The present study demonstrates that the G protein-coupled receptor EDG6 isolated from in vitro differentiated human and murine dendritic cells is expressed in hematopoietic and lymphoid tissues and cell lines as well as in lung. The mRNA expression pattern is identical in man and mouse and points to a putative function of the EDG6 receptor in the immune system. The human edg6 gene is encoded on chromosome 19p13.3, the murine homolog on chromosome 10. Both genes are located upstream of the of the Gna15/16 gene. The human and murine EDG6 receptors have been detected on the cell surface of transiently transfected cells in the correct orientation. Furthermore, three monoclonal antibodies that are directed against the N-terminus of the murine EDG6 receptor have been generated . They show distinct signals on transiently transfected cell lines, but they lack any specific signal on primary cells so far.
The generation of stably human EDG6 overexpressing cell lines has enabled the investigation of the ligand, the G protein-coupling, and the signal transduction of EDG6. Sphingosine 1-phosphate could be identified as a specific ligand for EDG6 with a moderate binding constant of 63nM. Stimulation of EDG6 leads to the activation of G alpha i and G alpha 12/13 subunits of trimeric G proteins. The mitogen-activated protein-kinases ERK1/2 as well as the phospholipase C are subsequently activated in a G alpha i-dependent manner. Moreover, stimulation of the EDG6 receptor leads to distinct cytoskeleton rearrangements resulting in increasing amounts of peripheral stress fibers, cell rounding and very long filopodia. These effects are induced presumably by G alpha 12/13 and Rho GTPase-driven signaling pathways. A certain pertussis toxin-sensitive and ligand-independent basal activity of the EDG6 receptor is demonstrable in stably human EDG6 overexpressing Jurkat cells that show a higher degree of spontaneous migration events. However, these experiments do not suggest an EDG6-induced migration towards a sphingosine 1-phosphate stimulus.
Experiments concerning the surface expression of EDG6 indicate a complex cell-specific regulation of the surface transportation. In HEK293 cells the internalization of the EDG6 receptor has been shown after stimulation with sphingosine 1-phosphate. In CHO-K1 cells EDG6 is transported towards the cell surface only several minutes after stimulation with sphingosine 1-phosphate, and in HeLa cells the addition of an unknown heat instable serum protein is critical for the surface transportation of EDG6. The mentioned regulation seems to be dependent on a free EDG6 N-terminus because N-terminal epitope-tagged EDG6 constructs lack the differential surface regulation in HeLa cells. They are constitutively present on the cell surface.
To further investigate the in vivo function of the EDG6 receptor, an embryonic stem cell clone has been generated to establish edg6-deficient mice. EDG6 possibly modulates the immunological response in an autocrine or paracrine fashion after binding free sphingosine 1-phosphate. This event may happen after an immunological challenge like an infection.
