TY - JOUR

T1 - Homologous desensitisation of the mouse leukotriene B4 receptor involves protein kinase C-mediated phosphorylation of serine 127.

AU - Mollerup, Jens

AU - Eriksen, Heidi N

AU - Albertsen, Janni

AU - Wulff, Tune

AU - Lambert, Ian H

AU - Hoffmann, Else K

N1 - Keywords: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amino Acid Sequence; Animals; Enzyme Activation; Fatty Alcohols; Female; Glycols; Leukotriene B4; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Oocytes; Pertussis Toxin; Phosphorylation; Protein Kinase C; Receptors, Leukotriene B4; Recombinant Proteins; Sequence Homology, Amino Acid; Serine; Signal Transduction; Tetradecanoylphorbol Acetate; Triterpenes

PY - 2007

Y1 - 2007

N2 - 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.

AB - 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.

U2 - 10.1159/000104162

DO - 10.1159/000104162

M3 - Journal article

C2 - 17595524

VL - 20

SP - 143

EP - 156

JO - Cellular Physiology and Biochemistry

JF - Cellular Physiology and Biochemistry

SN - 1015-8987

IS - 1-4

ER -