Fluorescent leukotriene B-4: potential applications
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/240081
- author
- Sabirsh, A ; Wetterholm, A ; Bristulf, Jesper LU ; Leffler, Hakon LU ; Haeggstrom, JZ and Owman, Christer LU
- organization
- publishing date
- 2005
- 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
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Molecular Neurobiology (0131000140), Division of Microbiology, Immunology and Glycobiology - MIG (013025200), LU Innovation System (018009040), Drug Target Discovery (013212045)
- id
- 10d526f2-7708-4a84-af52-e2a65420d237 (old id 240081)
- date added to LUP
- 2016-04-01 12:10:25
- date last changed
- 2022-03-28 21:18:27
@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}}, keywords = {{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}}, doi = {{10.1194/jlr.D500005-JLR200}}, volume = {{46}}, year = {{2005}}, }