Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequencespecific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.The effects of many growth factors and cytokines are mediated by high-affinity binding to receptor tyrosine kinases (RTKs) resulting in autophosphorylation of the cytoplasmic domain (1). Phosphotyrosine residues serve as binding sites for downstream signaling proteins that establish a complex network of interactions within the cell. Modular structures involved in binding of activated RTKs identified so far include Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains, which both bind specifically to phosphorylated tyrosine residues in a sequencespecific fashion (2).Recently, we isolated two Eph-related RTKs, named EphB3 and EphB2 (refs. 3 and 4; B.B., U.H., T.K., and K.S., unpublished data), which both bind specifically to the transmembrane subgroup of Eph-receptor ligands (5, 6). The genetic analysis of EphB2 and EphB3 revealed a physiological requirement of both receptors for pathfinding of specific commissural axons in the central nervous system (7,8). To date, the information about signaling molecules mediating Eph-receptor-specific responses is still limited to a few family members. Interactions have been described for the activated Eph-family members EphA2, EphA4, and EphB1 and the SH2-domain-containing proteins p85 subunit of phosphatidylinositol 3-kinase, the adapter protein SLAP, Grb2, Grb10, and Fyn (8-11). Lately, we found that activated EphB3 interacts with Crk, Fyn, and rasGAP in a SH2-dependent manner (12): Autophosphorylation of Tyr-614 in the juxtamembrane region of the receptor generates a multi-docking-site for these interactions...
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