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.