Murine leukotriene B4 (LTB4) receptor (mBLT1) cDNA was identified by searching the EST database using human LTB4 receptor as the query sequence. Expression of functional mBLT1 after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was demonstrated as LTB4-evoked, Ca2+-activated Cl- currents recorded by two-electrode voltage clamp. From mBLT1-expressing oocytes, a dose-dependent relationship between the Ca2+-activated Cl- current and LTB4 concentration was demonstrated with an apparent EC50 of 6.7 nM. Following LTB4 stimulation of mBLT1, we observed two transient, spatially distinct Ca2+-activated, inwardly directed Cl- currents in the oocytes: a fast peak current requiring relatively high LTB4 concentrations, and a slowly progressing Cl- current. Nucleotides, PGE2, 12R-hydroxy-5, 8, 14-cis-10-trans-eicosatetraenoic acid, and LTD4 did not activate mBLT1. U75302, specifically targeting BLT1, significantly reduced LTB4-evoked Cl- currents. Repetitive LTB4 administration desensitized the LTB4-evoked currents. Activation of protein kinase C (PKC) by PMA addition completely eliminated the LTB4-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.
Background/Aims: Signalling via CysLT1 is involved in activation of volume sensitive K+ channels and homologous desensitization of the LTD4 receptor impairs regulatory volume decrease (RVD). The aim is to illustrate the effect of mutation of putative PKC consensus phosphorylation sites in the CysLT1R on desensitization and RVD. Methods: mCysLT1 contains 4 putative PKC consensus phosphorylation sites, and four mutants were created: Thr151Gly, Thr323Gly, Thr151Gly plus Thr323Gly, and Thr236Gly plus Ser243Gly. Functional mCysLT1 receptor activity after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was visualized as a LTD4-evoked, Ca2+-activated Cl- currents recorded by two-electrode voltage clamp. Results: Repetitive LTD4 administration (100 nM) desensitized the LTD4-evoked currents in oocytes expressing wild type CysLT1. Single mutations as well as the double mutation Thr236Gly plus Ser243Gly had no or a slight effect on the LTD4 induced desensitization. However, double mutation Thr323Gly plus Thr151Gly prevented the desensitization. As a functional consequence we find that inhibition of PKC accelerates RVD and prevents the inhibitory effect of LTD4-pretreatment on RVD in Ehrlich ascites tumour cells. Conclusion: These data indicate that simultaneous PKC-mediated phosphorylation at the 2nd inner loop (Thr151) and at the C-terminal domain (Thr323) leads to mCysLT1 receptor desensitization and abrogates the RVD response following osmotic cell swelling.
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