The trafficking of amphiregulin in polarized epithelial cells

Abstract

Epithelial cells establish apical and basolateral (BL) membranes with distinct protein and lipid compositions. To achieve this spatial asymmetry, the cell utilizes a variety of mechanisms for differential sorting, delivery and retention of cell surface proteins. The EGF receptor (EGFR) and its ligand amphiregulin (AREG) are transmembrane proteins that are delivered to the BL membrane in polarized epithelial cells.

Herein, I demonstrate that the cytoplasmic domain of AREG contains dominant BL sorting information capable of redirecting an apical protein to the BL surface. By sequential truncations and site-directed mutagenesis of the AREG cytoplasmic domain, I identify a BL sorting motif consisting of a mono-leucine preceded by an acidic cluster (EExxxL). This sorting motif differs from the other reported mono-leucine BL sorting motif (EEDxxxxxL) in CD147 and stem cell factor. In LLC-PK1 and MDCK cells lacking AP-1B, AREG is detected on the apical surface, demonstrating that steady state BL distribution of AREG is AP-1B-dependent. In LLC-PK1 cells, AREG is transcytosed from the BL surface to the apical surface, suggesting AP-1B plays a role in recycling of AREG from an endosomal compartment to the BL membrane.

AREG is present in exosomes in a signaling competent topology and is enriched in exosomes from cells expressing mutant KRAS. Exosomal AREG appears to be post-translationally modified. I provide data supporting the hypothesis that this modification is ubiquitin and may be necessary for efficient delivery of AREG to exosomes. Signaling competent exosomal AREG may act as a novel EGFR signaling platform.

Using two methods, a split ubiquitin yeast two-hybrid screen and a crosslinked AREG IP mass spectral analysis, I identified potential AREG interacting proteins. Several of the proteins identified are interesting candidates for future work. Combined, the data presented in this dissertation demonstrates the mode of AREG BL delivery, provides insight into the regulation and effects of exosomal AREG localization, and reveals potential AREG interacting partners.