Rap1 is a small GTPase regulating cell-cell adhesion, cell-matrix adhesion, and actin rearrangements, all processes dynamically coordinated during cell spreading and endothelial barrier function. Here, we identify the adaptor protein ras-interacting protein 1 (Rasip1) as a Rap1-effector involved in cell spreading and endothelial barrier function. Using Förster resonance energy transfer, we show that Rasip1 interacts with active Rap1 in a cellular context. Rasip1 mediates Rap1-induced cell spreading through its interaction partner Rho GTPase-activating protein 29 (ArhGAP29), a GTPase activating protein for Rho proteins. Accordingly, the Rap1-Rasip1 complex induces cell spreading by inhibiting Rho signaling. The Rasip1-ArhGAP29 pathway also functions in Rap1-mediated regulation of endothelial junctions, which controls endothelial barrier function. In this process, Rasip1 cooperates with its close relative ras-association and dilute domain-containing protein (Radil) to inhibit Rho-mediated stress fiber formation and induces junctional tightening. These results reveal an effector pathway for Rap1 in the modulation of Rho signaling and actin dynamics, through which Rap1 modulates endothelial barrier function.

The small GTPase Rap1 controls the actin cytoskeleton by regulating Rho GTPase signaling. We recently established that the Rap1 effectors Radil and Rasip1, together with the Rho GTPase activating protein ArhGAP29, mediate Rap1-induced inhibition of Rho signaling in the processes of epithelial cell spreading and endothelial barrier function. Here, we show that Rap1 induces the independent translocations of Rasip1 and a Radil-ArhGAP29 complex to the plasma membrane. This results in the formation of a multimeric protein complex required for Rap1-induced inhibition of Rho signaling and increased endothelial barrier function. Together with the previously reported spatiotemporal control of the Rap guanine nucleotide exchange factor Epac1, these findings elucidate a signaling pathway for spatiotemporal control of Rho signaling that operates by successive protein translocations to and complex formation at the plasma membrane. The small GTPase Rap1 controls multiple processes linked to actin cytoskeletal dynamics, including integrin-mediated and cadherin-mediated cell adhesion. To this end, it translates spatial and temporal information into actin cytoskeleton modulation. This spatiotemporal information is received by Rap1 guanine nucleotide exchange factors (GEFs), such as Epac1 and PDZ-GEF. For instance, Epac1 responds to cyclic AMP (cAMP) by catalytic activation and translocation to specific sites at the plasma membrane resulting in the local activation of Rap1. Subsequently, actin cytoskeletal rearrangements occur, as exemplified in endothelial barrier regulation (1).The endothelial barrier is under tight control to allow passage of fluids, solutes, and immune cells, without losing tissue integrity and barrier function (2-4). Rap1 plays an important role in this by enhancing the endothelial barrier function (5-9). It does so by impinging on the family of Rho GTPases, the master regulators of the actin cytoskeleton. Once activated, Rap1 inhibits the small GTPase Rho, resulting in the relaxation and disappearance of stress fibers (1, 10-13), and activates the small GTPase Cdc42, resulting in the formation of junctional actin bundles (1, 10). As a result, barrier function is enhanced. Indeed, Rho GTPases are important players in adherens junction formation and maintenance, which is necessary to sustain the barrier integrity of both epithelial and endothelial monolayers (14-16).Recently, we have elucidated that one way for Rap1 to regulate Rho activity in endothelial barrier function is through the RhoGAP ArhGAP29. Rap1 does so through the highly related effectors Radil and Rasip1 (11, 17, 18), which have both been reported to interact with ArhGAP29 (19,20). Activation of this pathway by active Rap1 reduces radial stress fibers, which exert tension on the adherens junctions, resulting in increased endothelial barrier function. Radil, Rasip1, and ArhGAP29 also control cell spreading of epithelial cells, indicating that the pathway is not restricted to endothelial barrier regulation but functions in a more universal manner...

The small G-protein Rap1 plays an important role in the regulation of endothelial barrier function, a process controlled largely by cell–cell adhesions and their connection to the actin cytoskeleton. During the various stages of barrier dynamics, different guanine nucleotide exchange factors (GEFs) control Rap1 activity, indicating that Rap1 integrates multiple input signals. Once activated, Rap1 induces numerous signaling cascades, together responsible for the increased endothelial barrier function. Most notably, Rap1 activation results in the inhibition of Rho to decrease radial stress fibers and the activation of Cdc42 to increase junctional actin. This implies that Rap regulates endothelial barrier function by dual control of cytoskeletal tension. The molecular details of the signaling pathways are becoming to be elucidated.