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Fluorescent leukotriene B-4: potential applications

Sabirsh, A; Wetterholm, A; Bristulf, Jesper LU ; Leffler, Hakon LU ; Haeggstrom, JZ and Owman, Christer LU (2005) In Journal of Lipid Research 46(6). p.1339-1346
Abstract
Leukotriene B-4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and... (More)
Leukotriene B-4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a K-d of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
G-protein coupled receptor, pharmacology, method
in
Journal of Lipid Research
volume
46
issue
6
pages
1339 - 1346
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • wos:000229061100027
  • pmid:15805550
  • scopus:24944480063
ISSN
1539-7262
DOI
10.1194/jlr.D500005-JLR200
language
English
LU publication?
yes
id
10d526f2-7708-4a84-af52-e2a65420d237 (old id 240081)
date added to LUP
2007-08-13 14:36:19
date last changed
2017-10-22 03:47:13
@article{10d526f2-7708-4a84-af52-e2a65420d237,
  abstract     = {Leukotriene B-4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a K-d of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.},
  author       = {Sabirsh, A and Wetterholm, A and Bristulf, Jesper and Leffler, Hakon and Haeggstrom, JZ and Owman, Christer},
  issn         = {1539-7262},
  keyword      = {G-protein coupled receptor,pharmacology,method},
  language     = {eng},
  number       = {6},
  pages        = {1339--1346},
  publisher    = {American Society for Biochemistry and Molecular Biology},
  series       = {Journal of Lipid Research},
  title        = {Fluorescent leukotriene B-4: potential applications},
  url          = {http://dx.doi.org/10.1194/jlr.D500005-JLR200},
  volume       = {46},
  year         = {2005},
}