Eph Receptors and Ephrins Are Key Regulators of Morphogenesis 137
recently, using alkaline phosphatase tagged proteins, that the dissociation constants are different for the interactions between EphA3 and
ephrin-AS and between EphA3 and ephrin-A2. Ephrin-AS binds EphA3
with significantly greater efficacy than does ephrin-A2 (Monschau et al.
1997).
However, it is also important to compare the methods used to determine binding characteristics. For example, human EphA3 has similar
affinity constants for binding to human ephrin-A3 and ephrin-AS when
the interaction is assessed with the ligands as Fc-fusion proteins, but the
interaction is different when assessed by binding of monovalent ligands
and soluble receptor. Under these conditions ephrin-AS has a markedly
lower dissociation rate from the soluble receptor than ephrin-A3, making it more likely that ephrin-AS is the endogenous ligand (Lackmann et
al. 1997).
A further important issue with regard to receptor activation is the
observation by Stein et al. that the degree of oligomerisation of the
ligand can affect the nature of the downstream response of the receptor
(Stein et al. 1998). This goes some way towards an explanation for the
specific responses of receptor-expressing cells which encounter fields of
cells expressing graded distributions of ligand, such as in the retinotectal
system described above.
The Eph receptors have a standard structure which is illustrated in
Fig. 3. They have an uninterrupted catalytic domain intracellularly, and
a cysteine-rich domain and two fibronectin type III repeats in the extracellular region. At the extracellular N-terminus there is a globular
domain responsible for specificity of ligand binding (Labrador et al.
1997). This was shown by creating a series of soluble deletion and
domain substitution mutants of EphB2 as alkaline phosphatase-tagged
fusion proteins, and examining their binding to ephrin-B 1. In domain
deletion experiments, only EphB2 ectodomains containing the N-terminal globular domain bind to ephrin-B2. By switching the N-terminal
globular region of EphB2 with the corresponding domain of the orphan
receptor EphBS, it was shown that the EphB2 N-terminal globular
domain was sufficient to confer ephrin-B 1 specific binding. Also, the
globular domain of EphA3 renders the EphB2 receptor competent to
bind to the class A ligand, ephrin-A2 (Labrador et al. 1997). Furthermore, ephrin-B1-dependent transformation of NIH 3T3 cells was seen
with chimaeric receptors in which the ectodomain of EphB2 was fused
recently, using alkaline phosphatase tagged proteins, that the dissociation constants are different for the interactions between EphA3 and
ephrin-AS and between EphA3 and ephrin-A2. Ephrin-AS binds EphA3
with significantly greater efficacy than does ephrin-A2 (Monschau et al.
1997).
However, it is also important to compare the methods used to determine binding characteristics. For example, human EphA3 has similar
affinity constants for binding to human ephrin-A3 and ephrin-AS when
the interaction is assessed with the ligands as Fc-fusion proteins, but the
interaction is different when assessed by binding of monovalent ligands
and soluble receptor. Under these conditions ephrin-AS has a markedly
lower dissociation rate from the soluble receptor than ephrin-A3, making it more likely that ephrin-AS is the endogenous ligand (Lackmann et
al. 1997).
A further important issue with regard to receptor activation is the
observation by Stein et al. that the degree of oligomerisation of the
ligand can affect the nature of the downstream response of the receptor
(Stein et al. 1998). This goes some way towards an explanation for the
specific responses of receptor-expressing cells which encounter fields of
cells expressing graded distributions of ligand, such as in the retinotectal
system described above.
The Eph receptors have a standard structure which is illustrated in
Fig. 3. They have an uninterrupted catalytic domain intracellularly, and
a cysteine-rich domain and two fibronectin type III repeats in the extracellular region. At the extracellular N-terminus there is a globular
domain responsible for specificity of ligand binding (Labrador et al.
1997). This was shown by creating a series of soluble deletion and
domain substitution mutants of EphB2 as alkaline phosphatase-tagged
fusion proteins, and examining their binding to ephrin-B 1. In domain
deletion experiments, only EphB2 ectodomains containing the N-terminal globular domain bind to ephrin-B2. By switching the N-terminal
globular region of EphB2 with the corresponding domain of the orphan
receptor EphBS, it was shown that the EphB2 N-terminal globular
domain was sufficient to confer ephrin-B 1 specific binding. Also, the
globular domain of EphA3 renders the EphB2 receptor competent to
bind to the class A ligand, ephrin-A2 (Labrador et al. 1997). Furthermore, ephrin-B1-dependent transformation of NIH 3T3 cells was seen
with chimaeric receptors in which the ectodomain of EphB2 was fused