lysophospholipid receptors (signaling and biochemistry) || identification of direct intracellular...

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CHAPTER 4

Identification of Direct Intracellular Targets of Sphingosine 1-Phosphate (S1P)NITAI C. HAIT, SHELDON MILSTIEN, and SARAH SPIEGEL

4.1. INTRODUCTION

Sphingosine 1-phosphate (S1P) is a potent sphingolipid mediator that regu-lates diverse cellular processes important for cancer progression including cell growth and survival, invasion, angiogenesis, lymphocyte trafficking, and inflam-mation, among others (1). Formation of S1P inside cells is catalyzed by one of two closely related sphingosine kinases, SphK1 and SphK2, that have differ-ential cellular distributions as well as both overlapping and opposing functions and are activated by many different stimuli (2). Most of the research to date in the S1P field has been concentrated on its actions as a ligand for the five specific G protein-coupled cell surface receptors (GPCRs), termed S1PR1–5, that regulate diverse physiological and pathological processes. S1P produced inside cells can be transported out by adenosine triphosphate (ATP)-binding cassette multidrug-resistant transporter proteins, such as ABCC1 and ABCA1 (3, 4), and the recently identified transporter-like protein Spns2 (5). Erythro-cytes and thrombocytes contain high levels of intracellular S1P and contribute to the S1P in blood that circulates bound to albumin and low-density lipopro-tein (LDL). It is still somewhat puzzling why the S1P in serum, which is around 50-fold higher than the Kd values for binding to all of the S1PRs, does not constitutively downregulate all of them.

Growing evidence also supports the view that S1P has multiple intracellular functions independent of S1P receptors in organisms as diverse as yeast, plants, and even mammals (1, 6). Although it has long been known that intracellular

Lysophospholipid Receptors: Signaling and Biochemistry, First Edition. Edited by Jerold Chun, Timothy Hla, Sara Spiegel, and Wouter Moolenaar.© 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

72 DIRECT INTRACELLULAR TARGETS OF S1P

S1P regulates cell proliferation and protects against apoptosis (1), little prog-ress had been made in understanding the mechanisms involved. Recent tech-nological advances that are described in detail in the succeeding sections have spurred our recent efforts aimed at identifying physiologically important, direct intracellular targets of S1P.

4.2. INTRACELLULAR TARGETS FOR S1P

There is abundant evidence that activation of SphK1 and “inside-out signal-ing” by S1P regulates many cellular processes important in human diseases, such as cancer, atherosclerosis, inflammation, and autoimmune disorders including multiple sclerosis (7). However, much less is known of the functions of S1P produced by SphK2, which is expressed in various cellular compart-ments, including the endoplasmic reticulum, mitochondria, and nucleus, depending on the cell type.

We recently discovered a new role for S1P and SphK2 in the mitochondria (8). We found that S1P produced in the mitochondria by SphK2 binds with high affinity and specificity to prohibitin 2 (PHB2), a highly conserved protein that regulates mitochondrial assembly and function. Interestingly, although PHB2 has been reported to exist and function in the mitochondria as a large multimeric complex with the closely related protein PHB1, S1P was only associated with immunoprecipitates of PHB2 but not PHB1. Furthermore, the interaction of S1P with PHB2 in the mitochondria is important as depletion of either SphK2 or PHB2 induced a defect in mitochondrial respiration through cytochrome c oxidase. Furthermore, a new aberrant form of cyto-chrome c oxidase with low activity was present in mitochondria isolated from SphK2-null mice. These results indicate that association of S1P with homo-meric PHB2 is important for cytochrome c oxidase assembly and mitochon-drial respiration (8).

It has been shown in several studies that the nucleus contains sphingomy-elin and some enzymes of sphingolipid metabolism (9, 10) but their functions there beyond known roles in maintenance of membranes was not clear. In another study, we explored the function of S1P produced by SphK2, which is localized in the nucleus of many cells and to shuttle between the nucleus and the cytoplasm (11, 12). We have recently found that SphK2 was associated with chromatin and its expression increased acetylation of specific histone lysines, part of an epigenetic code that controls gene transcription (13). Several approaches were used to demonstrate that S1P itself directly bound to histone deacetylases, HDAC1 and HDAC2, and inhibited their ability to remove acetyl groups from histones, and regulate gene transcription. This study established that HDACs, which have emerged as key targets to reverse aberrant epigenetic changes associated with human diseases (14), are direct intracellular targets of S1P and was the first to link nuclear S1P and sphingolipid metabolism to epigenetic regulation of gene expression.

