期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 117, 期 49, 页码 31157-31165出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2010209117
关键词
cadherin; heterophilic binding; BioID; proteomics; atomic force microscopy
资金
- National Institute of General Medical Sciences of the NIH [R01GM121885]
- NIH [R03 EB021636]
- NSF [1562095]
- Directorate For Engineering [1562095] Funding Source: National Science Foundation
- Div Of Civil, Mechanical, & Manufact Inn [1562095] Funding Source: National Science Foundation
We combine proximity labeling and single molecule binding assays to discover transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a bait protein with BioID biotin ligase and identify proximal proteins that are biotin tagged on both their extracellular and intracellular regions. We then test direct binding interactions between proximal proteins and the bait, using single molecule atomic force microscope binding assays. Using this approach, we identify binding partners for the extracellular region of E-cadherin, an essential cell-cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-alpha 2 beta 1, the receptor tyrosine kinase ligand ephrin-B1, and the classical cadherin P-cadherin, all directly interact with E-cadherin ectodomains. Our data shows that combining extracellular and cytoplasmic proximal tagging with a biophysical binding assay increases the precision with which transmembrane ectodomain interactors can be identified.
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