title: Cross-linking of FcepsilonRI causes Ca2+ mobilization via a sphingosine kinase pathway in a clathrin-dependent manner.
abstract: 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 Ca(2+) response evoked by cross-linking of the high affinity receptors for IgE (FcepsilonRI) 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 FcepsilonRI-mediated Ca(2+) 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 (IP(3)) production. This suggests that the Ca(2+) 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 Cgamma 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/Ca(2+) pathway to be dependent on clathrin.
authors.affiliation: Department of Medicine, Division of Allergy and Clinical Immunology, The Johns Hopkins University School of Medicine, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
authors.forename: Seung-Duk
authors.initials: SD
authors.lastname: Ryu
authors.forename: Hyun Sil
authors.initials: HS
authors.lastname: Lee
authors.forename: Ho Young
authors.initials: HY
authors.lastname: Suk
authors.forename: Chang-Shin
authors.initials: CS
authors.lastname: Park
authors.forename: Oksoon Hong
authors.initials: OH
authors.lastname: Choi
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D015550:Cell Degranulation / Q000187:drug effects; D002460:Cell Line; D002746:Chlorpromazine / Q000494:pharmacology; D033922:Clathrin Heavy Chains / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000187:drug effects / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects*; D055785:Gene Knockdown Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism*; D000099157:Sphingosine Kinase
chemical_list: D002746:Chlorpromazine; D033922:Clathrin Heavy Chains; D003432:Cross-Linking Reagents; D015544:Inositol 1,4,5-Trisphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019000:Phosphotyrosine; D017455:Receptors, IgE; D000099157:Sphingosine Kinase