Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single-Molecule Diffusivity in the Membrane of Living Cells.
We present a single-molecule diffusional-mobility-shift assay (smDIMSA) for analyzing the interactions between membrane and water-soluble proteins in the crowded membrane of living cells. We found that ligand-receptor interactions decreased the diffusional mobility of ErbB receptors and β-adrenergic receptors, as determined by single-particle tracking with super-resolution microscopy. The shift in diffusional mobility was sensitive to the size of the water-soluble binders that ranged from a few tens of kilodaltons to several hundred kilodaltons. This technique was used to quantitatively analyze the dissociation constant and the cooperativity of antibody interactions with the epidermal growth factor receptor and its mutants. smDIMSA enables the quantitative investigation of previously undetected ligand-receptor interactions in the intact membrane of living cells on the basis of the diffusivity of single-molecule membrane proteins without ligand labeling.
School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang 790784 (Republic of Korea).|Center for Nano-Bio Convergence, KRISS, Daejeon 305340 (Republic of Korea).
Do-Hyeon
DH
Kim
Department of Life Sciences, POSTECH, Pohang 790784 (Republic of Korea).
Kai
K
Zhou
School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang 790784 (Republic of Korea).
Dong-Kyun
DK
Kim
Department of Life Sciences, POSTECH, Pohang 790784 (Republic of Korea).
Soyeon
S
Park
Department of Life Sciences, POSTECH, Pohang 790784 (Republic of Korea).
Jungeun
J
Noh
Department of Life Sciences, POSTECH, Pohang 790784 (Republic of Korea).
Yonghoon
Y
Kwon
Department of Life Sciences, POSTECH, Pohang 790784 (Republic of Korea).
Dayea
D
Kim
Center for Nano-Bio Convergence, KRISS, Daejeon 305340 (Republic of Korea).
Nam Woong
NW
Song
School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang 790784 (Republic of Korea).|Department of Physics, POSTECH, Pohang 790784 (Republic of Korea).
Jong-Bong
JB
Lee
School of Nano-Bioscience and Chemical Engineering, UNIST, Ulsan 689798 (Republic of Korea).
Pann-Ghill
PG
Suh
School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang 790784 (Republic of Korea). nklee@postech.ac.kr.|Department of Physics, POSTECH, Pohang 790784 (Republic of Korea). nklee@postech.ac.kr.
Nam Ki
NK
Lee
School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang 790784 (Republic of Korea). sungho@postech.ac.kr.|Department of Life Sciences, POSTECH, Pohang 790784 (Republic of Korea). sungho@postech.ac.kr.
Sung Ho
SH
Ryu
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D000068818:Cetuximab / Q000276:immunology; D002522:Chlorocebus aethiops; D004058:Diffusion; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008853:Microscopy; D009154:Mutation
D000911:Antibodies, Monoclonal; D008024:Ligands; D066246:ErbB Receptors; D000068818:Cetuximab
cell membranes; diffusion; ligand-receptor interactions; super-resolution microscopy; transmembrane proteins