Eph receptor-ephrin system: the link between adhesion and signaling

Introduction:

The Eph receptors constitute the largest family of tyrosine kinases receptors, and with their ligands the ephrins are implicated in many physiological and pathological processes, such as embryonic patterning, axon guidance, migration, adhesion, angiogenesis and cancer(Pasquale, 2010). The Eph/ephrin complexes are generally formed at sites of cell-cell contact where receptor and ligand reside in apposed cell membranes providing juxtacrine signaling and cell-cell anchorage (Marquardt et al., 2005; Qi et al., 2001).
In humans there are nine EphA receptors (EphA1-EphA9) that can interact with five GPI-linked ephrin-A ligands (ephrin-A1-A5) and five EphB receptors (EphB1-EphB5) that bind to three transmembrane ephrins-B ligands (ephrin-B1-B3), which in some cases one Eph receptor of one class binds ephrins of the other class(Himanen et al., 2004; Pasquale, 2008) as shown by the interaction and activation of the EphB2 receptor by the ephrin-A5 ligand.
The extracellular Eph region contains a conserved N-terminal ligand-binding domain (LBD), an adjacent cysteine-rich domain (CRD), followed by two fibronectin repeats (FN3)(Lackmann et al., 1998). The cytoplasmic region is formed by a regulatory juxtamembrane region that connects to the kinase domain, a sterile alpha motif (SAM) domain and a C-terminal PDZ-domain recognition motif. Crystal structures have shown that in addition to the two contact surfaces of the LBD, other protein interfaces at the level of the CRD are important for the generation and function of the Eph/ephrins signaling complexes. These regions have been shown important for the recruitment of non-ligand-bound Eph receptors via direct Eph-Eph contacts into Eph clusters (Himanen et al., 2010).
Each receptor binds an ephrin ligand, two Eph/ephrin dimers then join to form a tetramer (microclustering), in which each ligand interacts with two receptors and each receptor interacts with two ligands. The Eph and ephrin molecules are precisely positioned and orientated in these complexes, promoting high-order clustering(macroclustering)(Smith et al., 2004) and the initiation of bidirectional signaling(Himanen et al., 2001). Many studies suggest that the degree of Eph/ephrin clustering is important for the nature of the cellular response and that multiple protein-protein interaction domains of the Eph and the ephrins are responsible for the oligomerization of these tetramers into higher ordered clusters (Day et al., 2005; Poliakov et al., 2004). These motifs include the juxtamembrane region, the SAM domain, the CRD(Smith et al., 2004) and the PDZ recognition motif. Clustering may also be promoted via the ephrin ligands, which are present in lipid microdomains (rafts) (Bruckner et al., 1999; Gauthier and Robbins, 2003).

Not only in the case of ephrins, it is becoming clear that spatial organization of cell surface receptors can influence or regulate the associated signal transduction pathways(Hurtley, 2009; Qi et al., 2001) in addition mechanical forces acting on the ligands or ligand/receptor complexes can influence the spatial organization of receptors and therefore affect signaling(Scott and Pawson, 2009).

Recent studies reporting a spatio-mechanical regulation of the EphA signaling pathway have shown that upon stimulation with membrane-bound ligand, EphA2 receptors are transported radially inwards by an actomyosin contractile process, forming a large central receptor cluster. Restriction of the movement of the receptor, via nanopatterning of the range of ligand mobility evidences a link between Eph clustering and the cytoskeleton and the importance of the spatial organization of the ligand (Salaita et al., 2010). This radial inwards transport has a high correlation with the tissue invasion capacity of certain breast cancer cell lines.

Other groups have revealed that shorter or truncated splice variants of the Eph receptor inhibit repulsive signal transduction, while still enabling adhesive interactions; thus because both ephrins and Eph receptors are membrane anchored, suppression of repulsive signal transduction could hypothetically turn these proteins into cell-cell adhesion proteins(Holmberg et al., 2000). Tyrosine kinase receptors dimerize or oligomerize on ligand binding, enabling cross-phosphorylation and signal transduction. In turn, kinase-dead mutants of receptor tyrosine kinases act as dominant-negative inhibitors of signaling(van der Geer et al., 1994). The dimerization of a full-length (FL) Eph receptor with a truncated (T1) splice version blocks the phosphorylation of the Eph FL inhibiting the repulsive signal transduction, however, enabling an adhesive interaction with the ephrin expressing cell(Holmberg et al., 2000).

Hypothesis:

The Eph/ephrin complex does not only provide bi-directional signaling but provides also a mechanical link through the formation of micro and macro clusters. In addition, we propose that the CRD region in the extracellular domain as well as the kinase domain contain critical regions that can both affect lateral receptor clustering.
And finally that the spatio-mechanical regulation, define as the centripetal transport to form a high order central cluster which will be the signaling center, of the Eph/ephrin complexes is fundamental to the modulation of repulsive or adhesive responses.

Objectives:

Develop experimental strategies to test the putative link between the spatial distribution of the Eph receptors (micro versus macroclustering) and how this different spatial organization can switch between repulsive or adhesive responses.

