Rap1 Spatially Controls ArhGAP29 To Inhibit Rho Signaling during Endothelial Barrier Regulation

Abstract

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...

Rasip1 is essential to blood vessel stability and angiogenic blood vessel growth

“…This study suggested that Rasip1 primarily regulates the integrity of EC-EC adhesions and blood vessel permeability. More recent studies have confirmed aspects of Rasip1 signaling, placing it upstream of Rap1 and cell-cell junctions, but also identifying it as a key regulator of RhoA signaling via Arghap29 in control of the cytoskeleton and cell adhesion [15,16]. Together, these findings underscore the critical importance of Rasip1 for normal morphogenesis of blood vessels and open questions as to how it regulates formation of vessels across diverse vascular beds.…”

LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B

“…Indeed, overexpression of a spontaneously active RhoA leads to a decrease in proplatelets, indicating that RhoA activation inhibits proplatelet formation (28). Of note, it was reported that the Rho signaling pathway could be controlled by the small GTPase Rap1 (40). Interestingly, we have recently demonstrated in VWD-type 2B p.V1316M that the thrombopathy originates from an impaired activation of Rap1 (29).…”

Small Rho GTPase-mediated actin dynamics at endothelial adherens junctions

“…Interestingly, a study by the Bos lab showed that Rap1 activation results in inhibition of RhoA activity, releasing tension from the junction region and thereby promoting stabilization of endothelial cell-cell junctions. [82][83][84] Mechanistically they showed that the adapter protein Ras-interacting protein 1 (Rasip1) is involved in the formation of the endothelial barrier by forming a complex with Rap1 and promoting its activity. 83 This results in the recruitment of ARH-GAP29, a GAP that inactivates RhoA.…”

“…Thus, by recruiting ARHGAP29, RhoA activity is believed to be locally inhibited, releasing junctional tension and thereby promoting cell-cell junction interaction. 85,86 Endothelial Rho-GEFs and adherens junctions GEFs outnumber their target Rho-GTPase by more than 3-fold. 51 Therefore, different GEFs likely control different pools of the Rho GTPases, which directs local signaling pathways resulting in different outcomes.…”