homology can range from about 85% to about 95%, in still a further aspect, the percent homology can range from about 75% to about 85%, and in still another aspect, the percent homology can range from about 65% to about 75%. Derived receptors include those proteins having the same or similar affinity for a ligand as the parent but differ in their chemical structure. Their structure can be a truncated form or a structure that has been modified by the addition of one or more chemical moieties. Derived receptors include, but are not limited to, protein fragments that have a reduced complement as compared to the parent. Receptors of the present embodiment include those receptors that have been modified by, for example, the addition of one or more moieties. These moieties include, but are not limited to, nucleic acids, small organic molecules, protein-based molecules, lipids, and alike. Receptors of the present embodiment also include receptors that have been modified by mutations, such as replacing, deleting, or adding particular amino acid residues. 
     In one aspect, receptors of the present embodiment are labeled with a molecule capable of elaborating a signal. For example, a suitable label includes a fluorescence label. In a particular aspect, the fluorescent probe used is fluorescein. However, one skilled in the art will appreciate that other labels can be employed, see, e.g., Hermanson, G., 1996, Bioconjugate Techniques, Academic Press; Butcher, E. C. et al., 1960 J. Immunol. Methods 37:109; Chen, R. F. 1969 Arch. Biochem. Biophys. 133:263-276, the entire teachings of which are incorporated herein by reference. (For labeling procedures and an array of available fluorescent probes see: Haugland R. P. 2002, Molecular Probes, Handbook of fluorescent probes and research products. 9 th  edition, the teaching of which is hereby incorporated by reference.) 
     One embodiment of the present invention is directed to the measurement of retinoids, including, but not limited to, any and all derivatives such as all-trans-retinoic acid. The sensor proteins employed for this method include RAR, CRABP-I and II, and the like. 
     The biological activities of retinoids stem from their ability to regulate transcription of multiple target genes. Two classes of proteins are involved in these activities. One class is comprised of transcription factors that are activated by retinoids, i.e., the retinoic acid receptors (RAR). These receptors are activated by both all-trans-retinoic acid (RA) and 9-cis-retinoic acid (9cRA). The other class comprises retinoid X receptors (RXR), which are activated by 9cRA. 
     Exemplary receptors which are responsive to retinoids, natural or synthetic compounds as defined herein, include RAR-alpha, RAR-beta, RAR-gamma, and splicing variants encoded by the genes for such receptors, as well as various combinations thereof (i.e., homodimers, heterodimers, and the like), including combinations of such receptors with other members of the nuclear receptor super-family with which the retinoid receptors can interact by forming heterodimers. For example, the retinoic acid receptor-a can form a heterodimer with retinoid X receptor-α, the retinoic acid receptor-β can form a heterodimer with retinoid X receptor-α, retinoic acid receptor-γ can form a heterodimer with retinoid X receptor-α, retinoid X receptor-α can form a heterodimer with thyroid receptor, retinoid X receptor-β can form a heterodimer with vitamin D receptor, retinoid X receptor-γ can form a heterodimer with retinoic acid receptor-α, and the like. Another class of receptors that are responsive to retinoids are the iLBPs including CRABP-I, CRABP-II, the keratinocyte fatty acid binding protein. 
     In addition to the naturally occurring retinoids such as all-trans-retinoic acid, 9-cis-retinoic acid, 4-oxo-retinoic acid, and the like, various synthetic ligands that selectively activate either RAR (retinoids) or RXR (rexinoids) have been developed and are in current therapeutic use in a variety of disease states. In addition to associating with receptors, RA binds in cells to proteins known as cellular retinoic acid-binding proteins (CRABP-I and CRABP-II). The CRABPs bind RA, but they do not associate with rexinoids. The present methods exploit the ligand selectivities of retinoid receptors and binding proteins using CRABPs and RAR to quantify retinoids, and RXR to measure the concentrations of 9cRA and synthetic rexinoids. 
     In order to utilize these proteins (CRABPs, RAR, RXR, and alike) as ‘sensors’, the proteins are covalently labeled with, for example, environmentally-sensitive fluorescent probes using commercially available reagents. However, one skilled in the art will appreciate that other commercially available labels can also be employed such as labels having one or more radioactive moieties. The resulting labeled sensor proteins retain their ligand-binding properties and display absorption and emission peaks at long wavelengths (˜500 nm), a range in which optical interference from biological samples is minimal. Due to the environmental sensitivity of the probe (assuming that an appropriate label is employed), association of the label (e.g., fluorescent) sensor proteins with appropriate ligands leads to distinct changes in their signal (e.g., fluorescence intensity or spectrum), which are used to monitor the interaction. 
