The small GTPase Rap is best characterized as a critical regulator of integrin-mediated cell adhesion, although its mechanism of action is not understood. Rap also influences the properties of other cell-surface receptors and biological processes, although whether these are a consequence of effects on integrins is not clear. We show here that Rap also plays an important role in the regulation of cadherin-mediated cell-cell adhesion in epithelial cells. Expression of constitutively active Rap1A restored cadherin-mediated cell-cell contacts in mesenchymal Ras-transformed Madin-Darby canine kidney cells, resulting in reversion to an epithelial phenotype. Activation of endogenous Rap via the Rap exchange factor Epac1 also antagonized hepatocyte growth factor-induced disruption of adherens junctions. Inhibition of Rap signaling resulted in disruption of epithelial cell-cell contacts. Rap activity was required for adhesion of cells to recombinant E-cadherin extracellular domains, i.e. in the absence of integrinmediated adhesion. These findings suggest that Rap signaling positively contributes to cadherin-mediated adhesion and that this occurs independently of effects on integrin-mediated adhesion. Our results imply that Rap may function in a broader manner to regulate the function of cell-surface adhesion receptors.

Overexpression of a neural-specific rho family GTPase, cRac1B, selectively induces enhanced neuritogenesis and neurite branching in primary neurons. J. Cell Biol. 142, 815-825. Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y.and Kaibuchi, K. (1996). Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase).

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SummaryIn N1E-115 cells, neurite retraction induced by neurite remodelling factors such as lysophosphatidic acid, sphingosine 1-phosphate and semaphorin 3A require the activity of phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks). PIP5Ks synthesise the phosphoinositide lipid second messenger phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P 2 ], and overexpression of active PIP5K is sufficient to induce neurite retraction in both N1E-115 cells and cerebellar granule neurones. However, how PIP5Ks are regulated or how they induce neurite retraction is not well defined. Here, we show that neurite retraction induced by PIP5K is dependent on its interaction with the low molecular weight G protein Rac. We identified the interaction site between PIP5K and Rac1 and generated a point mutant of PIP5K that no longer interacts with endogenous Rac. Using this mutant, we show that Rac controls the plasma membrane localisation of PIP5K and thereby the localised synthesis of PtdIns(4,5)P 2 required to induce neurite retraction. Mutation of this residue in other PIP5K isoforms also attenuates their ability to induce neurite retraction and to localise at the membrane. To clarify how increased levels of PtdIns(4,5)P 2 induce neurite retraction, we show that mutants of vinculin that are unable to interact with PtdIns(4,5)P 2 , attenuate PIP5K-and LPA-induced neurite retraction. Our findings support a role for PtdIns(4,5)P 2 synthesis in the regulation of vinculin localisation at focal complexes and ultimately in the regulation of neurite dynamics.

Rac1 and Rac3 are highly homologous regulatory proteins that belong to the small GTPases of the Rho family. Previously, we showed that Rac3 induces cell rounding and prevents neuronal differentiation, in contrast to its close relative Rac1, which stimulates cell spreading and neuritogenesis. To explain these opposing effects, we investigated whether Rac1 and Rac3 interact with different proteins. Here, we show that both Rac1 and Rac3 interact with GIT1, a multifunctional Arf-GAP protein, which regulates cell-matrix adhesion, cell spreading and endocytosis. However, in contrast to Rac1, the Rac3-GIT1 interaction is not mediated by βPix. Interestingly, Rac3 expression severely attenuates the interaction between GIT1 and paxillin, accompanied by defective paxillin distribution, focal adhesion formation and disturbed cell spreading. Moreover, in Rac3-expressing cells, Arf6 activity is strongly reduced and the Arf6-GAP activity of GIT1 is required for Rac3 downstream signaling. Indeed, expression of wild-type Arf6 or the Arf6-GEF ARNO induced cell spreading in the otherwise rounded Rac3-expressing cells. Our data suggest that Rac3 and Rac1 oppose each other's function by differently modulating GIT1 signaling. Rac1 induces adhesion and differentiation by activating PAK1 and stimulating the GIT1-paxillin interaction, whereas Rac3 blocks this interaction and inactivates Arf6 by stimulating the GAP function of GIT1, thereby preventing cell spreading and differentiation.