Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with important roles in many cellular processes as well as in cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. How these dimers relate to higher-order EGFR oligomers seen in cell membranes, however, remains unclear. Here, we used single-particle tracking (SPT) and Förster resonance energy transfer imaging to examine how each domain of EGFR contributes to receptor oligomerization and the rate of receptor diffusion in the cell membrane. Although the extracellular region of EGFR is sufficient to drive receptor dimerization, we find that the EGF-induced EGFR slowdown seen by SPT requires higher-order oligomerization-mediated in part by the intracellular tyrosine kinase domain when it adopts an active conformation. Our data thus provide important insight into the interactions required for higher-order EGFR assemblies involved in EGF signaling.

Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) with important roles in many cellular processes as well as cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. However, it is not clear how these dimers relate to higher-order EGFR oligomers detected at the cell surface. We used single-particle tracking (SPT) and Förster resonance energy transfer (FRET) imaging to examine how each domain within EGFR contributes to receptor dimerization and the rate of its diffusion in the cell membrane. We show that the EGFR extracellular region is sufficient to drive receptor dimerization, but that the EGF-induced EGFR slow-down seen by SPT requires formation of higher order oligomers, mediated in part by the intracellular tyrosine kinase domain - but only when in its active conformation. Our data thus provide important insight into higher-order EGFR interactions required for EGF signaling.

EGFR transactivates RON to drive oncogenic crosstalk.

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation - arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.