Double mutation at the putative protein kinase C phosphorylation sites Thr¹⁵¹ plus Thr³²³ in the mouse leukotrieneD₄ receptor eliminates homologous desensitization

TY - JOUR

T1 - Double mutation at the putative protein kinase C phosphorylation sites Thr151 plus Thr323 in the mouse leukotrieneD4 receptor eliminates homologous desensitization

AU - Jørgensen, Sune T.

AU - Eriksen, H.N.

AU - Dausell, Janni

AU - Jespersen, Thomas

AU - Lambert, Ian Henry

AU - Hoffmann, Else Kay

PY - 2013/3

Y1 - 2013/3

N2 - Background/Aims: Signalling via CysLT1 is involved in activation of volume sensitive K + channels and homologous desensitization of the LTD 4 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 LTD 4 -evoked, Ca 2+ -activated Cl - currents recorded by two-electrode voltage clamp. Results: Repetitive LTD 4 administration (100 nM) desensitized the LTD 4 -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 LTD 4 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 LTD 4 - pretreatment on RVD in Ehrlich ascites tumour cells. Conclusion: These data indicate that simultaneous PKC-mediated phosphorylation at the 2 nd inner loop (Thr 151 ) and at the C-terminal domain (Thr 323 ) leads to mCysLT1 receptor desensitization and abrogates the RVD response following osmotic cell swelling.

AB - Background/Aims: Signalling via CysLT1 is involved in activation of volume sensitive K + channels and homologous desensitization of the LTD 4 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 LTD 4 -evoked, Ca 2+ -activated Cl - currents recorded by two-electrode voltage clamp. Results: Repetitive LTD 4 administration (100 nM) desensitized the LTD 4 -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 LTD 4 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 LTD 4 - pretreatment on RVD in Ehrlich ascites tumour cells. Conclusion: These data indicate that simultaneous PKC-mediated phosphorylation at the 2 nd inner loop (Thr 151 ) and at the C-terminal domain (Thr 323 ) leads to mCysLT1 receptor desensitization and abrogates the RVD response following osmotic cell swelling.

U2 - 10.1159/000343374

DO - 10.1159/000343374

M3 - Journal article

C2 - 23485744

SN - 1015-8987

VL - 31

SP - 366

EP - 378

JO - Cellular Physiology and Biochemistry

JF - Cellular Physiology and Biochemistry

IS - 2-3

ER -