METHODS TO IDENTIFy INTRACELLULAR S1P TARGETS 73

We have also recently demonstrated that TRAF2 (tumor necrosis factor [TNF] receptor-associated factor 2), a key component in nuclear factor-κB (NF-κB) signaling triggered by TNF-α, is another direct target of intracellular S1P produced by SphK1 (15), solving several long-standing puzzles associated with the cytoprotective effects of SphK1 and S1P (16). First, although S1P and dihydro-S1P are equally potent ligands for the five S1P receptors, only S1P suppresses apoptosis (17). Second, Xia et al. reported that TNF stimulated interaction of TRAF2 with SphK1 and this increased its activity (18). Although this was shown to be important for activation of prosurvival signaling of NF-κB downstream of the TNF receptor, neither the mechanism of this effect nor the involvement of S1P was discerned. Using the methodology described below, we demonstrated that S1P produced by SphK1 specifically bound to TRAF2 (15). Moreover, while TRAF2 is known to be important for Lys 63 polyubiquitination of receptor interacting protein 1 (RIP1), leading to activa-tion of NF-κB, previous attempts to demonstrate that TRAF2 is an E3 ligase for RIP1 failed. In this study, we also found that TRAF2 indeed had E3 ligase activity in the presence of S1P but not dihydro-S1P, establishing S1P as an essential cofactor required for TRAF2-catalyzed RIP1 polyubiquitination and for NF-κB activation (15). These findings also explain the many observations of the importance of S1P in inflammatory, antiapoptotic, and immune processes.

4.3. METHODS TO IDENTIFY INTRACELLULAR S1P TARGETS

Identification of receptors or targets of any bioactive lipid is a daunting task. Bioactive lipids are designed to bind to specific targets with very high affinity and they usually have low abundant targets. However, they are after all lipids and by their nature are very “sticky” and not stable. A few approaches were developed more than a decade ago that overcame some of these problems and were critical for the original identification of S1P as a specific ligand of the endothelial differentiation gene family of GPCRs (19). Despite these advances and solid hints that S1P had intracellular targets, until recently, little progress had been made since then. The recent characterization of PHB2 (8), HDAC1 and HDAC2) (13), and TRAF2 (15) as bona fide intracellular targets of S1P was made possible by the development of new affinity media coupled with highly sensitive technology to measure femtomole mass levels of S1P and other sphingolipids, coupled with knowledge gained in the identification of the S1P receptors. These are described in the succeeding sections.

4.3.1. S1P Immobilized on Agarose Beads

The approach that we found to be most useful to isolate unknown S1P targets is an agarose affinity matrix containing an immobilized derivative of S1P. The immobilized lipid approach has been successfully used to identify targets of


inositol trisphosphate that contain pleckstrin homology domains (20), includ-ing a novel phosphatidylinositol 3-kinase effector protein (21). A similar approach was previously utilized to identify a melanoma cell surface-binding protein for S1P (22). We have recently employed agarose beads that are coated with S1P for selective capture of several S1P binding proteins (Table 4.1). For example, we found a distinct protein band in nuclear extracts after pull down of proteins with S1P-conjugated beads that was not pulled down by control unconjugated or lysophosphatidic (LPA) agarose beads (8). This band was

TABLE 4.1. Identification of S1P-Binding Proteins

Method Advantages Disadvantages

S1P immobilized on agarose beads

Commercially available, simple, requires small amounts of protein, bound proteins can be identified by mass spectrometry

Some nonspecific binding, poor batch-to-batch reliability. Only S1P and LPA beads are available

Radioactive S1P binding

Simple, high sensitivity, can be used to identify analogues that specifically bind to targets and inhibitors of S1P binding, can be applied to other lipids

Requires preparation and purification of labeled S1P, target must be known and purified, need antibodies that can be used for immunoprecipitation

Pull down of proteins that bind S1P followed by LC-ESI-MS/MS

Simple and relatively rapid, quantitative binding assay, can measure endogenous S1P binding, can be applied to other lipids

Targets must be known, specific antibodies and isotype matched control IgG required that can be used for immunoprecipitation, requires sensitive mass spectrometer to quantitate bound S1P and other lipids

