Actin-binding protein filamin B regulates the cell-surface retention of endothelial sphingosine 1-phosphate receptor 1.

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor essential for vascular development and postnatal vascular homeostasis. When exposed to sphingosine 1-phosphate (S1P) in the blood of ∼1 µM, S1PR1 in endothelial cells retains cell-surface localization, while lymphocyte S1PR1 shows almost complete internalization, suggesting the cell-surface retention of S1PR1 is endothelial cell specific. To identify regulating factors that function to retain S1PR1 on the endothelial cell surface, here we utilized an enzyme-catalyzed proximity labeling technique followed by proteomic analyses. We identified Filamin B (FLNB), an actin-binding protein involved in F-actin cross-linking, as a candidate regulating protein. We show FLNB knockdown by RNA interference induced massive internalization of S1PR1 into early endosomes, which was partially ligand dependent and required receptor phosphorylation. Further investigation showed FLNB was also important for the recycling of internalized S1PR1 back to the cell surface. FLNB knockdown did not affect the localization of S1PR3, another S1P receptor subtype expressed in endothelial cells, nor did it affect localization of ectopically expressed ß2-adrenergic receptor. Functionally, we show FLNB knockdown in endothelial cells impaired S1P-induced intracellular phosphorylation events and directed cell migration and enhancement of the vascular barrier. Taken together, our results demonstrate that FLNB is a novel regulator critical for S1PR1 cell-surface localization and thereby proper endothelial cell function.

Apolipoprotein M supports S1P production and conservation and mediates prolonged Akt activation via S1PR1 and S1PR3.

Sphingosine 1-phosphate (S1P) is one of the lipid mediators involved in diverse physiological functions. S1P circulates in blood and lymph bound to carrier proteins. Three S1P carrier proteins have been reported, albumin, apolipoprotein M (ApoM) and apolipoprotein A4 (ApoA4). The carrier-bound S1P exerts its functions via specific S1P receptors (S1PR1-5) on target cells. Previous studies showed several differences in physiological functions between albumin-bound S1P and ApoM-bound S1P. However, molecular mechanisms underlying the carrier-dependent differences have not been clarified. In addition, ApoA4 is a recently identified S1P carrier protein, and its functional differences from albumin and ApoM have not been addressed. Here, we compared the three carrier proteins in the processes of S1P degradation, release from S1P-producing cells and receptor activation. ApoM retained S1P more stable than albumin and ApoA4 in the cell culture medium when compared in the equimolar amounts. ApoM facilitated theS1P release from endothelial cells most efficiently. Furthermore, ApoM-bound S1P showed a tendency to induce prolonged activation of Akt via S1PR1 and S1PR3. These results suggest that the carrier-dependent functional differences of S1P are partly ascribed to the differences in the S1P stability, S1P-releasing efficiency and signaling duration.