No abstract

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.

Several reports have demonstrated that SPP inhibits cell motility. SPP inhibited chemotactic motility of mouse melanoma B16, mouse fibroblast BALB/3T3 clone A31, and several tumor cell lines at nanomolar concentrations (17-19). Moreover, SPP immobilized on glass beads markedly inhibited melanoma cell motility. However, pertussis toxin treatment did not block the effect of SPP, suggesting that in these cells SPP acts through a cell surface receptor, independently of pertussis toxin-sensitive G-proteins (20). In contrast, SPP inhibits chemotaxis of human breast cancer cells only at high (micromolar) concentrations, acting independently of EDG-1 (21).We have recently identified SPP as a ligand for the G-protein-coupled receptor, endothelial differentiation gene-1 (EDG-1) (22). EDG-1 binds SPP with remarkable specificity and high affinity (K D ϭ 8 nM) (9,22). Binding of SPP to EDG-1 resulted in inhibition of adenylate cyclase and activation of mitogen-activated protein kinase (both G i -mediated), but did not mobilize calcium from internal stores (9, 23). In contrast, Okamoto et al. (12) found that in HEL cells overexpressing EDG-1, binding of SPP induced calcium mobilization (12).

Hypomorphic mutations in the human SPINT2 gene cause a broad spectrum of abnormalities in organogenesis, including organ and digit duplications, atresia, fistulas, hypertelorism, cleft palate and hamartoma. SPINT2 encodes the transmembrane serine protease inhibitor HAI2 (placental bikunin), and the severe developmental effects of decreased HAI2 activity can be hypothesized to be a consequence of excess pericellular proteolytic activity. Indeed, we show here that HAI2 is a potent regulator of protease-guided cellular responses, including motogenic activity and transepithelial resistance of epithelial monolayers. Furthermore, we show that inhibition of the transmembrane serine protease matriptase (encoded by St14) is an essential function of HAI2 during tissue morphogenesis. Genetic inactivation of the mouse Spint2 gene led to defects in neural tube closure, abnormal placental labyrinth development associated with loss of epithelial cell polarity, and embryonic demise. Developmental defects observed in HAI2-deficient mice were caused by unregulated matriptase activity, as both placental development and embryonic survival in HAI2-deficient embryos were completely restored by the simultaneous genetic inactivation of matriptase. However, neural tube defects were detected in HAI2-deficient mice even in the absence of matriptase, although at lower frequency, indicating that the inhibition of additional serine protease(s) by HAI2 is required to complete neural development. Finally, by genetic complementation analysis, we uncovered a unique and complex functional interaction between HAI2 and the related HAI1 in the regulation of matriptase activity during development. This study indicates that unregulated matriptase-dependent cell surface proteolysis can cause a diverse array of abnormalities in mammalian development.