Doctoral Dissertations

Date of Award

12-2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Francisco N. Barrera

Committee Members

Dan Roberts, Jennifer Morrell-Falvey, Rachel McCord, Bruce McKee

Abstract

Single-pass membrane receptor signaling plays vital roles in human development and maintaining homeostasis. These membrane receptors can also have causative functions in several diseases including cancer. Much is known about the structure and signaling outcomes of these receptors but the mechanistic details of how they pass an extracellular signal across the membrane and into cytoplasm via the transmembrane (TM) domain is unclear. It is further unknown how or if interactions with membrane lipids facilitate and/or regulate these events. Here we use the TYPE7 peptide to target the TM region of a receptor tyrosine kinase, EphA2. EphA2 engages in both tumorigenic (ligand-independent) and anti-tumorigenic (ligand-dependent) signaling making it an attractive drug target. From TYPE7 we learned that the activity of EphA2 could be modulated by interactions with a TM peptide. Findings from TYPE7 (Chapter II), lead to hypotheses about the signaling states of EphA2 and interactions with anionic lipids. We next demonstrated (Chapter III) that there is a TM conformation-specific coupling of juxtamembrane residues of EphA2 with PIP2 [phosphatidylinositol 4,5-bisphosphate]. Our data suggests that PIP2 promotes dimerization of EphA2 in the ligand-independent state, potentially regulating tumorigenic signaling. These findings add to the knowledge of the molecular events of EphA2 signal transduction which is vital to designing effective therapeutics. Finally, we investigated the effects that TM peptides can have on their lipid environments. We developed (Chapter IV) a fluorescence recovery after photobleaching (FRAP) protocol and an automated data analysis pipeline using programs written in Python and Mathematica languages for the determination of lipid diffusion coefficients. We used the pH responsive peptide (pHLIP) as a model TM domain and FRAP in supported lipid bilayers to investigate the effect of pHLIP on the rate of lipid diffusion.

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