Protein–lipid overlay

Simple and fast, ready-made lipid blots are commercially available, many different lipids on strips

Target must be known and specific antibodies available, S1P is not in a membrane environment, high nonspecific binding

ELISA Simple and fast, many different lipids can be immobilized

Not reproducible, high background, nonspecific binding to plastic

Liposome pull down

Potentially simple, quantitative

Not commercially available, preparation of liposomes is technically demanding, no consensus liposome composition, no reports on use with S1P


excised and sequencing by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identified this polypeptide as PHB2, an evolutionarily conserved and expressed protein essential for cell growth and development (23–25). Its identity was confirmed by Western blotting with a specific PHB2 antibody. Furthermore, another important control was that preincubation of extracts with exogenous S1P abolished binding of PHB2 to S1P immobilized on agarose beads.

We have also successfully employed this immobilized S1P approach to the identification of HDACs (HDAC1 and HDAC2) as direct targets of intracel-lular S1P (13). We found distinct HDAC1 and HDAC2 protein bands by Western blot analysis of the pull down of nuclear proteins with S1P-conjugated agarose beads but not with control or LPA-conjugated beads and confirmed these results with recombinant HDAC1 and HDAC2. Interestingly, we found that other classes of HDACS, including class IIa HDACs (HDAC4, HDAC5, HDAC7), class IIb HDAC (HDAC6), and the nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 did not bind to S1P agarose beads.

These S1P affinity beads were also critical for the discovery that TRAF2 is another direct target of intracellular S1P (15). TRAF2 was specifically pulled down from cell extracts with this affinity matrix and addition of excess S1P, but not dihydro-S1P, abolished binding of TRAF2. Moreover, a mutant form of TRAF2 with a deletion of its really interesting new gene (RING) domain, a predicted binding site of S1P, did not bind to S1P-conjugated beads, confirm-ing the specificity of this method.

Protocol for Pull Down of Proteins with S1P Agarose Beads

S1P-coated agarose beads obtained from Echelon Biosciences (Salt Lake City, UT) have S1P covalently linked to the beads through the terminus of its alkyl chain.

1. Equilibrate agarose beads in binding buffer containing 10 mM HEPES (pH 7.8), 150 mM NaCl, and 0.5% octylphenoxypolyethoxyethanol by gentle agitation followed by centrifugation at 1000 × g. Resuspend beads in two volumes of binding buffer at 4°C in borosilicate tubes with Teflon-lined caps (VWR, Atlanta, GA).

2. Typically, cell extracts or subcellular fractions (usually about 1 mg protein) in 1 mL are incubated with 50 μL of affinity beads for 2 hours at 4°C with gentle agitation.

3. Beads are washed four times with 0.9 mL of cold binding buffer by cen-trifugation at 1000 × g.

4. Beads are pelleted at 1000 × g, the supernatant removed, the beads boiled in sodium dodecyl sulfate (SDS) sample buffer, and bound proteins sepa-rated by sodium dodecyl sulfate polyacrylamide gel electrophoresis


4.3.2. Binding of 32P-Labeled S1P to Targets

Another useful quantitative method to confirm direct binding to identified targets is the evaluation of specific binding of 32P-labeled S1P to purified or immunoprecipitated proteins (13). It should be noted that this approach is only useful for confirming binding to identified targets and to determine binding affinities. However, it has the advantage that it can be used to identify and characterize inhibitors, agonists, and antagonists. As we recently reported, direct binding of PHB2 and S1P was assessed by incubating 32P-labeled S1P with nuclear extracts from MCF7 or HeLa cells, and PHB2 was immunopre-cipitated with specific anti-PHB2 antibodies. We found that a large amount of [32P]S1P was associated with the PHB2 immunoprecipitated complexes and as expected, preincubation of the binding reaction with excess unlabeled S1P, but not sphingosine or LPA, markedly reduced the amount of radioactivity in the immunoprecipitates. In sharp contrast, a very small amount of radioactivity was associated with the immunoprecipitates using control immunoglobulin G (IgG) antibodies or antibodies to the closely related protein PHB1, which shares >50% identical amino acid residues with PHB2. The displacement of 32P-labeled S1P bound to PHB2 with an excess concentration of S1P or dihydro-S1P, which lacks the trans double bond and is ligand of all S1P recep-tors, confirmed that the binding is specific (Kd < 1 μM) (8).

