Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

Abstract

Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membranelocalized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMPand Rap-mediated signaling to regulate adhesion to the extracellular matrix.Cyclic AMP (cAMP) is a second messenger that relays a wide range of hormone responses. The discovery of Epac as a direct effector of cAMP (15, 29) has triggered the elucidation of many cAMP-regulated processes that could not be explained by the previously known effectors protein kinase A (PKA) and cyclic nucleotide-regulated ion channels (21). Both Epac family members, Epac1 and Epac2, act as guanine nucleotide exchange factors (GEFs) for the small G proteins Rap1 and Rap2. Thereby, Epac functions in processes such as exocytosis (28,48,59), cell-cell junction formation (13,20,30,53,64), and cell-extracellular matrix (ECM) adhesion (55). Adhesion to the ECM induced by Epac1 and Rap is mediated by actin-linked integrin molecules and is implicated in diverse biological processes such as homing of endothelial progenitor cells to ischemic tissue (9), remodeling of the vasculature (10, 36), and transendothelial migration of leukocytes (37, 60).Epac1 and Epac2 are multidomain proteins containing a C-terminal catalytic region, which consists of a CDC25 homology domain responsible for GEF activity, a Ras exchange motif (REM), which stabilizes the CDC25 homology domain, and a Ras association (RA) domain. In the autoinhibited state, the catalytic site is sterically covered by the N-terminal regulatory region, which harbors a DEP (Dishevelled, Egl-10, and pleckstrin) domain and one or two cyclic nucleotide-binding domains in Epac1 and Epac2, respectively. As demonstrated by the crystal structures of both active and inactive Epac2, autoinhibition is released by a conformational change induced by the binding of cAMP (56, 57).After its production at the plasma membrane (PM) by adenylate cylases, cAMP becomes compartmentalized due to local degradation by spatially restricted phosphodiesterases (1). Further compartmentalization of cAMP signaling is established by the confined targeting of the cAMP effector proteins. Numerous adapt...

Ezrin is required for efficient Rap1-induced cell spreading

“…This interaction regulates cell adhesion because simultaneous knockdown of all three ERM proteins decreased, but did not block completely, Epac1-induced cell adhesion (Gloerich et al, 2010). Interestingly, although all three ERM family members are expressed in A549-Epac1 cells, we found that depletion of Ezrin blocked the 007 effect in more than 70% of cells (Fig.…”

“…Quantification of the cells expressing HA-RapV12 confirmed that Ezrin depletion caused a significant reduction in RapV12-induced spreading (Fig. 5C) and demonstrated that Ezrin is required for the focal adhesion formation and cell spreading induced by Rap activation, and would suggest that its role in spreading is not entirely upstream, via localisation of Epac1 (Gloerich et al, 2010), but also downstream of Rap1.…”

“…had to be depleted in to order block Epac-induced cell adhesion (Gloerich et al, 2010). In this study, through our analysis of the spreading induced by HA-RapV12 and YFP-Epac1 49, we have shown that Ezrin has an additional role downstream of Rap activation, independent from its ability to bind to Epac1.…”

“…Our group has demonstrated that active ERM family proteins can localise Epac1, and hence can regulate Epac-mediated cell adhesion (Gloerich et al, 2010), therefore it is possible that the major defect caused by Ezrin depletion occurs via disruption or mislocalisation of Epac1-Rap signalling complexes. To investigate whether Epac1-Rap1 signal transduction was still intact in cells depleted of Ezrin, we analysed the levels of Rap1-GTP, which were stimulated in response to 007 in cells transfected with siRNA against Ezrin.…”

“…In this protein, the first 49 amino acids that mediate the interaction with ERM proteins are deleted, producing a protein that is still activated by 007 and can induce cell adhesion, although to a lesser extent than the wild-type protein, but with any upstream regulation of Epac1-Rap1 signalling by Ezrin being abolished (Gloerich et al, 2010). Therefore, if Ezrin regulated cell spreading only by localising the Epac-Rap signalling complex, depletion of Ezrin would have no effect on the spreading induced by activation of YFP-Epac1 49 with 007.…”