Mapping transmembrane binding partners for E-cadherin ectodomains.

Area of Science:

• Cell Biology
• Biophysics
• Molecular Interactions

Background:

• Transmembrane proteins mediate crucial cell functions, but identifying their interaction partners, especially ectodomain interactions, remains challenging.
• Existing methods often lack the precision to distinguish direct binding events from proximity effects.
• E-cadherin, a key cell-cell adhesion molecule, has interaction partners that are not fully characterized.

Purpose of the Study:

• To develop and validate a novel dual proximity labeling and single-molecule biophysical assay for discovering transmembrane protein ectodomain interactions.
• To identify direct binding partners of the extracellular domain of E-cadherin.

Main Methods:

• Utilized BioID proximity labeling targeting both extracellular and cytoplasmic regions of a bait protein.
• Employed single-molecule atomic force microscopy (AFM) binding assays to confirm direct interactions.
• Screened for proteins biotinylated on both extracellular and intracellular regions using dual proximity labeling.

Main Results:

• Identified direct binding partners for the extracellular region of E-cadherin.
• Confirmed interactions with desmosomal proteins (desmoglein-2, desmocollin-3), focal adhesion protein (integrin-α2β1), receptor tyrosine kinase ligand (ephrin-B1), and classical cadherin (P-cadherin).
• Demonstrated that combined extracellular and cytoplasmic proximity tagging enhances the precision of ectodomain interactor identification.

Conclusions:

• The combined proximity labeling and single-molecule AFM assay is a powerful tool for precise identification of transmembrane protein ectodomain interactions.
• This study reveals novel direct interactors of E-cadherin, expanding our understanding of cell adhesion mechanisms.
• The developed methodology offers a significant advancement in studying protein-protein interactions at the cell surface.