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.

1 Somatostatin and the stable octapeptide analogues, octreotide and angiopeptin, were examined for their ability to stimulate the release of tritium from [ 3 H]-arachidonic acid pre-loaded CHO-K1 cells expressing human recombinant sst 2 , sst 3 or sst 5 receptors. 2 Somatostatin stimulated tritium release (pEC 50 ) through the sst 2 (7.8+0.1) and sst 5 (7.3+0.2), but not the sst 3 receptor. Octreotide behaved as a full (sst 2 receptor) or partial agonist (sst 5 receptor), whereas angiopeptin behaved as a weak partial agonist at both receptor types.3 Maximum responses to somatostatin through both receptor types were signi®cantly reduced by pertussis toxin, whereas pEC 50 estimates were una ected. 4 Inhibition of MEK1 or Src, but not PKA, PI 3-kinases or tyrosine kinases, by reportedly selective inhibitors reduced sst 2 -mediated responses by somatostatin, but not angiopeptin. A selective inhibitor of PKC (Ro-31-8220) reduced both somatostatin and angiopeptin responses. 5 These data provide further evidence for partial agonist activity of synthetic peptides of somatostatin. Furthermore, the somatostatin receptor signalling mechanisms which mediate arachidonic acid mobilization appear to be multiple and complex.

Somatostatin (SRIF) exerts antiproliferative effects, and angiopeptin (an sst2/sst5 receptor‐selective analogue) has recently been evaluated in clinical trials for the prophylaxis of restenosis following coronary angioplasty. Using an in vitro model of cell growth we have examined the effects of SRIF and angiopeptin on cell proliferation in CHO‐K1 cells stably transfected with the human or rat recombinant sst2 or sst5 receptor and compared these with their effects on rat aortic vascular smooth muscle cells (VSMC) expressing endogenous somatostatin receptors. In CHO‐K1 cells, expressing either human or rat recombinant sst2 or sst5 receptors, or in rat aortic VSMC, SRIF and angiopeptin (0.1–1000 nM) had no effect on basal re‐growth of cells into a denuded area of a previously confluent monolayer. In contrast, basic fibroblast growth factor (bFGF, 10 ng ml−1) stimulated re‐growth of these cells. SRIF (0.1–1000 nM) caused a concentration‐dependent inhibition of the bFGF‐stimulated re‐growth in CHO‐K1 cells expressing human sst2 (h sst2) or sst5 (h sst5) receptors (pIC50=8.05±0.03 and 8.56±0.12, respectively). In contrast, angiopeptin (0.1–1000 nM) acted as a partial agonist at the h sst2 receptor (44.6±2.7% inhibition of the bFGF‐stimulated re‐growth at 100 nM; pIC50=8.69±0.25) but was devoid of any agonist activity at the h sst5 receptor. In CHO‐K1 cells stably expressing rat recombinant sst2 (r sst2) or sst5 (r sst5) receptors, SRIF (0.1–1000 nM) was able to inhibit the bFGF‐stimulated re‐growth (pIC50=7.98±24 and 8.50±0.12, respectively). Angiopeptin (0.1–1000 nM) caused a concentration‐dependent inhibition of bFGF‐stimulated re‐growth at the r sst2 receptor (pIC50=8.08±0.24) but acted as a partial agonist at the r sst5 receptor (maximum response=57.7±3.6% inhibition of bFGF‐stimulated re‐growth at 100 nM; pIC50=8.60±0.16). Although angiopeptin was inactive as an agonist at the h sst5 receptor, 100 nM angiopeptin potently antagonized the SRIF‐induced inhibition of proliferation in CHO h sst5 (estimated pKB=10.4±0.3). 5‐Hydroxytryptamine (0.1 nM–10 μM) also inhibited bFGF‐stimulated re‐growth (pIC50=8.36±0.11) and angiopeptin had no effect on this response (pKB<7). SRIF (0.1–1000 nM) caused a concentration‐dependent (pIC50=8.04±0.08) inhibition of bFGF‐stimulated re‐growth in VSMC, whereas angiopeptin displayed weak agonist activity, only inhibiting bFGF‐stimulated re‐growth at concentrations greater than 100 nM. Angiopeptin (100 nM) caused a rightward displacement of the concentration‐effect curve to SRIF with an estimated pKB value of 7.70±0.12. These findings suggest that the low intrinsic activity of angiopeptin at the h sst2 receptor, combined with its lack of agonist activity at the h sst5 receptor, may explain the poor clinical efficacy of angiopeptin in trials for coronary artery restenosis, which contrasts with encouraging data found in equivalent in vivo animal studies.

We have investigated the actions of somatostatin (SRIF) and angiopeptin on cell proliferation of CHO-K1 cells expressing the recently cloned rat sst 2(b) receptor (CHOsst 2(b) ) and compared these to their eects in cells expressing the sst 2(a) receptor (CHOsst 2(a) ). In contrast to the sst 2(a) receptor, the sst 2(b) receptor did not mediate inhibition of bFGF (10 ng ml 71 )-stimulated re-growth and cell proliferation. Rather, SRIF (0.1 ± 1000 nM) and angiopeptin (0.1 ± 1000 nM) stimulated basal regrowth and proliferation of CHOsst 2(b) cells in a concentration-dependent manner (estimated pEC 50 values of 7.8 and 7.9, respectively). The opposite eects of SRIF on cell proliferation mediated through the two sst 2 receptor isoforms were both abolished by 18 h pre-treatment with pertussis toxin. The proliferative eect via the sst 2(b) receptor was also abolished by the tyrosine kinase inhibitor, genistein. In conclusion, the present study shows that the rat sst 2(a) and sst 2(b) receptor splice variants mediate opposite eects on cell proliferation.