ABSTRACT
Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of Gq-coupled receptors induced a profound, rapid (half-life 3-5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein alpha-subunits specifically of the Gq family. Classical Gq signalling pathways, or phosphorylation at Ser225, phospholipase D and Ca2+/calmodulin were not involved in M3 receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of Gq-coupled receptors, linking them via cross-activation of S1P receptors to G(i) and G12/13 signalling pathways.
Subject(s)
Cell Membrane/enzymology , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Receptors, Lysosphingolipid/metabolism , Animals , Cell Membrane/drug effects , Diglycerides/metabolism , Endocytosis/drug effects , Humans , Mice , Phospholipase D/metabolism , Phosphoserine/metabolism , Protein Kinase C/metabolism , Protein Transport/drug effects , Receptor, Muscarinic M3/metabolism , Sphingosine/analogs & derivatives , Sphingosine/pharmacologyABSTRACT
The lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), stimulate chemotaxis and induce differentiation of human keratinocytes. As Ca(2+) plays an important role in keratinocyte differentiation, we studied Ca(2+) signaling by S1P and LPA in these cells, known to express mRNA transcripts of the S1P(1-5) and LPA(1-3) receptors, and the receptor subtypes involved in this process. S1P and LPA caused transient increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)), with pEC(50) values of 8.5+/-0.11 and 7.5+/-0.23, respectively. The [Ca(2+)](i) increases are apparently mediated by stimulation of phospholipase C and involve Ca(2+) mobilization from thapsigargin-sensitive stores and subsequent Ca(2+) influx. The LPA-induced [Ca(2+)](i) increases were not inhibited by the LPA(1/3) receptor antagonist, dioctanoylglycerol pyrophosphate. The S1P-induced [Ca(2+)](i) increases were largely inhibited by the putative S1P(3) antagonist, BML-241, and the S1P(1/3) antagonist, VPC23019. The S1P(1)-specific agonist, SEW2871, did not increase [Ca(2+)](i) but stimulated chemotaxis of keratinocytes, which was fully blocked by S1P(1) antisense oligonucleotides. The data indicate that LPA and S1P potently increase [Ca(2+)](i) in human keratinocytes and that the effect of LPA is mediated by LPA(2), whereas that of S1P is mediated at least to a large part by S1P(3). The S1P(1) receptor, without stimulating [Ca(2+)](i) increases, mediates chemotaxis of keratinocytes.
