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 ...

The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst 2 receptor isoforms, which couple to identical G␣ subunit pools (G␣ i3 > G␣ i2 >> G␣ 0 ), were both inhibited following ␤␥ sequestration. The sst 2(a) receptormediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70 rsk induced by sst 2(b) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst 2(a) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21 cip1 was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst 2(a) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst 2(a) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst 2 receptor isoforms may determine the selection of appropriate ␤␥-pairings necessary for interaction with distinct kinase cascades.Mitogen-activated protein (MAP) kinases are proline-directed serine/threonine kinases that play important roles as mediators of cellular responses to a variety of stimuli such as growth factors, cytokines, hormones, and environmental stresses (18,23). MAP kinases in mammalian cells have been classified into at least four subfamilies: extracellular signalregulated kinases (ERKs), stress-activated protein kinases/cJun NH 2 -terminal kinase (SAPKs/JNK), p38 kinases, and BMK1/ERK5 (51). ERK is activated by many growth factors and cytokines and is implicated in cell growth as well as differentiation (32). Various stressors such as chemical agents and UV irradiation, tumor necrosis factor, interleukin-1, CD40 ligand, and Fas/CD95 ligand stimulate the activities of SAPKs and p38 (10, 24) which appear to play a decisive role in the control of cell death. Thus, the SAPK pathway is critical during ceramide-induced (49) and stress-induced (56) apoptosis as well as in the Daxx-mediated Fas cascade (55), whereas transfection of a constitutively active mutant of MKK3/6, the physiological ac...

G protein-coupled receptors can stimulate the p38 kinase cascade, but the effect this has on cell growth remains poorly characterized. Here we show human somatostatin sst(2) and sst(4) receptors inhibit basic fibroblast growth factor (bFGF)-induced proliferation, via a mechanism that was blocked by the p38 inhibitor PD 169316. The sst(4) receptor could also induce a proliferative activity in the absence of bFGF, which was unaffected by PD 169316. In contrast, the sst(3) receptor had no effect on basal cell growth or on the proliferation evoked by bFGF. The extracellular signal-regulated kinase activity stimulated by the sst(3) receptor was transient in duration compared with a sustained activity induced by the sst(2) and sst(4) receptors and which was critical for the proliferative response of the latter receptor. In addition, activated sst(2) and sst(4) but not sst(3) receptors evoked a prolonged phosphorylation of p38 that was amplified by bFGF. The accumulation of the cell cycle inhibitor p21(cip1) was only apparent after sst(2) and sst(4) receptor activation in the presence of bFGF, which was sensitive to PD 169316 or pertussis toxin. Thus, the contrasting antiproliferative effects evoked by the human sst(2), sst(3), and sst(4) receptors can be accounted for by their differential abilities to activate p38. This activity is critical for p21(cip1) induction, blockade of entry into S phase, as indicated by the lack of retinoblastoma protein phosphorylation, and the associated antiproliferative activity of somatostatin. Furthermore, by changing the intracellular signaling threshold of p38 through cooperative effects of somatostatin and bFGF, the sst(4) receptor can mediate opposing effects on cell proliferation.

Several di erent molecular species of phosphatidic acid (PA) bind to a G-protein coupled receptor (GPCR) to induce activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway in HEK 293 cells. PA is active at low nanomolar concentrations and the response is sensitive to pertussis toxin (which uncouples GPCRs from G i/o ). The de-acylated product of PA, lysophosphatidic acid (LPA), which binds to members of the endothelial di erentiation gene (EDG) family of receptors also stimulated p42/p44 MAPK in a pertussis toxin sensitive manner, but with an *100 ± 1000 fold lower potency compared with the di erent molecular species of PA. RT ± PCR using gene-speci®c primers showed that HEK 293 cells express EDG2 and PSP24, the latter being a lipid binding GPCR out with the EDG cluster. We conclude that PA is a novel high potency GPCR agonist.