This method was also successfully used to confirm the physical interaction between S1P and HDAC1/2. As expected, it was evident that 32P-labeled S1P was specifically bound to purified recombinant his-tagged HDAC1 or HDAC2

(SDS-PAGE). Proteins on gels are stained with silver, Coomassie, or SyproRuby and specific protein bands that are only bound to S1P beads are excised and sequenced by MALDI-TOF mass spectrometry. The cor-responding areas in the control gels are also subjected to peptide sequenc-ing as controls.

5. Identification of S1P-conjugated bound proteins should be confirmed by Western blotting with specific antibodies.

This S1P affinity bead approach is simple to use in principle and has several advantages. However, a major disadvantage of this approach is the tendency of nonspecific proteins to stick to lipids and to uncoated control agarose beads. In these experiments, extensive washing of the protein-bound beads, selecting the most effective ratio of beads to total proteins as well as the total amount of protein necessary to detect a protein of interest are all required to success-fully utilize this approach. Furthermore, many other experiments are needed to confirm the binding of specific proteins to the S1P, including direct binding assays to purified proteins and binding of endogenous S1P in vivo as described in the succeeding paragraphs, and demonstrations that the association of S1P and with its target is biologically relevant.


and displaced specifically with excess unlabelled S1P or dihydro-S1P but not with sphingosine or LPA (13). Similar experiments also confirmed that sube-roylanilide hydroxamic acid (SAHA) and trichostatin A, potent inhibitors of HDACs, also completely displaced bound [32P]S1P from HDAC1 and HDAC2, supporting the notion that S1P and these inhibitors bind to the same binding pocket on HDACs.

We also evaluated the interaction between TRAF2 and S1P using the 32P-labeled S1P binding method. 32P-labeled S1P specifically bound to TRAF2 isolated from cells or to purified TRAF2 but not to TRAF3 and binding was completely abolished by the presence of excess unlabeled S1P but not with dihydro-S1P, sphingosine, or LPA. This approach was also utilized to deter-mine the Kd value for binding of S1P to TRAF2 (15).

Protocol to Determine Binding of 32P-Labeled S1P to Proteins

1. [32P]-Labeled S1P is prepared by phosphorylation of sphingosine with recombinant SphK1 in the presence of ATP[g32P] and purified exactly as described (26).

2. Suspected targets of S1P, either recombinant or epitope-tagged proteins purified by affinity methods from cell extracts, are incubated for 60 minutes at 4°C in borosilicate tubes with Teflon-lined caps with [32P]-labeled S1P (0.1 nM, 6.8 μCi/pmol) in buffer containing 50 mM Tris (pH 7.5), 137 mM NaCl, 1 mM MgCl2, 2.7 mM KCl, 15 mM NaF, and 0.5 mM NaV3O4 for 25 minutes at 30°C or 60 minutes at 4°C. To demonstrate specificity, control samples contain a 100-fold molar excess of unlabeled sphingosine or related lipids.

3. Proteins with bound [32P]S1P are isolated by immunoprecipitation with specific antibodies or pulled down with immobilized affinity media and washed three times with cold binding buffer. Radioactivity associated with immunoprecipitates or affinity media can be determined by scintil-lation counting. We found that background nonspecific binding was decreased by elution of bound His-tagged proteins from Ni-NTA-aga-rose or from anti-FLAG-agarose with imidazole or FLAG peptide, respectively.

4.3.3. Mass Measurement of Endogenous S1P in Immunoprecipitates of Target Proteins

To quantify mass levels of endogenous S1P bound to specific target proteins in vivo, endogenous proteins are pulled down from cell extracts with specific antibodies and lipids in the immunoprecipitates determined by liquid chroma-tography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). For example, we used LC-ESI-MS/MS to confirm that endogenous S1P specifically bound to PHB2 but not to PHB1 (8) and to verify the interaction


between nuclear S1P and endogenous HDAC1 and HDAC2 (13). We also showed by LC-ESI-MS/MS that of all the sphingolipids present in the cells, only S1P bound to TRAF2 immunoprecipitated complexes and this interac-tion was significantly enhanced by TNF-α (15). Furthermore, much more S1P was bound to ectopically overexpressed TRAF2. In contrast, no S1P was detected by LC-ESI-MS/MS in immunoprecipitates of mutant TRAF2 that lacked the N-terminal 87 amino acids containing the RING domain, suggesting that the binding site of S1P to TRAF2 is within the RING domain (15).

