Formation of inositol 1,4,5‐trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G‐protein‐coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC–IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine‐1‐phosphate (SPP), is involved in GPCR‐mediated Ca2+ signalling as proposed for platelet‐derived growth factor and FcϵRI antigen receptors. Inhibition of sphingosine kinase by dl‐ threo ‐dihydrosphingosine and N,N ‐dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK‐293 cells without affecting mAChR‐induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK‐293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK‐293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase–SPP pathway in addition to PLC–IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR‐induced Ca2+ signalling.

Sphingosine-1-phosphate (SPP) has attracted much attention as a possible second messenger controlling cell proliferation and motility and as an intracellular Ca-releasing agent. Here, we present evidence that SPP activates a G protein-coupled receptor in the plasma membrane of various cells, leading to increase in cytoplasmic Ca concentration ([Ca]), inhibition of adenylyl cyclase, and opening of G protein-regulated potassium channels. In human embryonic kidney (HEK) cells, SPP potently (EC, 2 nM) and rapidly increased [Ca] in a pertussis toxin-sensitive manner. Pertussis toxin-sensitive increase in [Ca] was also observed with sphingosylphosphorylcholine (EC, 460 nM), whereas other sphingolipids, including ceramide-1-phosphate, N-palmitoyl-sphingosine, psychosine, and D-erythro-sphingosine at micromolar concentrations did not or only marginally increased [Ca]. Furthermore, SPP inhibited forskolin-stimulated cAMP accumulation in HEK cells and increased binding of guanosine 5′-3-O-(thio)triphosphate to HEK cell membranes. Rapid [Ca] responses were also observed in human transitional bladder carcinoma (J82) cells, monkey COS-1 cells, mouse NIH 3T3 cells, Chinese hamster ovary (CHO-K1) cells, and rat C6 glioma cells, whereas human HL-60 leukemia cells and human erythroleukemia cells failed to respond to SPP. In guinea pig atrial myocytes, SPP activated G protein-regulated inwardly rectifying potassium channels. Activation of these channels occurred strictly when SPP was applied at the extracellular face of atrial myocyte plasma membrane as measured in cell-attached and inside-out patch clamp current recordings. We conclude that SPP, in addition to its proposed direct action on intracellular Ca stores, interacts with a high affinity G protein-coupled receptor in the plasma membrane of apparently many different cell types.

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well‐studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X‐linked IAP (XIAP) and cellular IAP1 (c‐IAP1) directly bind to Rac1 in a nucleotide‐independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c‐IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1‐dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis. There is a [Have you seen?][1] (January 2012) associated with this Article. [1]: http://dx.doi.org/10.1038/emboj.2011.452