cDNA cloning of human LTB4 receptor
Please refer to our Nature paper(1)
.
Leukotriene B4 (LTB4)is a potent chemoattractant primarily involved in inflammation,
immune responses, and host defense. LTB4 activates inflammatory cells by
binding to its cell-surface receptor (BLTR).
Despite extensive efforts towards receptor cloning, BLTR cDNA has not so
far been isolated. Recently, LTB4 was reported to bind and activate the
intranuclear transcription factor PPAR alpha, resulting in the activation
of genes that terminate inflammatory processes(2).
Here we report(1) the first cDNA cloning of a cell-surface LTB4 receptor
that is highly expressed in human leukocytes. Using a subtraction strategy,
we isolated two cDNA clones (HL-1 and -5) from
retinoic acid (RA)-differentiated HL-60 cells. These two clones contain
identical open reading frames (ORFs), encoding a 352 amino acid protein
predicted to contain seven transmembrane spanning domains,
but different 5'-untranslated regions (UTRs), showing that they are differentially
transcripted variants.
Norhtern blotting shows that this receptor
mRNA is expressed highest in leukocytes, followed by spleen and thymus.
In HL-60 cells, BLTR receptor mRNAs corresponding to two transcripts are
induced following exposure to 1 micro M retinoic
acid.
Membrane fractions of Cos-7 cells transfected with an expression construct
of BLTR containing the ORF of HL-5 showed specific
LTB4 binding with a Kd (0.154 nM) comparable to that observed in RA-differentiated
HL-60 cells. In CHO cells stably expressing this receptor, LTB4 induced
increases in intracellular calcium levels,
D-myo-inositol 1, 4, 5-triphosphate (IP3) accumulation, and inhibition
of adenylyl cyclase. Furthermore, CHO cells expressing exogenous
BLTR exhibited marked chemotactic responses
toward low concentrations of LTB4 in a pertussis-toxin
(PTX) sensitive manner.
Our findings together with previous reportsshow that LTB4 is a unique lipid
mediator that interacts with both cell surface and nuclear receptors.
Ref.
(1)Yokomizo, T. et al. A G-protein coupled receptor
for leukotriene B4 that mediates chemotaxis. Nature
387, 620-624 (1997)
(2)Devchand, P.R., et al. The PPAR alpha-leukotriene
B4 pathway to inflammation control. Nature
384, 39-43 (1996).
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