Kinetic analysis of the binding of monomeric and dimeric ephrins to Eph receptors: correlation to function in a growth cone collapse assay

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.

Figure 1.

Purification of dimeric and monomeric human ephrin-A5. The protein was expressed as an Fc fusion in HEK293 cells and was purified from culture supernatant by Protein-A Sepharose affinity chromatography (lane 1) and Superdex-200 size exclusion chromatography (lane 2). Following thrombin cleavage, the free Fc tag and the uncleaved ephrin-A5-Fc were pulled down using Protein-A beads (lane 3) to obtain monomeric ephrin-A5 in the supernatant (lane 4). (LMW) Low Molecular Weight markers—from top: 97.4, 66.2, 45.0, 31.0, 21.5, and 14.4 kDa.

Figure 2.

SPR sensorgrams of binding of ephrin-B2 to EphB2 and of ephrin-A5 to EphA3: (A) ephrin-B2-Fc interacting with EphB2-Fc, (B) ephrin-B2-ECD interacting with EphB2-Fc and the R_eq vs. C plot, (C) ephrin-A5-Fc interacting with EphA3-Fc, (D) ephrin-A5-ECD interacting with EphA3-Fc.

Figure 3.

Effect of different forms of ephrin-B2 (A) and ephrin-A5 (B) on the growth cones from rat hippocampus neurons in a growth cone collapse assay. (*) Statistical significance from the control; (**) statistical significance from ephrin-B2-Fc.

Scheme 1.