Research Plan:

In order to address this question we propose a system in which we can measure the mechanical adhesion between cells transfected either with the Eph receptor or with its correspondent ephrin ligand. By applying shear stress forces using the “Spinning disc” device, we can directly quantify adhesion in response to receptor clustering. In addition, we will test receptors mutated in the CRD to identify the residues that are important for clustering, and how this altered clustering can modulate the adhesive response.

Specific Aim 1: Identifying regions essential for clustering

Rational

Structural studies have pointed three different interfaces as key regions for the correct Eph/ephrin interaction, the first two located within the LBD, one of higher affinity responsible of the heterodimerization the other of lower affinity responsible of the heterotetramerization(Lackmann et al., 1998) and a third region within the CRD with less affinity but still essential for the Eph activation and downstream signaling, probably because its role in laterally recruiting other Eph receptors(Day et al., 2005; Himanen et al., 2010; Smith et al., 2004)
We will focus on human EphA2, which is expressed during embryonic development and in adult epithelia, where it regulates cell adhesion (Carter et al., 2002). EphA2 also acts as a powerful oncogene in many tumors by promoting vascularization and metastasis (Kinch and Carles-Kinch, 2003; Zelinski et al., 2001).

We will create EphA2 point mutants tagged with the GFP (green fluorescent protein) molecule, the mutations will cover the three crucial interaction domains and we will transfect either the wt or the mutants into HEK293 cells. We will then seed the cells onto a coverslip which is coated with a supported membrane prepared as described in(Salaita et al., 2010), displaying laterally mobile mRFP tagged ephrin-A1 ligands, because they are reported to have the highest affinity with EphA2(Kullander and Klein, 2002). We will perform time-lapse with the Total Internal Reflection Fluorescent (TIRF) microscope, which allows us to look the receptor-ligand interface, to determine the degree of clustering of each mutant. For that we will analyze the TIRF images and we will quantify the average radial distribution of the receptor in each cell at defined time points. We will mimic the procedure with mutations at the kinase region of the cytoplasmic domain, to determine the importance of this domain in the normal clustering.

Finally we will cotransfect the wt EphA2 with a cytoplasmic truncated version to confirm if the mutant lacking kinase activity can act as dominant-negative inhibitor of signaling.

Specific Aim 2: Measuring the correlation between clustering and adhesion.

Rational

Eph receptors and ephrins have emerged as key players in regulation of migration and adhesive interactions between cells and have a number of intriguing and distinctive properties that may underlie the mechanisms by which they control cell adhesion and the assembly of the actin cytoskeleton(Poliakov et al., 2004). We propose that the formation of big clusters of Eph receptors will increase the cell-cell adhesion. To try to measure this, we will calibrate the cell-cell adhesion by measuring the resistance to shear-stress by using the “spinning-disc” (Boettiger, 2007) of a co-culture of COS cells expressing the ephrin-A1 and HEK293 expressing either the wt or the significant EphA2 mutants that we would have identify in the clustering analysis. Experimental procedure

Cos cells expressing Ephrin-A1-RFP will be plated in glass coverslips and incubated for 2 hours until they form a monolayer all over the glass surface. Then HEK293 expressing the wt or the mutated EphA2 receptor will be seed on top of the monolayer and incubated for 60 minutes, so the high order cluster can be formed. Then the coverslips will be placed on the spinning device(Boettiger, 2007) and spun during 5 minutes. Cells at the periphery of the disc, were the shear stress is higher, will be detached, and there will be a density radial gradient depending on the stability of the interaction between the cells. Then the coverslip will be fixed and analyze for radial distribution of the cells. We then can correlate the resistance to shear stress with the adhesive or repulsive interaction between the Eph receptor and its ephrin ligand.
The advantage of the spinning disc is that we can apply high forces to the whole cell surface(Garcia et al., 1997) rather than applied at a point. Therefore we can measure the detachment forces for the individual cell. The spinning disc produces shear stress as a linear function of radial position (distance from the axis of rotation). The linear shear gradient allows us to plot shear stress as a function of cell detachment in a cell population from a single disc. This detachment we then can correlate it to the level of receptor clustering of each mutant.

Specific Aim 3: Separating adhesive from signaling functions of Eph/ephrin complexes

Rational

The lack of kinase signaling can increase the adhesion by blocking the repulsive response (Holmberg et al., 2000). It has been described that a truncated version of the Eph receptor will dimerize with the full length and block its cross-phosphorylation, thus blocking its signaling (van der Geer et al., 1994). We would like to analyze the importance of this signaling in real cell-cell adhesion with our spinning disc system. For that, we will proceed similarly as previously described; with a COS cell monolayer expressing the ephrin-A1-RFP and we will seed HEK293 expressing wt + truncated EphA2 or expressing the mutant + truncated EphA2 versions. We will then asses the importance of this signaling in the adhesive resistance to shear stress and that if this increase in adhesion can overcome the less adhesive response of the clustering mutants. References:

