No abstract

Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). Together with other sphingolipid metabolizing enzymes, SphKs regulate the balance of the lipid mediators, ceramide, sphingosine, and S1P. The ubiquitous mediator S1P regulates cellular functions such as proliferation and survival, cytoskeleton architecture and Ca(2+) homoeostasis, migration, and adhesion by activating specific high-affinity G-protein-coupled receptors or by acting intracellularly. In mammals, two isoforms of SphK have been identified. They are activated by G-protein-coupled receptors, receptor tyrosine kinases, immunoglobulin receptors, cytokines, and other stimuli. The molecular mechanisms by which SphK1 and SphK2 are specifically regulated are complex and only partially understood. Although SphK1 and SphK2 appear to have opposing roles, promoting cell growth and apoptosis, respectively, they can obviously also substitute for each other, as mice deficient in either SphK1 or SphK2 had no obvious abnormalities, whereas double-knockout animals were embryonic lethal. In this review, our understanding of structure, regulation, and functional roles of SphKs is updated and discussed with regard to their implication in pathophysiological and disease states.

Sphingosine-1-phosphate (SPP) has attracted much attention as a possible second messenger controlling cell proliferation and motility and as an intracellular Ca(2+)-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 Ca2+ concentration ([Ca2+]i), inhibition of adenylyl cyclase, and opening of G protein-regulated potassium channels. In human enbryonic kidney (HEK) cells, SPP potently (EC50, 2 nM) and rapidly increased [Ca2+]i in a pertussis toxin-sensitive manner. Pertussis toxin-sensitive increase in [Ca2+]i was also observed with sphingosylphosphorylcholine (EC50, 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 [Ca2+]i. 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 [Ca2+]i 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 Gi 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 Ca2+ stores, interacts with a high affinity Gi protein-coupled receptor in the plasma membrane of apparently many different cell types.

No abstract

Sphingolipid breakdown products are now being recognized to play a dual role in cellular signalling, acting as intracellular as well as extracellular signalling molecules. Both types of action may even be found with one sphingolipid species. The recent demonstration of G protein-coupled receptors with high affinity for sphingosine 1-phosphate and sphingosylphosphorylcholine has been followed by the discovery of several novel sphingolipid actions, such as regulation of heart rate, oxidative burst, neurite retraction or platelet activation. Ligand profiles and concentration-response relationships suggest the existence of putative sphingolipid receptor subtypes. Against this background, several observations on supposed sphingolipid second messenger actions deserve a new evaluation.

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic ␤-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the ␤ 2 -adrenergic receptor have demonstrated that ␤-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of ␤-arrestin in mAChR sequestration, we determined the effect of overexpressing ␤-arrestin-1 and the dominant-negative inhibitor of ␤-arrestin-mediated receptor sequestration, ␤-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60 -75% in cells overexpressing ␤-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of ␤-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrincoated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50 -70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a ␤-arrestin-and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.Exposure of many G protein-coupled receptors (GPCRs) 1 to their agonists results within seconds to minutes in attenuation of receptor responsiveness. An important step in this process of receptor desensitization is the rapid phosphorylation of agonist-bound receptors by G protein-coupled receptor kinases (1). These kinases phosphorylate serine and threonine residues located in the third cytoplasmic loop (i.e. m1 and m2 muscarinic acetylcholine receptors (mAChRs)) (2, 3) or in the cytoplasmic carboxyl-terminal tail of the receptors (for example, ␤ 2 -adrenergic receptors) (4). After phosphorylation, cytosolic ␤-arrestins bind with increased affinity to the receptors and sterically inhibit further coupling of the receptors with G proteins (1). To date, two ␤-arrestin isoforms, ␤-arrestin-1 (arrestin 2) and ␤-arrestin-2 (arrestin 3) have been identified, each undergoing alternative splicing (1). Both isoforms are ubiquitously expressed, with ␤-arrestin-1 being the major ␤-arrestin expressed in many tissues (1). Recent evidence indicates that ␤-arrestins do not only bind to GPCRs but also associate with nanomolar affinity with clathrin heavy chains and target ␤-arrestin-bound GPCRs to the clathrin-coated pits, leading to receptor internalization (5, 6). This process ...

Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the G i proteincoupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF 4 Ϫ also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca 2؉ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through G i -type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.During the last few years, it has become clear that sphingolipids, in addition to being structural constituents of cell membranes, are sources of important signaling molecules. Particularly, the sphingolipid metabolites, ceramide and sphingosine-1-phosphate (SPP), 1 have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation (1-4). Interest in SPP focused recently on two distinct cellular actions of this lipid, namely its function as extracellular ligand activating specific G protein-coupled membrane receptors and its role as intracellular second messenger (5). Important clues to a specific intracellular action of SPP were the following findings. First, activation of various plasma membrane receptors, such as the platelet-derived growth factor receptor (6, 7), the Fc⑀RI (8), and the Fc␥RI antigen receptors (9), was found to rapidly increase intracellular SPP production through stimulation of sphingosine kinase. Second, inhibition of sphingosine kinase stimulation strongly reduced or even prevented cellular events triggered by these tyrosine kinase-linked receptors, such as receptor-stimulated DNA synthesis, Ca 2ϩ mobilization, and vesicular trafficking (6,8,9). Finally, intracellular SPP was found to mimic the receptor responses, i.e. it stimulated DNA synthesis and mobilized Ca 2ϩ from internal stores (10 -14). We recently reported that the G protein-coupled muscarinic acetylcholine receptor subtypes m2 and m3 expressed in HEK-293 cells also induce a rapid and transient SPP production by sphingosine kinase. Furthermore, intracellular in...

1. Activation of muscarinic K+ current (IK(ACh)) by sphingosine-1-phosphate (Sph-1-P) was studied in isolated cultured guinea-pig atrial myocytes using whole-cell voltage clamp.2. Sph-1-P caused activation of IK (ACh)