Characterization of a Novel Sphingosine 1-Phosphate Receptor, Edg-8

Abstract

Three G protein-coupled receptors (Edg-1, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator sphingosine 1-phosphate have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized of a fourth, high affinity, rat brain sphingosine 1-phosphate receptor, Edg-8. When HEK293T cells were co-transfected with Edg-8 and G protein DNAs, prepared membranes showed sphingosine 1-phosphate-dependent increases in [35 S]guanosine 5-(3-O-thio)triphosphate binding with an EC 50 of 90 nM. In a rat hepatoma Rh7777 cell line that exhibits modest endogenous responses to sphingosine 1-phosphate, this lipid mediator inhibited forskolin-driven rises in cAMP by greater than 90% when the cells were transfected with Edg-8 DNA (IC 50 0.7 nM). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G i/o ␣ proteins. Furthermore, Xenopus oocytes exhibit a calcium response to sphingosine 1-phosphate after injection of Edg-8 mRNA, but only when oocytes are co-injected with chimeric G q/i ␣ protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (K D ‫؍‬ 2 nM) binding for radiolabeled sphingosine 1-phosphate. Rat Edg-8 RNA is expressed in spleen and throughout adult rat brain where in situ hybridization revealed it to be associated with white matter. Together our data demonstrate that Edg-8 is a high affinity sphingosine 1-phosphate receptor that couples to G i/o ␣ proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.Sphingosine 1-phosphate (S1P) 1 is a potent, extracellular lysolipid phosphoric acid mediator that is released, for example, during platelet activation (1). S1P elicits a wide variety of responses by cells; prominent among these are cell proliferation (2-4) and anti-apoptosis (5, 6) as well as a wide variety of other effects. S1P and the structurally related lysolipid mediator, lysophosphatidic acid (LPA), are recognized now to signal cells through a set of G protein coupled receptors known colloquially as the "Edg" receptors. Discovered initially as "orphan" receptors (7, 8), three members of the group, Edg-1, Edg-5, and Edg-3, have been shown to be S1P receptors. For example, Edg-1 mediates S1P activation of mitogen-activated protein kinase and inhibition of adenylyl cyclase in a pertussis toxindependent manner (9, 10). S1P activation of Edg-3 results calcium mobilization in a pertussis toxin-independent manner (11), while others found that this receptor coupled also, for example, to inhibition of adenylyl cyclase via G i/o ␣ protein (12). Likewise, several groups have shown that a third S1P receptor, Edg-5, couples also to G q/11 ␣ proteins (13, 14). All three S1P receptors signal in Xenopus oocytes, although Edg-1 signaling was dependent on co-injection with a chimeric G q ␣/G i ␣ protein (15). These data suggest that the three known S1P receptors interact with different signa...

Lysosphingolipid receptor‐mediated diuresis and natriuresis in anaesthetized rats

“…As intercellular messengers lysosphingolipids act on speci®c receptors, some of which couple to pertussis toxin (PTX) sensitive G-proteins (Goetzl & An, 1998;Meyer zu Heringdorf et al, 1997). While some members of the Edg receptor family are preferentially activated by lysophosphatidic acid, Edg1, Edg3, Edg5, Edg6 and Edg 8 are preferentially activated by SPP (Im et al, 2000; renal function. In the present study we report that systemically infused SPP can alter renovascular and tubular function in anaesthetized rats in a PTX-sensitive manner, whereas SPPC, SPH and GLU alter tubular functions in the absence of detectable renovascular e ects.…”

Inhibitory Regulation of Rac Activation, Membrane Ruffling, and Cell Migration by the G Protein-Coupled Sphingosine-1-Phosphate Receptor EDG5 but Not EDG1 or EDG3

“…Recent identification of cell surface heptahelical receptors for S1P and its structurally related lysophospholipid, lysophosphatidic acid (LPA), which are collectively designated EDG (for endothelial differentiation gene) or LP (for lysophospholipid) receptors, strongly suggests that a diversity of S1P-induced responses are mediated through the EDG receptors (5,15,26,49,65,66,68), although some biological activities of S1P were reported to be mediated through its intracel-lular actions (16,54,57,58,65,66,70,74). Among the EDG receptors, EDG1, EDG3, EDG5 (AGR16 or H218), and EDG8 are identified as receptors specific for S1P (8,20,30,34,35,44,46,47,49,65,66,71), while EDG2, EDG4, and EDG7 are receptors specific for LPA (5,15,26,49). EDG1, EDG3, and EDG5 are widely expressed in various tissues (25,53,76), whereas expression of EDG8 is confined to the central nervous system (30).…”

“…Among the EDG receptors, EDG1, EDG3, EDG5 (AGR16 or H218), and EDG8 are identified as receptors specific for S1P (8,20,30,34,35,44,46,47,49,65,66,71), while EDG2, EDG4, and EDG7 are receptors specific for LPA (5,15,26,49). EDG1, EDG3, and EDG5 are widely expressed in various tissues (25,53,76), whereas expression of EDG8 is confined to the central nervous system (30). We and others have shown that the S1P receptors exhibit distinct signaling characteristics.…”

Sphingolipids and membrane biology as determined from genetic models

“…Although all these receptors are activated by S1P binding, studies with receptor inhibitors suggest variations in the S1P binding site among these receptors [152,158,159]. There are also differences in the coupling of receptor activation to downstream G proteins [160][161][162][163]. S1P 1 couples to G i , S1P 2 and S1P 3 couple to Gi, Gq, and G 13 .…”