Abstract
Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase, a member of the epidermal growth factor receptor (EGFR) family of proteins. The receptor activates various cellular signaling pathways by dimerization with other family members, which results in cell growth and proliferation. Upregulation of HER2 can lead to tumorigenesis, frequently observed in breast cancer. HER2 has been identified as an important drug target for HER2 positive breast cancer wherein anti-HER2 therapeutic antibodies have been developed and are in use. However, the mechanisms of HER2 activation and how the antibodies interact with the receptor and inhibit the downstream signaling events are not fully understood. It has been suggested that the membrane content around HER2 plays an important role in its activation, or inhibition. In order to understand the receptor’s mode of operation, structural observation of HER2 in its native environment is required. Previous structural analysis of HER2 revealed the protein’s architecture: an extracellular domain and a cytoplasmic domain linked by a single α-helix transmembrane domain. However, the structure of the intact receptor embedded in its native environment is still elusive, presumably due to technical limitations. In this thesis, I have developed the technical means and carried out the investigation of HER2 in its native membrane environment by cryo-electron microscopy (cryo-EM) and tomography (cryo-ET). To this end, a HER2 positive breast cancer cell line, SKBR3, was utilized due to its native over-expression of HER2, at over a million copies per cell. Tomograms were acquired from the thin periphery regions of SKBR3 cells cultured on the galectin-8 coated substrates. Using immunogold labeling of the receptor, we identified two oligomeric states of HER2 which indicates that there is a mixed population of monomers and dimers on the cell surface. In the second part of the work, I used the native properties of SKBR3 cells to produce extracellular vesicles (EVs) and compared these vesicles to mechanically induced membrane vesicles. Here, we developed a purification strategy to enrich HER2 containing vesicles. Designed ankyrin repeat proteins (DARPins) that bind to HER2 were attached to magnetic beads to produce affinity-based isolation of HER2 containing vesicles, from which tomograms were acquired. They revealed a crowded HER2 local membrane environment. In this part I 5 characterized the different vesicles and showed that the mechanically induced vesicles are more homogeneous and therefore they are the potentially better samples to conduct future structural analysis for the receptor. In the third part of this thesis, I utilized the above strategy to determine the structure of HER2 in its native membrane environment. Two methods were used to elucidate the structure: firstly, cryo-ET followed by subtomogram averaging (STA); and, secondly, single particle analysis (SPA) from cryo-EM. STA 3D classification proved difficult due to the lower signal to noise ratio of subtomograms and the limited information caused by the ‘missing wedge’ effect. SPA proved to be the more suitable method to reconstruct the structure of the receptor. Although only reconstructed to a modest resolution, the final 3D density map had morphology consistent with the extracellular domain (ECD) of HER2 monomer, suggesting that HER2 monomers are the most abundant state for the receptor in the plasma membrane of the SKBR3 cells. Additional verification of our structural analysis was conducted by comparing the 3D density map with the AlphaFold2 predicted structures of all membrane proteins detected in the vesicle sample. HER2 fitted the best and the structure of subdomains I, II and III of HER2 ECD can be properly docked into the reconstructed map. In summary, this thesis was designed to develop a platform for the structural analysis of receptors in their native membranes. The high structural flexibility of HER2, together with the large diversity of membrane proteins in the eukaryotic plasma membrane, made the structural determination of HER2 in the native membrane still a challenging task. Nevertheless, the developed approach can be used as an integrative approach, where high-resolution in vitro structural analysis can be combined with functional knowledge to understand the receptor’s mode of operation and organization in its native environment, and can also be applied to resolve structures of other membrane proteins in their native environments.
Wang, Chunyang