Selective activation of G alpha i mediated signalling of S1P3 by FTY720-phosphate

doi:10.1016/j.cellsig.2008.01.019

Cellular Signalling

Volume 20, Issue 6, June 2008, Pages 1125-1133

https://doi.org/10.1016/j.cellsig.2008.01.019 Get rights and content

Abstract

The immune modulator FTY720 is phosphorylated in vivo to FTY720 phosphate (FTY-P), which activates four sphingosine 1-phosphate (S1P) receptors including S1P₃. Upon activation with S1P, S1P₃ couples to G_i- and G_q-protein-dependent signalling pathways. Here we show that FTY-P selectively activates the S1P₃-mediated and G_i-coupled inhibition of adenylyl cyclase. Contemporaneously, it antagonizes the S1P-induced activation of G_q via S1P₃ in intracellular calcium flux measurements, GTP-binding experiments, and flow cytometric analyses of activation-induced receptor down-regulation. In contrast to S1P, pre-treatment with FTY-P did not desensitize S1P-induced calcium flux or chemotaxis via S1P₃. The lack of receptor desensitization prevented S1P₃-mediated migration to FTY-P. Human umbilical vein endothelial cells express S1P₁ and S1P₃, and respond to S1P and FTY-P by ERK1/2 phosphorylation and by intracellular calcium release in a pertussis toxin-sensitive manner. But whereas a mixture of S1P and FTY-P was not affecting ERK1/2 phosphorylation, the intracellular calcium flux was hampered with increasing amounts of FTY-P, which points to a cross-talk between S1P₁ and S1P₃. FTY-P is therefore one of the rare ligands which bind to a receptor that couples multiple G-proteins but selectively activates only one signalling pathway.

Introduction

The immune modulator Fingolimod (2-amino-2-(2-[4-octylphenyl]ethyl]-1,3-propanediol, FTY720) is phosphorylated in vivo by sphingosine kinase type 2 to FTY-P [1], [2], which activates, except of S1P₂, all known sphingosine 1-phosphate (S1P) receptors [3], [4]. Its beneficial effect was attributed to the onset of lymphopenia by blocking S1P and S1P₁-mediated lymphocyte egress from lymphoid tissues [5], [6], [7]. One of its reported side-effects is the incidence of bradycardia, which was linked to activation of S1P₃ [8], [9]. The compound was shown to have clinical efficacy in phase II clinical studies in multiple sclerosis (MS) patients [10] and is currently tested in phase III clinical studies for treatment of MS.

FTY-P mimics S1P which is constitutively present in blood [3], [4], [11]. Blood-borne S1P and S1P₁ expression on lymphocytes are critical for lymphocyte circulation [6], [7]. FTY-P is thought to be a superagonist for S1P₁ which induces receptor down-regulation leading to its inhibition [10]. On the other hand it was shown that S1P₁ agonists activate S1P₁ on sinus lining endothelial cells of lymphoid tissues [12], [13]. Activation of S1P₁ leads to the closure of postulated endothelial cell portals, which consequently generate a barrier for exiting lymphocytes [12]. The contribution of S1P₁ activation on endothelial cells and its down-regulation and subsequent inhibition on lymphocytes for FTY-P induced lymphopenia is unclear. One of the major questions that need to be answered is why S1P₁ deficiency on lymphocytes and administration of S1P₁ agonists like FTY-P share the same phenotype of lymphopenia, whereas S1P₁ antagonists are ineffective [14], [15], [16].

FTY720′s mode of action is complex. It was shown to inhibit cytosolic phospholipase A2 independently from S1P receptors by an intracellular mechanism [17]. After phosphorylation it acts as an extracellular agonist on four S1P receptors [3], [4]. But FTY-P also induces receptor internalization and degradation [5], which has opposite effects to its agonistic activity. S1P receptor activation by FTY-P induces bradycardia and protects capillary integrity amongst others [8], [9]. The internalization and degradation of S1P receptors after FTY-P treatment mediate distinct phenotypes of S1P receptor deficiency, like the observed block of lymphocyte emigration in lymphocyte-specific conditional S1P₁ knockout mice [6], [18]. Some of its effects are considered to be beneficial. For example, the down-regulation of S1P₁ cell surface expression on lymphocytes is important for the onset of lymphopenia and for the immunosuppressive activity [6]. Others however are unintentional side-effects. Bradycardia induced by S1P₃ activation is only one example of an unwanted activity of FTY-P [8], [9]. The quest for compounds with similar efficacy and lesser side-effects than FTY720 requires detailed information about its interplay with receptors and their signalling pathways.

