Dynamin internalizes tyrosine phosphorylated sphingosine 1 phosphate receptor 1 and impair downstream signaling

Abstract

We recently showed that S1P induces S1PR1 phosphorylation at tyrosine 143 residue impairing S1PR1 cell‐surface expression. Here we determined if phosphorylation alters receptor trafficking to the cell‐surface and if so what is the mechanism. The GTPase dynamin is essential for both clathrin‐dependent and clathrin‐independent‐endocytosis of receptors and thereby regulates the abundance of the receptor on the plasma membrane and downstream signaling. Thus, we inhibited dynamin using dynasore, which blocks dynamin GTPase activity in HEK cells transducing native S1PR1, phosphodefective (Y143F)‐S1PR1 and phosphomimicking (Y143D)‐S1PR1 and assessed S1PR1 spatial localization using confocal imaging. Interestingly, we found that inhibition of dynamin function blocked the internalization of Y143D‐S1PR1 leading to cell surface retention of the receptor. Cell‐surface retained S1PR1 induces signaling events by binding Gi heterotrimeric protein which includes calcium mobilization, cAMP generation, activation of MAP kinases. Thus, we assessed if phosphorylation altered S1PR1 downstream signaling. We observed that S1P‐induced enhanced Ca2+ entry in HEK cells expressing Y143D‐S1PR1 in Gi‐dependent manner indicating phosphorylated receptor binds Gi and dynamin and the internalized complex is functional. Further studies are under way to characterize the role of dynamin interaction with Y143 phosphorylated receptor in regulating S1PR1 signaling and endothelial barrier repair function.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.