     One embodiment of the present invention is directed to the measurement of rexinoids including 9-cis-retinoic acid, including, but not limited to, any and all derivatives or other compounds that bind to RXR such as docosahexaenoic acid. The sensor proteins employed for this method include RXR, and alike. Protocols for using RXR as a sensor for rexinoids are essentially identical to the one described below except for the usage of labeled RXR in place of CRABP. 
     In one embodiment, sensor proteins were generated as mutants viz. wild-type in order to optimize labeling conditions. Although representative calibration curves are illustrated herein, the dynamic range of the measurements can be adjusted to accommodate different concentration ranges by changing the concentration of sensor proteins used in a particular assay. For example, using a sensor protein concentration of ˜1 μM allows for reliable measurements of one or more ligands at concentrations ranging from 100 nM to about 800 nM (a range that is appropriate for measurements of serum levels of retinoids observed during therapy, Lanvers, C. et al. 2003, Med. Pediatr. Oncol., 40:293-301, the entire teaching of which is incorporated herein by reference). However, scaling the concentrations of sensor proteins used in the present methods can accommodate a much wider range of retinoid concentrations. For example, using a sensor protein concentration of 50 nM allows for measurements of ligand concentration in the range of 5 nM to 40 nM. 
     In one embodiment, the sensor protein is selected from the family of intracellular lipid binding proteins (iLBP), for example, cellular CRABP-I or CRABP-II. The iLBPs, including CRABPs, lack reactive residues that allow for efficient labeling. To overcome this difficulty, mutants (derivatives) of these proteins can be generated. As shown in  FIGS. 1  ( a ) and ( b ), L29 is an amino acid residue of the CRABP-I sensor protein that undergoes conformation change upon ligand-receptor interaction.  FIGS. 2  ( a ) and ( b ) shows the amino acid sequences of mutant CRABPs and other intracellular lipid binding proteins and highlights the residues that undergo conformation change upon ligand-protein interactions. In the mutants, the native residue in this position is replaced by a cysteine to allow for efficient labeling. 
     Residue L29 in both CRABP-I and CRABP-II was replaced with a cysteine. Recombinant mutant proteins, tagged with either GST or hexahistidine, are over-expressed in  E. coli  and purified by affinity chromatography using standard methodologies. (For purification of hexahistidine-tagged proteins see: The Recombinant Protein Handbook: Protein Amplification and Simple Purification, 2002, Amersham Biosciences, 18-1142-75, pp. 41-58; Nieba, L. et al., 1997, Anal Biochem 252, 217-228 (1997). For purification of GST-fusion proteins see: Smith, D. B. and Johnson, K. S., 1988, Gene 67, 31. Parker, M. W. et al., 1990, J. Mol. Biol. 213:221; Ji, X. et al., 1992, Biochemistry 31, 10169; the entire teachings of which are incorporated herein by reference). 
     Purified proteins can be labeled with a label such as the fluorescent probe fluorescein using a commercially available reagent (bromomethyl fluorescein), see, Stefanova, et al., 1993, Biochemistry 32:6095-6103, the entire teaching of which is incorporated herein by reference.  FIG. 3  depicts a representative assay in which labeled-CRABP-I is used to detect RA at the 10-100 nM range. Similar results can be obtained using labeled CRABP-II. 
     In another embodiment, the sensor protein is RAR. RAR can be used as an additional tool for measuring retinoid concentrations. For high-yield bacterial expression, the protein of choice is a truncated form of the receptor comprised of its ligand-binding domain. This is labeled with, for example, fluorescein. In the case of RAR, the ‘readout’ may be comprised of monitoring fluorescence energy transfer between the bound RA and the fluorophore (λexcitation=360 nm; λemission=520 nm), or monitoring changes in the direct fluorescence of the probe (λexcitation=490 nm, λemission=515 nm). 