Here we report that FTY-P activates G_i proteins via S1P₃, and simultaneously blocks G_q-mediated signalling pathways of S1P₃ after S1P-stimulation. Thus FTY-P does not mimic S1P as the natural ligand for S1P₃. It stabilizes signalling events via G_i proteins and contemporaneously antagonizes S1P-mediated G_q coupling at S1P₃.

Section snippets

Chemicals

S1P and pertussis toxin (PTx) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Chemicals and solvents were purchased from Roth (Karlsruhe, Germany) if not stated otherwise. FTY720 and FTY-P were kindly provided by Dr. Volker Brinkmann (Novartis, Basel, Switzerland). FTY-P was dissolved in DMSO/50 mM HCl (stock concentration of 50 mM) and diluted with methanol to 1 mM FTY-P. FTY720 and S1P were dissolved directly in methanol to final concentrations of 1 mM.

Cell culture

Serum, cell culture media,

FTY-P induced activation of G_i, but not G_q via S1P₃

It has been shown that S1P₃ couples to G_i and G_q proteins upon S1P stimulation [21]. To test whether or not FTY-P is also capable to activate both signalling pathways, we established cAMP accumulation assays with transiently transfected COS-7 cells expressing S1P₃ to test for the G_i-dependent inhibition of forskolin-activated adenylyl cyclase (AC). FTY-P was able to inhibit stimulation of AC with similar efficiency as compared to S1P (Fig. 1A). Forskolin-induced AC stimulation was reduced by

Discussion

FTY720 is the first compound related to sphingolipids with clinical relevance. It was shown to alleviate the effects of experimental autoimmune encephalomyelitis (EAE) in rodents, and multiple sclerosis (MS) in humans [10], [26], [27]. Its immunosuppressive effect was attributed to the onset of lymphopenia by blocking lymphocyte egress from lymphoid tissues [28]. Inhibition of S1P₁ on lymphocytes, and stimulation of S1P₁ on lymphatic sinus lining endothelial cells were proposed as potential

Acknowledgements

The authors thank Anika Münk for her excellent technical assistance, Dr. Evi Kostenis for the G_sq5 construct, and Dr. Volker Brinkmann (Novartis, Basel, Switzerland) for generously providing us with FTY720 and FTY-P. This work was supported by the German Research Foundation (DFG), grant GR-1943/1-3 of the Emmy Noether Program, grant LE-940/4-1, and grant LE-940/3-1.

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Because our data suggests that S1P signals through Gαi to induce StAR mRNA expression (Fig. 3A) and ACTH activates a Gαs-cAMP-PKA signaling pathway (Mountjoy et al., 1992), it is tempting to speculate that S1P-mediated Gαi activation downregulates ACTH-dependent Gαs signaling by inhibiting adenylyl cyclase (Taussig et al., 1993). Gαi-mediated downregulation of cAMP production by S1P has been previously reported (Jongsma et al., 2009; Jun et al., 2006; Means et al., 2008; Sensken et al., 2008). Many reports support the activation of Ca2+ mobilization by S1P through GPCR-mediated PLC activation (Bornfeldt et al., 1995; Noh et al., 1998; Okajima et al., 1996; Suhaiman et al., 2010).
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Selective activation of G alpha i mediated signalling of S1P3 by FTY720-phosphate

Abstract

Introduction

Section snippets

Chemicals

Cell culture

FTY-P induced activation of Gi, but not Gq via S1P3

Discussion

Acknowledgements

J. Biol. Chem.

Blood

J. Biol. Chem.

Am. J. Transplant.

J. Biol. Chem.

Bioorganic Med. Chem. Letters

Chem. Biol.

Blood

J. Biol. Chem.

Trends Pharmacol. Sci.

J. Biol. Chem.

Life Sci.

Methods Enzymol.

Prostaglandins Other Lipid Mediat.

Biochem. Biophys. Res. Commun.

Trends Pharmacol. Sci.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Science

Selective activation of G alpha i mediated signalling of S1P₃ by FTY720-phosphate

FTY-P induced activation of G_i, but not G_q via S1P₃