Cross-linking of FcεRI causes Ca²⁺ mobilization via a sphingosine kinase pathway in a clathrin-dependent manner

Clathrin-coated pits are now recognized to be involved in cell signaling in addition to receptor down-regulation. Here we tried to identify signaling pathways that might be dependent on clathrin. Our initial data with pharmacological inhibitors of formation of clathrin-coated pits or lipid-rafts indicated that Ca²⁺ response evoked by cross-linking of the high affinity receptors for IgE (FcεRI) was dependent on clathrin. To confirm this finding, we created clathrin-knockdown cells by transfecting the mast cell line RBL-2H3 with a shRNA-clathrin heavy chain construct. In these cells, the FcεRI-mediated Ca²⁺ response was almost completely abolished, which was accompanied by the inhibition of sphingosine 1-phosphate (S1P) production with no changes in inositol 1,4,5-trisphosphate (IP₃) production. This suggests that the Ca²⁺ signaling pathway via a sphingosine kinase (SK) is dependent on clathrin. Furthermore, antigen-induced tyrosine phosphorylation of p85 and p110 subunits of PI3K was almost completely inhibited in clathrin-knockdown cells. In contrast, antigen-induced tyrosine phosphorylation of phospholipase Cγ was not affected by clathrin-knockdown and tyrosine phosphorylation of Syk and degranulation were partially inhibited in clathrin-knockdown cells. The present study identifies the SK/Ca²⁺ pathway to be dependent on clathrin.