     In yet another embodiment, the sensor protein is RXR. RXR can serves as a selective tool for measuring rexinoids. A complication in utilizing this protein is that the wild-type receptor self-associates into high affinity homotetramers, which, in turn, dissociates upon binding of ligand. Consequently, titrations of labeled RXR with rexinoids result in complex curves. To overcome this difficulty, an RXR mutant that does not form tetramers is used, see, RXRαF443A/F444A, see Kersten, S. et al. 1995, Biochemistry, 34:13717-13721; Kersten, S. et al. 1995, Biochemistry, 34:14263-14269, the entire teachings of which are incorporated herein by reference. The recombinant mutant is purified from over-expressing  E. coli  as a GST-tagged or hexa-histidine-tagged protein and labeled with fluorescein.  FIG. 4  shows a representative calibration curve for the natural rexinoid 9-cis-RA. Ligand-binding was followed by monitoring changes in the fluorescence of the labeled sensor protein (λ excitation=490 nm, and λ emission=517 nm). Additional experiments showed that this reagent can be similarly used to measure concentrations of the synthetic RXR ligand bexarotene (Targretin). 
     Example 
     An example of performing a method of the present invention for measuring retinoic acid concentrations is detailed below. In this example, the CRABP-I mutant CRABP-I-L29C was employed and labeled with BMF. The labeled receptor protein was used to measure retinoic acid concentrations in cultured cells. 
     MCF-7 cells were cultured on 60 mm plates in DMEM containing 5% charcoal-treated FBS until reaching 75-90% confluence. Media was then changed to serum free DMEM and cells were treated with retinoic acid. Following treatment, media were removed and replenished with media devoid of retinoic acid. At different time points, cells washed twice in 2 ml phosphate-buffered saline (PBS, pH 7.4). Cells were scraped, resuspended in 1 ml PBS and pelleted by centrifugation. PBS was removed and the cell pellet resuspended in ethanol. The suspension was placed at −20° C. overnight. Cells were then centrifuged, and the supernatant (ethanol extract), containing retinoic acid was stored at 20° C. until use. Cell pellet was resuspended in 1 M NaOH and protein content was measured by the Bradford assay. 
     BMF-CRABP-I-L29C (in 20 mM Hepes, pH 8.0, 100 mM KCl, 1 mM EDTA, 1 mM DTT) was placed in a cuvette and titrated with a standard retinoic acid solution in ethanol. RA was added, the cuvette was mixed, and the fluorescence recorded at room temperature. Different points on the titration curve were obtained by sequential addition of RA to the same cuvette. The titration was monitored using a spectrofluorometer by following the retinoic-acid induced change in the fluorescence of the protein-bound label (λex=494 nm; λem=519 nm). To obtain a calibration curve that is consistent with the samples, standard retinoic acid solutions were obtained as follows: MCF-7 mammary carcinoma cells were plated at the same time and density as the test plates, and extracted like the test samples with the exception that known concentrations of retinoic acid were added to the ethanol prior to extraction to yield standard solutions. BMF-CRABP-I-L29C was titrated with each of the standard solutions to obtain individual titration curves similar to the one shown in  FIG. 3 . The resulting data were analyzed to obtain the initial linear slope for the progress of each of the standard titrations. These slopes were then plotted against the concentrations of RA in each standard solution to obtain a calibration curve ( FIG. 5 ). 
     The labeled protein was titrated with each test sample to obtain an initial slope. Using the calibration curve, the total retinoic acid in the test sample was then calculated. The amount of RA in each sample was expressed as pmoles retinoic acid per mg protein. A time course for degradation of retinoic acid in MCF-7 cells following a 1 hour-term treatment with RA is shown in  FIG. 6 . 
     Another embodiment is directed towards testing the association of ligands with a particular receptor. In one aspect of this embodiment, the method is used to determine the ability of potential ligands to bind to a receptor. Test compounds include known receptor ligands and novel potential ligands obtained from natural sources or chemically synthesized. The sensor proteins employed for this method include nuclear receptors, such as RXR, estrogen receptor, glucocorticoid receptor and the like, and intracellular binding proteins, such as cellular retinoic acid- and retinol-binding proteins, fatty acid binding proteins and the like. Protocols for screening ligands are similar to those described above except that different sensor proteins are used, as appropriate. 
     Although the invention has been described with respect to various embodiments, it should be realized this invention is also capable of a wide variety of further and other embodiments.