PDZ-DOMAIN-MEDIATED INTERACTION OF THE EPH-RELATED RECEPTOR TYROSINE KINASE EPHB3 AND THE RAS-BINDING PROTEIN AF6 DEPENDS ON THE KINASE-ACTIVITY OF THE RECEPTOR

Eph-related receptor tyrosine kinases (RTKs) have been implicated in i ntercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid sy stem. We could demonstrate that the carboxyl termini of the Eph-relate d RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ dom ain of the ras-binding protein AF6. A mutational analysis revealed tha t six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this P DZ 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 activi ty of EphB3, suggesting a regulated mechanism for the PDZ-domain-media ted interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic ef fects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by E phB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homo logy-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate s ignal transduction through their ability to mediate the interaction wi th RTKs.