Protocol for Measurement of Bound Lipids by Mass Spectrometry

1. Endogenous proteins or overexpressed epitope-tagged proteins are pulled down form cell extracts with specific antibodies or epitope affinity media, respectively, as previously described.

2. After washing immunoprecipitates or affinity media bound proteins three times with binding buffer previously described, lipids are extracted with methanol.

3. Internal mass spectrometry standards (500 pmol of each sphingolipid in 20 μL of ethanol, Avanti Polar Lipids, Alabaster, AL) are added to the methanol extracts in borosilicate glass tubes with Teflon-lined caps and evaporated under nitrogen.

4. Samples are resuspended in 0.3 mL of mobile phase for LC-ESI-MS/MS analysis and centrifuged at 16,000 × g for 30 seconds to remove particu-lates prior to transferring into autoinjector vials.

5. The mass spectrometry system consists of a Shimadzu (Columbia, MD) LC-20 AD binary pump system coupled to a SIL-20AC autoinjector and DGU20A3 degasser interfaced with an AB Sciex 4000 QTRAP mass spectrometer (Applied Biosystems, Carlsbad, CA) operating in a triple quadrupole mode. Q1 and Q3 were set to pass molecularly distinctive precursor and product ions (mass-to-charge ratio [m/z] from 380.4 to 264.4 for S1P), due to N2-induced dissociations in Q2. LC separations utilized a binary solvent system at a flow rate of 1.5 mL/min and a Discovery C18 column (Supelco 2.1 × 50 mm, Sigma, St. Louis, MO). The column was preequilibrated for 0.5 minute with 80% solvent A (methanol : water : formic acid, 58:41:1, containing 5 mM ammonium formate) and 20% solvent B (methanol : formic acid 99:1, v/v, containing 5 mM ammonium formate). The A : B ratio was maintained at 80:20 for 0.8 minute after sample injec-tion, followed by a linear gradient to 100% B over 1.2 minutes, held at 100% B for 1.4 minutes, and then a 0.3 minute gradient return to 80:20 A : B. For more details on quantification of sphingolipids and mass spec-trometry parameters, see Reference 27.

The advantages of mass spectrometry are that all sphingolipids present in the immunoprecipitates can be detected and quantitated simultaneously.

OTHER POTENTIALLy USEFUL METHODS TO IDENTIFy LIPID BINDING PROTEINS 79

4.4. OTHER POTENTIALLY USEFUL METHODS TO IDENTIFY LIPID BINDING PROTEINS

Several other methods have been used in the past to detect proteins that spe-cifically bind lipids. Although none of these methods have been used yet for detection of proteins that bind S1P, we will describe how these methods have been used to identify intracellular targets of other lipids, particularly phos-phoinositides, and readers are referred to an excellent review on these methods (28).

4.4.1. Lipid Strips for Identification of Binding Proteins (Protein–Lipid Overlay)

This is a very simple and easy to use method that has successfully been applied to identify proteins that bind to lipids other than sphingolipids. These are com-mercially available hydrophobic membranes that have been spotted with many biologically active lipids and sphingolipids, and bound proteins can be visualized by standard immunodetection methods (29). However, this approach can only be used to confirm lipid–protein interactions, not to identify unknown targets.

This method was used recently to identify a role for a conserved polybasic amino acid motif in an N-terminal domain of lipin1β in selective binding to phosphatidic acid. The major disadvantage of this method is that it is two-dimensional and important residues of the lipid for binding to proteins might not be available. Persistent high background is also an issue; however, various blocking agents and many washing steps with detergents may improve the quality of binding information. Unfortunately, we were not able to successfully use this approach to identify S1P target proteins. However, our protocol is described below.

1. Lipid strips, hydrophobic nylon membranes spotted with equal amount of different biologically active lipids (100 pmol, Echelon Biosciences, Salt Lake City, UT ) are blocked by incubating for 1 hour at room tempera-ture in a solution containing 1% nonfat dry milk, 3% fatty acid-free bovine serum albumin (Sigma) (or 0.1% ovalbumin) in TBST (50 mM Tris-buffered saline, pH 7.5, containing 150 mM NaCl and 0.1% Tween-20).

