Platelet-derived growth factor (PDGF) and sphingosine 1-phosphate (S1P) act via PDGF␤ receptor-S1P 1 receptor complexes in airway smooth muscle cells to promote mitogenic signaling. Several lines of evidence support this conclusion. First, both receptors were co-immunoprecipitated from cell lysates with specific anti-S1P 1 antibodies, indicating that they form a complex. Second, treatment of airway smooth muscle cells with PDGF stimulated the phosphorylation of p42/p44 MAPK, and this phosphorylated p42/p44 MAPK associates with the PDGF␤ receptor-S1P 1 receptor complex. Third, treatment of cells with antisense S1P 1 receptor plasmid construct reduced the PDGF-and S1P-dependent activation of p42/p44 MAPK. Fourth, S1P and/or PDGF induced the formation of endocytic vesicles containing both PDGF␤ receptors and S1P 1 receptors, which was required for activation of the p42/ p44 MAPK pathway. PDGF does not induce the release of S1P, suggesting the absence of a sequential mechanism. However, sphingosine kinase 1 is constitutively exported from cells and supports activation of p42/p44 MAPK by exogenous sphingosine. Thus, the presentation of sphingosine from other cell types and its conversion to S1P by the kinase exported from airway smooth muscle cells might enable S1P to act with PDGF on the PDGF␤ receptor-S1P 1 receptor complex to induce biological responses in vivo. These data provide further evidence for a novel mechanism for G-protein-coupled receptor and receptor tyrosine kinase signal integration that is distinct from the transactivation of receptor tyrosine kinases by G-proteincoupled receptor agonists and/or sequential release and action of S1P in response to PDGF. Sphingosine 1-phosphate (S1P)1 is a bioactive lysolipid that has been proposed to have both intracellular and extracellular actions (1). To date, five closely related G-protein-coupled receptors (GPCR), termed S1P 1 -S1P 5 (2) (and formerly named EDG1, EDG5/AGR16/H218, EDG3, EDG6 and EDG8/nrg-1, respectively) have been identified as high affinity S1P receptors (3-9). Further characterization studies confirmed the S1P 1 receptor to be a GPCR with high affinity for S1P that stimulates p42/p44 MAPK and inhibits adenylyl cyclase in cells (10 -13). The S1P 2 and S1P 3 receptors also have high affinity for S1P (14) and are linked via G q to phospholipase C and calcium mobilization and p42/p44 MAPK activation (14, 15) and via G 12 and G 13 to Rhoguanine nucleotide factor and Rho activation. The S1P 4 receptor is lymphoid specific and, in common with the S1P 5 receptor, uses G i/o and G 12 to signal (6, 8).The S1P 1 receptor is implicated in regulating smooth muscle cell migration, proliferation, and vascular maturation. Insight into the function of the S1P 1 receptor was obtained by studies showing that disruption of the s1p 1 gene by homologous recombination in mice results in extensive intra-embryonic hemorrhaging and intrauterine (16). This is caused by incomplete vascular maturation due to the failure of mural cells, vascular smooth muscle cells, and per...

Sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid bind to G-protein-coupled receptors to stimulate intracellular signaling in mammalian cells. Lipid phosphate phosphatases (1, 1a, 2, and 3) are a group of enzymes that catalyze de-phosphorylation of these lipid agonists. It has been proposed that the lipid phosphate phosphatases exhibit ecto activity that may function to limit bioavailability of these lipid agonists at their receptors. In this study, we show that the stimulation of the p42/p44 mitogen-activated protein kinase pathway by sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid, all of which bind to G i/ocoupled receptors, is substantially reduced in human embyronic kidney 293 cells transfected with lipid phosphate phosphatases 1, 1a, and 2 but not 3. This was correlated with reduced basal intracellular phosphatidic acid and not ecto lipid phosphate phosphatase activity. These findings were supported by results showing that lipid phosphate phosphatases 1, 1a, and 2 also abrogate the stimulation of p42/p44 mitogen-activated protein kinase by thrombin, a peptide G i/o -coupled receptor agonist whose bioavailability at its receptor is not subject to regulation by the phosphatases. Furthermore, the lipid phosphate phosphatases have no effect on the stimulation of p42/p44 mitogen-activated protein kinase by other agents that do not use G-proteins to signal, such as serum factors and phorbol ester. Therefore, these findings show that the lipid phosphate phosphatases 1, 1a, and 2 may function to perturb G-proteincoupled receptor signaling per se rather than limiting bioavailability of lipid agonists at their respective receptors.

Here we provide evidence to show that the plateletderived growth factor ␤ receptor is tethered to endogenous G-protein-coupled receptor(s) in human embryonic kidney 293 cells. The tethered receptor complex provides a platform on which receptor tyrosine kinase and G-proteincoupled receptor signals can be integrated to produce more efficient stimulation of the p42/p44 mitogen-activated protein kinase pathway. This was based on several lines of evidence. First, we have shown that pertussis toxin (which uncouples G-protein-coupled receptors from inhibitory G-proteins) reduced the platelet-derived growth factor stimulation of p42/p44 mitogen-activated protein kinase. Second, transfection of cells with inhibitory G-protein ␣ subunit increased the activation of p42/p44 mitogenactivated protein kinase by platelet-derived growth factor. Third, platelet-derived growth factor stimulated the tyrosine phosphorylation of the inhibitory G-protein ␣ subunit, which was blocked by the platelet-derived growth factor kinase inhibitor, tyrphostin AG 1296. We have also shown that the platelet-derived growth factor ␤ receptor forms a tethered complex with Myc-tagged endothelial differentiation gene 1 (a G-protein-coupled receptor whose agonist is sphingosine 1-phosphate) in cells co-transfected with these receptors. This facilitates platelet-derived growth factorstimulated tyrosine phosphorylation of the inhibitory G-protein ␣ subunit and increases p42/p44 mitogenactivated protein kinase activation. In addition, we found that G-protein-coupled receptor kinase 2 and ␤-arrestin I can associate with the platelet-derived growth factor ␤ receptor. These proteins play an important role in regulating endocytosis of G-protein-coupled receptor signal complexes, which is required for activation of p42/p44 mitogenactivated protein kinase. Thus, platelet-derived growth factor ␤ receptor signaling may be initiated by G-proteincoupled receptor kinase 2/␤-arrestin I that has been recruited to the platelet-derived growth factor ␤ receptor by its tethering to a G-protein-coupled receptor(s). These results provide a model that may account for the co-mitogenic effect of certain G-protein-coupled receptor agonists with platelet-derived growth factor on DNA synthesis.Mitogenic stimuli initiate cell proliferation via different classes of cell surface receptors that include growth factor receptor tyrosine kinase receptors and G-protein-coupled receptors (GPCRs). 1 This involves stimulation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) cascade (1). For many years it has been known that certain GPCR agonists can function as co-mitogens with growth factors to stimulate DNA synthesis. However, the molecular mechanism for this interaction has not been fully defined. It is known that both growth factors and GPCR agonists stimulate the tyrosine phosphorylation of Shc (SH2-containing protein) and the sequential activation of Grb-2-mSos (son of sevenless), Ras, c-Raf, MEK1, and p42/p44 MAPK. GPCR agonists also activate non-receptor tyrosine kinases ...

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