Three distinct molecular surfaces in Ephrin-A5 are essential for a functional interaction with EphA3

Memorial Sloan-Kettering Cancer Center, New York, New York, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2005; 280(28):26526-32. DOI: 10.1074/jbc.M504972200
Source: PubMed


Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to
direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite
sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal
initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random
library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the
assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure
of the homologous EphB2·ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E α-helix, the E-F loop,
the underlying H β-strand, as well as the nearby B-C loop, which define a distinct third surface required for oligomerization
and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal
ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct
protein-interaction interfaces to assemble functional EphA3 signaling complexes.

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    • "The majority of in vitro and in vivo studies on Eph receptor function point to the induction of repulsive interaction upon the binding of Eph or ephrin ligands (O'Leary and Wilkinson, 1999); although attractive interactions mediated by each group are known to exist (Davy and Robbins , 2000; Gao et al., 2000; Zhou et al., 2001; Hindges et al., 2002; Mann et al., 2002; Palmer et al., 2002; Davy and Soriano, 2005). Eph receptors are unique among receptor tyrosine kinases in that, in addition to interacting with a cell-bound ligand, significant activation of the receptor does not occur in the dimeric state; but instead requires higher order association (Day et al., 2005; Pabbisetty et al., 2007). Binding of ephrin ligands to the Eph receptor ectodomain, results in the induction of conformational changes which induce receptor activation. "
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    ABSTRACT: Previously, we have demonstrated that EphB2 activity is required for proper development of the posterior branch of the anterior commissure (ACpp) within the mammalian forebrain. In the present study, using magnetic resonance imaging (MRI), immunohistochemistry, and in vivo stereotactic fluorescence tracing of EphB2, B3, A4 and combinatorial Eph receptor mutants, we have developed a detailed three-dimensional model of how EphB-class receptors interact to regulate commissural formation within the forebrain. The results demonstrate that EphB2 and EphA4 each regulate distinct aspects of axon guidance within the ACpp. Specifically, while EphB2 is required to retard ACpp axons from projecting aberrantly into the ventral forebrain, EphA4 is required to restrict axons from entering the anterior branch of the anterior commissure (ACpa). Together, EphB2 and EphA4 act synergistically to prevent a subpopulation of axons within the anterior branch of the AC from mis-projecting caudally. Analysis of EphA4 null mice using high resolution MRI reveals for the first time that, in addition to errors in midline guidance, loss of EphA4 results in aberrant lateral and ventral displacement of the ACpa tract. In addition, tracing studies in alpha-chimerin null mice reveal that EphA4-mediated effects are not regulated through this pathway. Taken together, the results demonstrate that each of the principal guidance decisions within both anterior and posterior tracts of the anterior commissure can be accounted for by the individual and combinatorial actions of EphB2/A4 receptors.
    Neuroscience 04/2009; 160(4):784-95. DOI:10.1016/j.neuroscience.2009.03.013 · 3.36 Impact Factor
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    • "Binding of representative ephrin ligands to Eph receptors was carried out on a Biacore 3000 SPR biosensor (Biacore International AB) using EphB2-Fc-and EphA3-Fc-immobilized CM5 chips essentially as described (Lackmann et al. 1997; Day et al. 2005). Briefly, EphB2-Fc and EphA3-Fc (6 mg/mL) were coupled onto CM5 chip surfaces at 10 mL/min using a standard amine coupling protocol with EDC (N-ethyl-N9-[dimethyl- aminopropyl]carbodiimide)/NHS (N-hydroxysuccinimide). "
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    ABSTRACT: Eph receptors and ephrins play important roles in regulating cell migration and positioning during both normal and oncogenic tissue development. Using a surface plasma resonance (SPR) biosensor, we examined the binding kinetics of representative monomeric and dimeric ephrins to their corresponding Eph receptors and correlated the apparent binding affinity with their functional activity in a neuronal growth cone collapse assay. Our results indicate that the Eph receptor binding of dimeric ephrins, formed through fusion with disulfide-linked Fc fragments, is best described using a bivalent analyte model as a two-step process involving an initial monovalent 2:1 binding followed by a second bivalent 2:2 binding. The bivalent binding dramatically decreases the apparent dissociation rate constants with little effect on the initial association rate constants, resulting in a 30- to 6000-fold decrease in apparent equilibrium dissociation constants for the binding of dimeric ephrins to Eph receptors relative to their monomeric counterparts. Interestingly, the change was more prominent in the A-class ephrin/Eph interactions than in the B-class of ephrins to Eph receptors. The increase in apparent binding affinities correlated well with increased activation of Eph receptors and the resulting growth cone collapse. Our kinetic analysis and correlation of binding affinity with function helped us better understand the interactions between ephrins and Eph receptors and should be useful in the design of inhibitors that interfere with the interactions.
    Protein Science 04/2007; 16(3):355-61. DOI:10.1110/ps.062608807 · 2.85 Impact Factor
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    ABSTRACT: We identified 1113 articles (103 reviews, 1010 primary research articles) published in 2005 that describe experiments performed using commercially available optical biosensors. While this number of publications is impressive, we find that the quality of the biosensor work in these articles is often pretty poor. It is a little disappointing that there appears to be only a small set of researchers who know how to properly perform, analyze, and present biosensor data. To help focus the field, we spotlight work published by 10 research groups that exemplify the quality of data one should expect to see from a biosensor experiment. Also, in an effort to raise awareness of the common problems in the biosensor field, we provide side-by-side examples of good and bad data sets from the 2005 literature.
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