Sphingosine 1-phosphate and dioleoylphosphatidic acid are low affinity agonists for the orphan receptor GPR63.

Five high affinity G-protein-coupled receptors for sphingosine 1-phosphate (S1P) have been characterised so far (S1P(1,2,3,4,5) formerly referred to as edg1,5,3,6,8). In this study, we show that S1P, dihydro-sphingosine 1-phosphate (dihydro-S1P) and dioleoylphosphatidic acid (doPA) are agonists for the orphan receptor GPR63. All three phospholipids mobilise intracellular calcium in CHO cells transiently transfected with GPR63. Calcium signals required cotransfection of a chimeric Galpha(q/i) protein in a fluorometric imaging plate reader (FLIPR) assay but did not require overexpressed G proteins in an aequorin assay, using a green fluorescent protein (GFP)-aequorin fusion protein as a bioluminescent Ca(2+) reporter. GPR63 expression in CHO cells confers proliferative responses to S1P in a pertussis toxin (PTX)-insensitive manner. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) indicated highest expression in brain, especially in the thalamus and the nucleus caudatus. In peripheral tissues, highest expression was observed in thymus, stomach and small intestine; lower abundance of transcripts was detected in kidney, spleen, pancreas and heart. The discovery that S1P, dihydro-S1P and dioleoylphosphatidic acid activate GPR63 will facilitate the identification of agonists and antagonists, and help to unravel the biological function of this receptor.

Comparative analysis of functional assays for characterization of agonist ligands at G protein-coupled receptors.

A variety of functional assays are available for agonist or antagonist screening of G protein-coupled receptors (GPCRs), but it is a priori not predictable which assay is the most suitable to identify agonists or antagonists of GPCRs with therapeutic value in humans. More specifically, it is not known how a given set of GPCR agonists compares in different functional assays with respect to potency and efficacy and whether the level of the signaling cascade that is analyzed has any impact on the detection of agonistic responses. To address this question, the authors used the recently cloned human S1P(5) receptor as a model and compared a set of 3 lipid ligands (sphingosine 1-phosphate [S1P], dihydro sphingosine 1-phosphate [dhS1P], and sphingosine) in 5 different functional assays: GTPgammaS binding, inhibition of adenylyl cyclase activity, mobilization of intracellular Ca(2+) via the FLIPR and aequorin technology, and MAP kinase (ERK1/2) activation. S1P induced agonistic responses in all except the ERK1/2 assays with EC(50) values varying by a factor of 10. Whereas dhS1P was identified as a partial agonist in the GTPgammaS assay, it behaved as a full agonist in all other settings. Sphingosine displayed partial agonistic activity exclusively in GTPgammaS binding assays. The findings suggest that assays in a given cellular background may vary significantly with respect to suitability for agonist finding and that ligands producing a response may not readily be detectable in all agonist assays.

Regulated and constitutive activation of specific signalling pathways by the human S1P5 receptor.

1 We tested the hypothesis, whether G Protein-coupled receptors (GPCRs) may differentially regulate specific signalling pathways by constitutive and agonist-induced activation using the human sphingosine 1-phosphate receptor S1P(5) as a model. 2 S1P(5) receptor-expressing HEK293 cells exhibited a high degree of basal activity for both inhibition of adenylyl cyclase and extracellular signal regulated kinase (ERK) when cultured in serum, which contains high levels of sphingosine 1-phosphate (S1P). However, basal activity was independent of the presence of S1P: (i) constitutive activity remained when cells were cultured in delipidated serum, (ii) addition of S1P to delipidated serum did not increase basal S1P(5) receptor signalling. 3 Conversely, constitutive inhibition of forskolin-stimulated adenylyl cyclase was further enhanced by S1P in S1P(5)-HEK293 cells. 4 Transfection of several mammalian cell lines (CHO-K1, HEK293, NIH-3T3, RH7777) with the S1P(5) receptor induced cell rounding, which was more pronounced in the presence of S1P-containing serum. Rounded cell morphology did not correlate with apoptotic cell death, but led to detachment of cells. 5 Cell surface ELISA assays showed that a fraction of plasma membrane S1P(5) receptors were dose-dependently internalized with S1P. 6 These data reveal that intrinsic inhibition of unstimulated adenylyl cyclase or ERK activity by the S1P(5) receptor is insensitive to ligand modulation. Conversely, effects on forskolin-stimulated adenylyl cyclase, cell morphology and internalization can be further augmented with S1P. Our results suggest that different signal transduction pathways are not equally activated through constitutively active GPCRs with promiscuous signalling characteristics.

