Regulation of receptor tyrosine kinases by cholesterol on the plasma membrane of living cells

Abstract

Most cellular communications through the PM rely on cell surface receptors. Under the stimulation of an intrinsic ligand, the membrane receptor generates intracellular signals that facilitate corresponding functional responses. Numerous PM receptors have been discovered and thoroughly characterized in this classic ligand-receptor model, even though the lipids on the PM have not received an equivalent level of consideration. In the first chapter, I introduced the roles of lipids in the receptor tyrosine kinase (RTK) activation model and the challenge of technology limitation for the lipid–protein regulation study. In Chapter 2, I described a newly developed tool to study biological processes in living cells using fluorescence imaging technology. Here, I discovered an ultrastable blue-converted organic dye (BAss550), which is super stable under multiple conditions. I utilized this ultrastable dye to directly visualize and characterize the clathrin-mediated endocytosis process of EGFR on the PM of live Cos7 cells. In Chapter 3, I discovered the function of inner leaflet cholesterol in EGFR activation with the assistance of my newly developed tool in Chapter 2. Here, I discovered a direct interaction between the inner leaflet cholesterol and ligated EGFR using coimmunoimmobilization assay (CoII). Furthermore, by using the BAss550 organic dye, I found that the EGFR dimer is only sufficiently stabilized for autophosphorylation in the presence of inner leaflet cholesterol. Ultimately, I proposed a new activation model of EGFR. Under the stimulation of an intrinsic ligand, monomeric tethered EGFR is extended, enabling the asymmetric dimer of two extended EGFRs. The asymmetric dimer of EGFR is stabilized only in the presence of inner leaflet cholesterol. This stabilized asymmetric dimer of EGFR allows the approach of the EGFR tyrosine kinase domain (TKD) for autophosphorylation. Moreover, based on the findings obtained from my study of lipid regulation (especially cholesterol) on RTK activity, in chapter 4, I also identified that the different activation mechanisms between insulin receptor (IR) isoforms on the PM occur in a cholesterol-dependent manner. In this study, I provide strong evidence indicating differences between the activation of IR isoforms, which critically depend on the level of inner leaflet cholesterol. Lastly, my study provides fundamental knowledge and state-of-the-art single-molecule techniques for studying the function of lipids in RTK activities on the PM of living cells. By understanding the activation mechanism of RTK in different lipid contexts, we can develop improved strategies for drug discovery or combination therapy for each target cell surface receptor with respect to different lipidomics in different diseases or different individuals.