Double Mutation at the Putative Protein Kinase C Phosphorylation Sites Thr151 Plus Thr323 in the Mouse LeukotrieneD4 Receptor Eliminates Homologous Desensitization

Abstract

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 2^nd inner loop (Thr¹⁵¹) and at the C-terminal domain (Thr³²³) leads to mCysLT1 receptor desensitization and abrogates the RVD response following osmotic cell swelling.