Homologous desensitisation of the mouse leukotriene B4 receptor involves protein kinase C-mediated phosphorylation of serine 127.
Murine leukotriene B(4) (LTB(4)) receptor (mBLT1) cDNA was identified by searching the EST database using human LTB(4) receptor as the query sequence. Expression of functional mBLT1 after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was demonstrated as LTB(4)-evoked, Ca(2+)-activated Cl(-) currents recorded by two-electrode voltage clamp. From mBLT1-expressing oocytes, a dose-dependent relationship between the Ca(2+)-activated Cl(-) current and LTB(4) concentration was demonstrated with an apparent EC(50) of 6.7 nM. Following LTB(4) stimulation of mBLT1, we observed two transient, spatially distinct Ca(2+)-activated, inwardly directed Cl(-) currents in the oocytes: a fast peak current requiring relatively high LTB(4) concentrations, and a slowly progressing Cl(-) current. Nucleotides, PGE(2), 12R-hydroxy-5, 8, 14-cis-10-trans-eicosatetraenoic acid, and LTD(4) did not activate mBLT1. U75302, specifically targeting BLT1, significantly reduced LTB(4)-evoked Cl(-) currents. Repetitive LTB(4) administration desensitized the LTB(4)-evoked currents. Activation of protein kinase C (PKC) by PMA addition completely eliminated the LTB(4)-evoked currents, whereas down-regulation of PKC by prolonged PMA exposure (20 h) impaired mBLT1 desensitisation. In addition, Ser-127-Ala substitution of the PKC consensus phosphorylation site on the second intracellular loop prevented the mBLT1 desensitisation. These data indicate that PKC-mediated phosphorylation at Ser-127 leads to mBLT1 desensitisation.
Mollerup
Jens
J
Department of Molecular Biology, University of Copenhagen, Copenhagen, Denmark. jmollerup@my.molbio.ku.dk
Eriksen
Heidi N
HN
Albertsen
Janni
J
Wulff
Tune
T
Lambert
Ian H
IH
Hoffmann
Else K
EK
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D005233:Fatty Alcohols / Q000494:pharmacology; D005260:Female; D006018:Glycols / Q000494:pharmacology; D066298:In Vitro Techniques; D007975:Leukotriene B4 / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D018102:Receptors, Leukotriene B4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014315:Triterpenes
D005233:Fatty Alcohols; D006018:Glycols; C503704:Ltb4r1 protein, mouse; D018102:Receptors, Leukotriene B4; D011994:Recombinant Proteins; D014315:Triterpenes; C062137:U 75302; D007975:Leukotriene B4; D012694:Serine; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C087107:platanic acid; D037342:Pertussis Toxin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate