Willem-Jan Pannekoek

Cell-cell junction formation: The role of Rap1 and Rap1 guanine nucleotide exchange factors

Rap proteins are Ras-like small GTP-binding proteins that amongst others are involved in the control of cell-cell and cell-matrix adhesion. Several Rap guanine nucleotide exchange factors (RapGEFs) function to activate Rap. These multi-domain proteins, which include C3G, Epacs, PDZ-GEFs, RapGRPs and DOCK4, are regulated by various different stimuli and may function at different levels in junction formation. Downstream of Rap, a number of effector proteins have been implicated in junctional control, most notably the adaptor proteins AF6 and KRIT/CCM1. In this review, we will highlight the latest findings on the Rap signaling network in the control of epithelial and endothelial cell-cell junctions.

8-pCPT-2′-O-Me-cAMP-AM: An Improved Epac-Selective cAMP Analogue

Cyclic adenosine monophosphate (cAMP) is a common second messenger involved in the regulation of many different cellular processes through the activation of protein kinase A (PKA), exchange protein directly activated by cAMP (Epac) and cyclicnucleotide-regulated ion channels. Adenylyl cyclases are ACHTUNGTRENNUNGresponsible for catalysing the formation of cAMP from ATP. Levels of cAMP can be raised in cells in response to a large variety of extracellular stimuli, which act via receptors coupled to heterotrimeric G proteins, which stimulate the activity of adenylyl cyclase. In addition, cAMP levels are controlled by phosphodiesterases (PDE), which catalyse the degradation of cAMP to AMP. In cells, cAMP levels can be artificially elevated by forskolin, which activates adenylyl cyclase directly. Furthermore, cAMP levels can be raised by inhibiting PDEs. These approaches are commonly used in tissue culture experiments, but, by generating cAMP, they do not discriminate between the various target proteins that are activated. Alternatively, membrane-permeable cAMP analogues, which selectively interact with particular receptor proteins, can be applied. For example, signalling pathways activated by Epac and PKA can be ACHTUNGTRENNUNGdistinguished by using 8-pCPT-2’-O-Me-cAMP and 6-Bnz-cAMP, respectively. Epac is a guanine nucleotide exchange factor for the small G protein Rap. Rap cycles between a signalling-inactive GDPbound state and a signalling-active GTP-bound state. cAMP-activated Epac catalyses the exchange of Rap-bound GDP for GTP. Epac and Rap function in a number of different cellular processes including insulin secretion, inhibition of cell scattering, neurotransmitter release and cAMP-induced barrier function in endothelial cells. Even though 8-pCPT-2’-O-Me-cAMP has become a widely used tool in Epac-related research, its biological application is limited by its low membrane permeability, caused by the negatively charged phosphate. However, the negatively charged singly bonded oxygen on the phosphate group can be masked by labile esters. Such a precursor is expected to enter the cell efficiently, where the ester is hydrolysed either directly by water or by cellular esterases to liberate the active compound. We therefore synthesised 8-pCPT-2’-O-Me-cAMP-AM from 8pCPT-2’-O-Me-cAMP, whereby acetoxymethyl bromide was used as a donor for the AM group. The product that was obtained had a purity exceeding 97% and consisted of a mixture of the equatorial and the axial isomers of the ester (Figure S1 in the Supporting Information, Scheme 1). Even though the isomers could be resolved by repetitive analytical HPLC runs, efficient separation on a preparative scale was not possible. Orange peel acetylesterase and esterase from porcine liver cleaved the equatorial isomer about five times more efficiently than the axial isomer within minutes (data not shown). The pharmacokinetics of both isomers are thus expected to be similar, justifying the application of a mixture of both isomers to cells. In any case, the isomeric ratio of an individual synthesis can be easily quality controlled by P NMR (Figure S1). To compare the efficiency of 8-pCPT-2’-O-Me-cAMP-AM and 8-pCPT-2’-O-Me-cAMP in activating Epac1 in vivo, an Epac1based fluorescence resonance energy transfer (FRET) probe was used. In this assay, activation of Epac1 by the binding of cAMP to the Epac1-FRET probe is measured as a reduction in the FRET signal. A431 cells transfected with the FRET probe were stimulated with 8-pCPT-2’-O-Me-cAMP-AM or 8-pCPT-2’O-Me-cAMP (Figure 1). Stimulation of cells with 100 mm 8pCPT-2’-O-Me-cAMP resulted in a decrease of the FRET signal that was approximately one order of magnitude slower than the decrease obtained upon stimulation with 1 mm 8-pCPT-2’O-Me-cAMP-AM. Furthermore, activation of Epac1 following stimulation with 100 mm 8-pCPT-2’-O-Me-cAMP could be further enhanced by the addition of forskolin, whereas 1 mm 8pCPT-2’-O-Me-cAMP-AM induced maximal activity of Epac1 under the given conditions. The activation of Epac by 8-pCPT2’-O-Me-cAMP-AM occurs within one minute after application. This is comparable with the kinetics of forskolin-induced Epac activation, and thus 8-pCPT-2’-O-Me-cAMP-AM mimics the “natural” response time of the signalling pathway. The activity of endogenous Epac can be monitored by isolating selectively Rap·GTP from cell lysates. Primary human umbilical vein endothelial cells (HUVEC) were stimulated with different concentrations of 8-pCPT-2’-O-Me-cAMP and 8-pCPT-2’-OMe-cAMP-AM (Figure 2A). Partial activation of Rap was induced by 10 mm 8-pCPT-2’-O-Me-cAMP, and full activation of the G protein was stimulated by 100 mm 8-pCPT-2’-O-Me-cAMP. In contrast, treatment of the cells with just 0.1 mm 8-pCPT-2’-OMe-cAMP-AM was sufficient to induce full Rap activation. [a] M. J. Vliem, W.-J. Pannekoek, Dr. J. Riedl, M. R. H. Kooistra, Prof. Dr. J. L. Bos, Dr. H. Rehmann Department of Physiological Chemistry Centre for Biomedical Genetics and Cancer Genomics Centre University Medical Center Utrecht Universiteitsweg 100, 3584CG Utrecht (The Netherlands) Fax: (+31)88-75-68101 E-mail : j.l.bos@umcutrecht.nl h.rehmann@umcutrecht.nl [b] B. Ponsioen, Dr. K. Jalink Division of Cell Biology, The Netherlands Cancer Institute Amsterdam (The Netherlands) [c] Dr. F. Schwede, Dr. H.-G. Genieser BIOLOG Life Science Institute Flughafendamm 9a, 28071 Bremen (Germany) [] These authors contribute equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Rasip1 mediates Rap1 regulation of Rho in endothelial barrier function through ArhGAP29

