Abstract
AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131–135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro. Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway.
Highlights
We evaluated the specificity of the peptide in binding to and affinity purifying monomeric or dimerized Anterior Gradient-2 (AGR2) protein from human cell lysates
When lysates from untreated cells are incubated with streptavidin beads coated with Peptide A4, monomeric AGR2 protein can be affinity purified from the crude lysate (Fig. 1A, lane 4 versus Load, Flow-though, and wash, lanes 1–3)
Another yeast-two hybrid screen revealed that AGR2 could bind to membrane receptors but many nuclear proteins such as RIP140 and Reptin, which might reflect a role for the form of AGR2 that escapes the endoplasmic reticulum (ER) through its non-canonical KDEL retention sequence [47]
Summary
Human codon optimized extracellular domain of EpCAM (amino acids 24–265). KanR pEpCAM-Y251A-C-eGFP Tyrosine251 of pEpCAM-C-eGFP mutated to Ala. AmpR of proteins destined for the plasma membrane. AmpR of proteins destined for the plasma membrane During these studies, a clinical proteomics analysis using resected biopsies from patients with esophageal adenocarcinoma identified AGR2 and EpCAM as highly expressed in primary adenocarcinoma as well as cancer-associated lymph nodes [27]. A clinical proteomics analysis using resected biopsies from patients with esophageal adenocarcinoma identified AGR2 and EpCAM as highly expressed in primary adenocarcinoma as well as cancer-associated lymph nodes [27] This allows scientific information acquired on AGR2 and EpCAM pathway regulation to be translated into novel therapeutics that target the AGR2:EpCAM axis for improved cancer management
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