Monday, May 6, 2024
5/6/2024
PROJECT ABSTRACT There is no doubt that the complex lipid makeup of the membrane bilayer and the associated peripheral membrane proteins dictate integral membrane protein (IMP) structure, function, localization, and dynamics in vivo. Yet, all this is replaced with detergents in traditional in vitro studies of IMPs due to technological limits. Indeed, the rapidly advancing G protein-coupled receptor and ion channel fields are showing us that both tightly bound and loosely-associated membrane lipids play crucial roles in defining receptor behavior. Current structural understanding of receptor tyrosine kinases (RTKs) seems extensive; however, we lack the fundamental knowledge of how RTKs are regulated by and transmit signals though the plasma membrane. Recent data have revealed this as a major shortcoming, as lipid-like antidepressants are thought to bind and modulate tropomyosin receptor kinase B and to quell aberrant epidermal growth factor receptor (EGFR) signaling in glioblastoma (by altering sphingolipid metabolism). EGFR, is an RTK that plays crucial roles in cellular and lipid metabolism, differentiation, motility, and proliferation in cancer. EGFR is a member of the ErbB family which has an extracellular-ligand binding domain linked to its intracellular kinase signaling domain. Monomeric EGFR is canonically regarded as inactive and ligand binding activates the receptor by driving formation of an asymmetric kinase dimer; both the active and inactive states are associated with different lipid microenvironments. Numerous structures have shown how individual RTK domains function in isolation and full-length EGFR (FL-EGFR) studies have revealed only another ectodomain structure due to being performed in a non-native membrane environment, omitting crucial lipid-cofactors. Ideally, one would study (FL-EGFR) in the context of the appropriate endogenous plasma membrane environment. However, the necessity to replace the bilayer with detergent during membrane protein isolation has made this historically impossible. To address this, I will combine newly available key technologies to define in vivo membrane protein environments and investigate lipid co-factor impacts on FL-EGFR structure and function. Aim 1 seeks to understand how the bilayer environment controls EGFR function with kinetic assays of FL-EGFR in defined lipid compositions will be used to understand the enzymatic influence of literature-identified lipid co-factors. Lipid compositions that significantly alter EGFR’s enzymatic properties will then be used to develop a comprehensive “real” structural study. The results in artificial bilayer systems will be validated with FL-EGFR in native nanodiscs which allow for study of membrane proteins along with their native lipid environment. Aim 2 asks what are the bilayer environments of EGFR? Numerous studies propose EGFR to reside in multiple poorly-defined bilayer environments: one in the ligand-bound active dimer state, and another in the inactive monomer state. Native nanodiscs and mass spectrometry-based lipidomics will characterize and compare the membrane and protein compositions in these two distinct environments.
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