Comparative analysis of human and rat S1P(5) (edg8): differential expression profiles and sensitivities to antagonists.

Five guanine nucleotide-binding protein-coupled receptors (S1P(1-5)) for the lysophospholipid mediator sphingosine 1-phosphate (S1P) have thus far been described. Whereas tissue distribution and functional properties of the human S1P(1-4) genes are well characterized, only limited functional and expression data are available for S1P(5), todate. Northern blot analysis indicated that human S1P(5) (hS1P(5)) is an alternatively spliced gene, with a 5.4-kb transcript that is predominantly expressed in peripheral tissues, and a 2.4-kb transcript expressed in brain, spleen, and peripheral blood leucocytes. In contrast, rat S1P(5) (rS1P(5)) was exclusively detected in brain and skin. Expression of hS1P(5) and rS1P(5) in mammalian CHO-K1 or HEK293 cells conferred onto the cells the ability to mobilize intracellular calcium as determined by a functional Fluorometric Imaging Plate Reader assay, when challenged with S1P and dihydro S1P, respectively. Applying a lipid library with 200 bioactive lipids in a functional Fluorometric Imaging Plate Reader assay did not reveal additional agonists. However, both receptors exhibited differential sensitivity towards the S1P- and lysophosphatidic acid-receptor antagonist, suramin: rS1P(5)-mediated intracellular calcium mobilization was partly inhibited by suramin (IC(50): 5800 microM), whereas hS1P(5) was completely antagonized (IC(50): 130 microM). Both receptors were sensitive towards inhibition with the related drug (8,8'-(carbonylbis(imino-3,1-phenylene))bis(1,3,5-naphthalenetrisulfonic acid)) but IC(50) values differed significantly (340 microM for hS1P(5), 4000 microM for rS1P(5)). In addition, rS1P(5) displayed antiproliferative effects in transfected CHO-K1 and HEK293 cells in contrast to hS1P(5). Taken together, our data imply that differences between hS1P(5) and rS1P(5) will be an important point to be considered in the development of selective receptor antagonists.

Modulation of myocardial contractility by lysophosphatidic acid (LPA).

Lysophosphatidic acid (LPA) is a phospholipid messenger, which is released from activated platelets and leukocytes. This study examined the effects of LPA on myocardial contractility and characterized the signal transduction pathway involved in these effects. Functional effects of LPA were determined in isolated, electrically driven human myocardial preparations and rat cardiac myocytes. In human atrial and ventricular myocardial preparations, LPA (100 micromol/l) decreased isoprenaline (0.03 micromol/l) enhanced force of contraction by 17 +/- 2% and 28 +/- 3%, respectively. The effect of LPA was attenuated by suramin (1 mmol/l). In isolated rat cardiomyocytes, LPA (1-100 micromol/l) concentration dependently abolished isoprenaline (0.03 micromol/l) induced increase in cell shortening. This antiadrenergic effect was blunted after pretreatment with pertussis toxin (5 microg/ml, 12 h). Forskolin (10 micromol/l) stimulated adenylyl cyclase activity was inhibited by LPA in human myocardial membranes. PCR analysis of human atrial and ventricular cDNAs revealed the expression of two cognate LPA receptors: EDG-2 and EDG-7. Our results suggest that LPA exerts antiadrenergic effects on force of contraction in human and rodent myocardium via a Galpha(i/o) protein-mediated mechanism, most probably by LPA binding to the mammalian LPA receptors EDG-2 and/or EDG-7. This newly discovered action of LPA might be of pathophysiological importance in conditions like myocardial ischemia or inflammatory disorders when LPA release is enhanced.