While mass spectrometry is more sensitive than other methods and can measure sphingolipids in the femtomole range, the utility of this method depends on the availability of high quality and specific antibodies for immu-noprecipitations. It is important to carry out the appropriate control of pull downs with nonimmune isotype-matched antibodies to exclude nonspecific binding.


2. Cell lysates are incubated with lipid strips in blocking solution at 4°C overnight on a rocking platform.

3. Membranes are washed three times with TBST.4. The lipid strips containing bound proteins are incubated for 1 hour in

blocking solution with a specific antibody to the protein of interest, a control antibody, or no antibody as an additional control.

5. Membranes are washed three times with TBST and incubated with con-jugated secondary antibody for 1 hour at room temperature in blocking solution.

6. After washing four times for 5 minutes with TBST, the bound protein of interest is visualized by chemiluminescence.

4.4.2. Detection of Lipid Binding Proteins by Enzyme-Linked Immunadsorbent Assays

A method similar in principle to lipid strips is based on immobilizing lipids on microtiter plates and detecting bound proteins by enzyme-linked immunad-sorbent assays (ELISAs). Although this method has been extensively used to characterize binding of proteins to gangliosides (30), we were unsuccessful in obtaining consistent results detecting specific binding of proteins to S1P adsorbed microwells. This protocol is below.

1. Polystyrene plates (96 well) are washed with butanol and ethanol, then air dried.

2. Increasing concentrations of S1P (10, 100, 1000, 10,000 pmol) or other lipids in ethanol are added to the plates together with an equal volume of water and incubated uncovered for 90 minutes at 37°C.

3. The liquid is aspirated and wells washed three times with water.4. One hundred microliters (100 μL) of blocking solution (phosphate-

buffered saline [PBS] containing 0.1% bovine serum albumin [BSA] and 0.1% Tween-20) is then added to each well and incubated for 60 minutes.

5. Cell extracts diluted in blocking solution are added to wells and incu-bated for 90 minutes at room temperature.

6. Wells are aspirated and washed four times with phosphate-buffered saline with Tween-20 (PBST).

7. Specific primary antibodies are then added and plates rocked at 4°C overnight.

8. Wells are aspirated and washed four times with PBST.9. Secondary antibodies (horseradish peroxidase [HRP] conjugated) are

added and plates incubated at room temperature for 1 hour.10. Wells are aspirated and washed six times with PBST.

REFERENCES 81

11. One hundred microliters (100 μL) of substrate (enhanced chemilumi-nescence substrate, Promega, Madison, WI) solution is added. Reactions are stopped by adding 50 μL of sulfuric acid to each well.

12. Absorbance is measured with an ELISA plate reader.13. Blank wells, wells without lipids, and wells that received only a second-

ary antibody are included as negative controls.

4.4.3. Liposome Pull Down

Liposome pull down is another method that has been extensively used to characterize proteins that contain various phosphatidylinositol-specific binding domains (28). Proteins bound to phosphatidylinositol incorporated in lipo-somes are recovered by centrifugation (in the case of heavy multilamellar liposomes). A recent report used this method to demonstrate that phosphatidylinositol-3,5-bisphosphate binds specifically to endolysosome-localized mucolipin transient receptor potential channels (31). Liposome-bound proteins can be analyzed by immunoblotting or by mass spectroscopic peptide sequencing to identify unknown target proteins. So far, there are no reports that have used this method for identification of S1P binding proteins.

4.5. CONCLUDING REMARKS

In this review, we have summarized the methods utilized in our lab to identify and characterize proteins that specifically bind S1P. We have provided detailed protocols with suggestions for proper controls and discussions of some pitfalls. Although several methods are now available for those interested in interac-tions of S1P with its protein companions, we recommend that multiple approaches be utilized as each has its own specific drawbacks. There is still room for considerable improvement of the existing methods and perhaps for the development of fundamentally different approaches.

ACKNOWLEDGMENTS

This work was supported by NIH grants, R01CA61774, R37GM043880, R01AI50094, 1U19AI077435 (S.S.), and supported, in part, by funding from NIH-NCI Cancer Center Support Grant 5 P30 CA016059.

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lysophospholipid receptors (signaling and biochemistry) || identification of direct intracellular...

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