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.

Epac1 and PDZ-GEF cooperate in Rap1 mediated endothelial junction control

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.

The RapGEF PDZ-GEF2 is required for maturation of cell–cell junctions

The small G-protein Rap1 is a critical regulator of cell-cell contacts and is activated by the remodeling of adherens junctions. Here we identify the Rap1 guanine nucleotide exchange factor PDZ-GEF2 as an upstream activator of Rap1 required for the maturation of adherens junctions in the lung carcinoma cells A549. Knockdown of PDZ-GEF2 results in the persistence of adhesion zippers at cell-cell contacts. Activation of Rap1A rescues junction maturation in absence of PDZ-GEF2, demonstrating that Rap1A is downstream of PDZ-GEF2 in this process. Moreover, depletion of Rap1A, but not Rap1B, impairs adherens junction maturation. siRNA for PDZ-GEF2 also lowers the levels of E-cadherin, an effect that can be mimicked by Rap1B, but not Rap1A siRNA. Since junctions in Rap1B depleted cells have a mature appearance, these data suggest that PDZ-GEF2 activates Rap1A and Rap1B to perform different functions. Our results present the first direct evidence that PDZ-GEF2 plays a critical role in the maturation of adherens junctions.

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.

Rap1 signaling in endothelial barrier control

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.

Rap1 and Rap2 antagonistically control endothelial barrier resistance.

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.

Semaphorin Signaling Meets Rap

Semaphorin binding stimulates the ability of plexin receptors to inhibit the GTPase Rap1, thereby enabling neurite retraction. Plexins are transmembrane receptors for semaphorins that serve as guidance cues for neurite outgrowth. The intracellular region of plexins contains a guanosine triphosphatase (GTPase)–activating protein (GAP) domain for Ras. New evidence shows that the GAP activity is specific for Rap proteins, small GTPases involved in the regulation of processes that are potentially important for axon guidance, including cell adhesion and migration. Semaphorin-induced dimerization stimulates plexin GAP activity, thereby locally inhibiting Rap1 and enabling neurite retraction. This important finding connects semaphorin signaling to Rap-mediated signaling and is another intriguing example of how small GTPases are used for spatial and temporal control of cell behavior.

Multiple Rap1 effectors control Epac1-mediated tightening of endothelial junctions

ABSTRACT Epac1 and Rap1 mediate cAMP-induced tightening of endothelial junctions. We have previously found that one of the mechanisms is the inhibition of Rho-mediated tension in radial stress fibers by recruiting the RhoGAP ArhGAP29 in a complex containing the Rap1 effectors Rasip1 and Radil. However, other mechanisms have been proposed as well, most notably the induction of tension in circumferential actin cables by Cdc42 and its GEF FGD5. Here, we have investigated how Rap1 controls FGD5/Cdc42 and how this interconnects with Radil/Rasip1/ArhGAP29. Using endothelial barrier measurements, we show that Rho inhibition is not sufficient to explain the barrier stimulating effect of Rap1. Indeed, Cdc42-mediated tension is induced at cell-cell contacts upon Rap1 activation and this is required for endothelial barrier function. Depletion of potential Rap1 effectors identifies AF6 to mediate Rap1 enhanced tension and concomitant Rho-independent barrier function. When overexpressed in HEK293T cells, AF6 is found in a complex with FGD5 and Radil. From these results we conclude that Rap1 utilizes multiple pathways to control tightening of endothelial junctions, possibly through a multiprotein effector complex, in which AF6 functions to induce tension in circumferential actin cables.