RESUMO
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.
Assuntos
Endotélio Vascular/fisiologia , Proteínas Ativadoras de GTPase/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas rap1 de Ligação ao GTP/fisiologia , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Ligação Proteica , Transdução de SinaisRESUMO
PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.
Assuntos
Polaridade Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mucosa Intestinal/metabolismo , Microvilosidades/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ácidos Fosfatídicos/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Mucosa Intestinal/ultraestrutura , Microvilosidades/genética , Microvilosidades/ultraestrutura , Proteínas do Tecido Nervoso/genética , Ácidos Fosfatídicos/genética , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/metabolismoRESUMO
Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2.
Assuntos
Endotélio/metabolismo , Proteínas rap de Ligação ao GTP/antagonistas & inibidores , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rap1 de Ligação ao GTP/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , RNA Interferente Pequeno/genética , Proteínas rap de Ligação ao GTP/deficiência , Proteínas rap de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/deficiência , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Epac1 and its effector Rap1 are important mediators of cAMP induced tightening of endothelial junctions and consequential increased barrier function. We have investigated the involvement of Rap1 signalling in basal, unstimulated, barrier function of a confluent monolayer of HUVEC using real time Electric Cell-substrate Impedance Sensing. Depletion of Rap1, but not Epac1, results in a strong decrease in barrier function. This decrease is also observed when cells are depleted of the cAMP independent Rap exchange factors PDZ-GEF1 and 2, showing that PDZ-GEFs are responsible for Rap1 activity in control of basal barrier function. Monolayers of cells depleted of PDZ-GEF or Rap1 show an irregular, zipper-like organization of VE-cadherin and live imaging of VE-cadherin-GFP reveals enhanced junction motility upon depletion of PDZ-GEF or Rap1. Importantly, activation of Epac1 increases the formation of cortical actin bundles at the cell-cell junctions, inhibits junction motility and restores barrier function of PDZ-GEFs depleted, but not Rap1 depleted cells. We conclude that PDZ-GEF activates Rap1 under resting conditions to stabilize cell-cell junctions and maintain basal integrity. Activation of Rap1 by cAMP/Epac1 induces junctional actin to further tighten cell-cell contacts.