S1PR1 interacts with endoplasmic reticulum stress sensor, BiP, to regulate calcium homeostasis and endothelial barrier function

Abstract

Endothelial–cell surface localized sphingosine 1 phosphate receptor 1 (S1PR1) is well known to enhance barrier function upon ligating S1P. We recently showed that S1P phosphorylation of S1PR1 at tyrosine (Y) 143 residue induces S1PR1 internalization and compromises barrier function. Here, we assessed the mechanism internalizing the Y143‐S1PR1 and how Y143‐S1PR1 disrupts barrier function. Herein, using TIRF and confocal imaging, we show that phosphorylated (Y143D‐S1PR1) S1PR1 mutant localizes at endoplasmic reticulum (ER). Inhibition of dynamin activity, a GTP‐dependent pinchase, rescued Y143D‐S1PR1 expression at the cell surface. However, dynamin inhibition had no effect on cell‐surface retention of S1PR1 or Y143F‐S1PR1. In other studies, we show that Y143D‐S1PR1 bound WT‐dynamin strongly while weakly with GTPase defective dynamin. Cell‐surface retained S1PR1 induces signaling including calcium mobilization and activation of MAP kinases by binding Gi heterotrimeric protein. Intriguingly, we show that internalized Y143D‐S1PR1 augmented Ca2+ entry which was blocked by pertussis toxin. Furthermore, phosphorylated receptor bound Gi indicating that internalized S1PR1 binds cytosolic Gi which then increases Ca2+ entry. We further showed Y143D‐S1PR1 mutant augmented Ca2+ entry by organizing stromal‐interaction molecule 1 (STIM1) dependent ER‐PM junction formation. To address the mediator involved in retaining Y143D‐S1PR1 at the ER, we performed mass spectrometric analysis and identified GRP78 (BiP), an endoplasmic resident protein, as the novel interacting partner of S1PR1. Depletion of BiP or STIM1 rescued barrier function even in presence of Y143D‐S1PR1. Thus, BiP recruits phosphorylated Y143D‐S1PR1 at the ER where S1PR1 mutant organizes ER‐PM junctions to mediate Ca2+ entry disrupting endothelial barrier function.Support or Funding InformationNIH