Boettiger, D. 2007. Quantitative measurements of integrin-mediated adhesion to extracellular matrix. Methods Enzymol. 426:1-25.
Bruckner, K., J. Pablo Labrador, P. Scheiffele, A. Herb, P.H. Seeburg, and R. Klein. 1999. EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains. Neuron. 22:511-524.
Carter, N., T. Nakamoto, H. Hirai, and T. Hunter. 2002. EphrinA1-induced cytoskeletal re-organization requires FAK and p130(cas). Nat Cell Biol. 4:565-573.
Day, B., C. To, J.P. Himanen, F.M. Smith, D.B. Nikolov, A.W. Boyd, and M. Lackmann. 2005. Three distinct molecular surfaces in ephrin-A5 are essential for a functional interaction with EphA3. J Biol Chem. 280:26526-26532.
Garcia, A.J., P. Ducheyne, and D. Boettiger. 1997. Quantification of cell adhesion using a spinning disc device and application to surface-reactive materials. Biomaterials. 20:2417-2433.
Gauthier, L.R., and S.M. Robbins. 2003. Ephrin signaling: one raft to rule them all? One raft to sort them? One raft to spread their call and in signaling bind them? Life Sci. 74:207-216.
Himanen, J.P., M.J. Chumley, M. Lackmann, C. Li, W.A. Barton, P.D. Jeffrey, C. Vearing, D. Geleick, D.A. Feldheim, A.W. Boyd, M. Henkemeyer, and D.B. Nikolov. 2004. Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling. Nat Neurosci. 7:501-509.
Himanen, J.P., K.R. Rajashankar, M. Lackmann, C.A. Cowan, M. Henkemeyer, and D.B. Nikolov. 2001. Crystal structure of an Eph receptor-ephrin complex. Nature. 414:933-938.
Himanen, J.P., L. Yermekbayeva, P.W. Janes, J.R. Walker, K. Xu, L. Atapattu, K.R. Rajashankar, A. Mensinga, M. Lackmann, D.B. Nikolov, and S. Dhe-Paganon. 2010. Architecture of Eph receptor clusters. Proc Natl Acad Sci U S A. 107:10860-10865.
Holmberg, J., D.L. Clarke, and J. Frisen. 2000. Regulation of repulsion versus adhesion by different splice forms of an Eph receptor. Nature. 408:203-206.
Hurtley, S. 2009. Location, Location, Location. Science. 326.
Kinch, M.S., and K. Carles-Kinch. 2003. Overexpression and functional alterations of the EphA2 tyrosine kinase in cancer. Clin Exp Metastasis. 20:59-68.
Kullander, K., and R. Klein. 2002. Mechanisms and functions of Rph and ephrin signalling. Nat. Rev. Mol. Cell. Biol. . 3:475-486.
Lackmann, M., A.C. Oates, M. Dottori, F.M. Smith, C. Do, M. Power, L. Kravets, and A.W. Boyd. 1998. Distinct subdomains of the EphA3 receptor mediate ligand binding and receptor dimerization. J. Biol. Chem. 273:20228-20237.
Marquardt, T., R. Shirasaki, S. Ghosh, S.E. Andrews, N. Carter, T. Hunter, and S.L. Pfaff. 2005. Coepressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains. Cell. 121:127-139.
Pasquale, E.B. 2008. Eph-Ephrin Bidirectional Signaling in Physiology and Disease. Cell. 133.
Pasquale, E.B. 2010. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer. 10:165-180.
Poliakov, A., M. Cotrina, and D.G. Wilkinson. 2004. Diverse roles of eph receptors and ephrins in the regulation of cell migration and tissue assembly. Dev Cell. 7:465-480.
Qi, S.Y., J.T. Groves, and A.K. Chakraborty. 2001. Synaptic pattern formation during cellular recognition. Proc Natl Acad Sci U S A. 98:6548-6553.
Salaita, K., P.M. Nair, R.S. Petit, R.M. Neve, D. Das, J.W. Gray, and J.T. Groves. 2010. Restriction of Receptor Movement Alters Cellular Response: Physical Force Sensing by EphA2. Science. 327:1380-1385.
Scott, J.D., and T. Pawson. 2009. Cell signaling in space and time:where proteins come together and when they're apart. Science. 326:1220-1224.
Smith, F.M., C. Vearing, M. Lackmann, H. Treutlein, J. Himanen, K. Chen, A. Saul, D. Nikolov, and A.W. Boyd. 2004. Dissecting the EphA3/Ephrin-A5 interactions using a novel functional mutagenesis screen. J Biol Chem. 279:9522-9531. van der Geer, P., T. Hunter, and R.A. Lindberg. 1994. Receptor protein-tyrosine kinases and their signal transduction pathways. Annu. Rev. Cell Biol. 10:251-337.
Zelinski, D.P., N.D. Zantek, J.C. Stewart, A.R. Irizarry, and M.S. Kinch. 2001. EphA2 overexpression causes tumorigenesis of mammary epithelial cells. Cancer Res. 61:2301-2306.