EDG-1 is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for sphingosine-1-phosphate (SPP). Cell migration toward platelet-derived growth factor (PDGF), which stimulates sphingosine kinase and increases intracellular SPP, was dependent on expression of EDG-1. Deletion of edg-1 or inhibition of sphingosine kinase suppressed chemotaxis toward PDGF and also activation of the small guanosine triphosphatase Rac, which is essential for protrusion of lamellipodia and forward movement. Moreover, PDGF activated EDG-1, as measured by translocation of beta-arrestin and phosphorylation of EDG-1. Our results reveal a role for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility.
Polarized cell migration results from the transduction of extracellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting G␥ and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/ threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.P-Rex1 and P-Rex2 are Rac guanine exchange factors connecting G protein-coupled receptors, through G␥ and phosphatidylinositol 3-kinase, to Rac activation. In particular, P-Rex1 has been associated with the activation of Rac2, generating reactive oxygen species in neutrophils. P-Rex2 (showing two splice variants) is similarly regulated by G␥ and phosphatidylinositol 3-kinase. Northern blot assays revealed a differential distribution of the two members of the P-Rex 3 family, suggesting that they exert equivalent functions in different cellular populations (1-3, 7-11). The complex architecture of this family of proteins, constituted by a catalytic DH domain, followed by a phosphatidylinositol 3,4,5-trisphosphate-sensitive pleckstrin homology domain, two DEP and two PDZ domains in tandem, and a long carboxyl terminus (except for P-Rex2b, which is the short version, having a reduced carboxyl terminus), suggests that these Rac guanine exchange factors might be regulated by diverse protein-protein interactions modulating signal transduction pathways associated with the activation of Rac. In fact, in the developing brain, P-Rex1 is associated with neuronal migration in response to nerve growth factor (12, 13).The mammalian target of rapamycin, mTOR, a highly conserved serine-threonine k...
Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of G␣ q , G␣ i , G␣ 12/13 , and G␥ subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G 12/13 -mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.Sphingosine 1-phosphate (S1P), 1 a sphingolipid metabolite found in organisms as diverse as plants, yeast, worms, flies, and mammals, has been linked to a wide spectrum of biological processes, among which cell growth, survival, and motility are prominent (1, 2). S1P is formed by sphingosine kinase (SphK), a highly conserved enzyme that is activated by numerous stimuli (1, 3).The most well known actions of S1P are mediated by binding to a family of specific G protein-coupled receptors (GPCRs). To date, five members, EDG-1/S1P 1 , EDG-5/S1P 2 , EDG-3/S1P 3 , EDG-6/S1P 4 , and EDG-8/S1P 5 , have been identified (1, 2, 4, 5). S1P receptors (S1PRs) are differentially expressed; coupled to a variety of G proteins; and regulate angiogenesis, vascular maturation, cardiac development, neuronal survival, and immunity (1, 2, 4). In particular, S1PRs have been shown to play critical roles in cell migration (6 -10). Activation of S1P 1 or S1P 3 by S1P in many cell types increases directional or chemotactic migration (6, 8, 10 -12), whereas binding to S1P 2 abolishes chemotaxis and membrane ruffling (13).Downstream of heterotrimeric G proteins, the S1PRs regulate tyrosine kinases such as focal adhesion kinase (FAK) and Src, which reside in focal adhesions, and the small GTPases of the Rho family that are important for cytoskeletal rearrangements (14). Whereas binding of S1P to S1P 1 mediates cortical ...
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