S1P1 Receptor Regulation by Phosphorylation

Abstract

Sphingosine‐1‐phosphate (S1P) is a bioactive molecule that has been implicated in regulating cellular processes. S1P exerts its extracellular effects as a ligand of a GPCR family. S1P1 receptors are coupled to heterotrimeric Gi/o proteins. The importance of S1P and its receptor S1P1 in multiple cellular and physiological processes (vascular tone, permeability, angiogenesis, lymphocyte egress) makes them and their regulation, an interesting pharmacological target. It has been demonstrated that S1P1 activation by its natural ligand and the activation of PKC by phorbol myristate acetate leads to receptor phosphorylation and internalization. However, little is known about the identity of the kinases involved in this process. The primary objective of this study is to characterize the molecular processes that lead to receptor phosphorylation and identify the PKC isoform/s implicated.A human prostate cancer cell line stably expressing the S1P1 fused to the Green Fluorescence Protein was used for this work. We conducted functional analysis by measuring intracellular calcium mobilization in the presence or absence of PMA and distinct PKC selective inhibitors for the different isoforms. S1P1‐PKC interaction in the course of stimulation with PMA was analyzed by co‐immunoprecipitation. Our results indicate that of two isoforms participate in the PMA‐mediated desensitization, PKC α and to a lesser extent PKC β.