Transmembrane association of DDR1 and DDR2 mediated by Leucine zipper motifs

Discoidin domain receptors (DDRs) are single-pass transmembrane proteins belonging to receptor tyrosine kinases (RTKs) family, which are activated by collagen ligands with unusual slow, sustained kinetics, distinguishing them from canonical RTKs. While DDRs play critical roles in cell adhesion, differentiation, and cancer progression, their activation mechanisms remain partly understood. Here, we investigated the transmembrane domains (TMDs) of DDR1 and DDR2 to elucidate their interaction dynamics in membrane. Using bacterial adenylate cyclase two-hybrid (BACTH) assays, we demonstrated robust homotypic interactions and even stronger heterotypic associations between DDRTMDs. NMR spectroscopy of DDR1TMD and DDR2TMD reconstituted in lipid bilayer-mimetic bicelles showed obvious chemical shift alterations, further validating the stability of their heterocomplex formation. Systematic mutagenesis identified leucine zipper motifs rather than GXXXA motifs mediated both homo- and hetero-associations of DDR1TMD and DDR2TMD. These findings demonstrated the TMD as a critical mediator of DDRs oligomerization and revealed their interaction patterns within membrane. Our study advances the understanding of DDR signaling regulation and highlights transmembrane domain interactions as potential targets for modulating DDR-related pathologies.