Molecular basis for multimerization in the activation of the epidermal growth factor receptor

AbstractMolecular basis for multimerization in the activation of the epidermal growth factor receptorbyYongjian HuangDoctor of Philosophy in BiophysicsUniversity of California, BerkeleyProfessor John Kuriyan, ChairThe epidermal growth factor receptor (EGFR), and its relatives HER2, HER3 and HER4, are receptor tyrosine kinases that couple the binding of extracellular ligands to the initiation of intracellular signaling pathways that control cell growth and proliferation. Aberrant signaling from EGFR family members underlies the onset of many human cancers. The canonical view of EGFR activation is that the receptor is activated by dimerization. It is known, however, that EGFR activation also generates higher-order multimers, whose structural details and functional consequences are poorly understood. Due to the incomplete understanding of the significance of multimerization for EGFR activity, the monomer/dimer model has remained the paradigm for EGFR activation mechanism. In collaboration with Shashank Bharill, a postdoc in Ehud Isacoff’s group, we now have characterized ligand-induced dimerization and multimerization of human EGFR using single-molecule based analysis, specifically, stepwise fluorescence photobleaching using EGFR transfected into Xenopus oocytes and fluorescence cross-correlation spectroscopy of EGFR expressed in mammalian cells. This single-molecule technique was first developed in Isacoff lab back in 2007, for counting subunits of membrane proteins in live-cell by observing photobleaching steps of GFP that is fused to a protein of interest. Without disrupting their membrane environment, we have now been able to determine the stoichiometry of EGFR complexes before and after activation. Using mutations identified in this study that are specifically important for multimerization of EGFR, I uncovered a functional role for multimerization in EGFR activation. These mutations reduce EGFR autophosphorylation in mammalian cells, particularly for sites in the C-terminal tail that are proximal to the kinase domain, and they also attenuate phosphorylation of phosphatidyl inositol 3-kinase, which binds to a proximal site on the EGFR C-terminal tail. In collaboration with Deepti Karandur, a postdoc in Kuriyan lab, we propose a structural model for EGFR multimerization that is consistent with our experimental observations, which might be applicable to other human EGFR family members as well. During the investigation of EGFR stoichiometry in the presence of EGF using Xenopus oocytes, we made an unexpected discovery. Upon binding to EGF, EGFR shows linear back-and-forth motion on the oocyte surface, which is dependent on EGFR kinase activity and the actin cytoskeleton. To fully uncover the mechanism behind the observed linear motion of EGFR, more extensive studies are needed