Spatial control of EGF receptor activation by reversible dimerization on living cells.
Epidermal growth factor receptor (EGFR) is a type I receptor tyrosine kinase, the deregulation of which has been implicated in a variety of human carcinomas. EGFR signalling is preceded by receptor dimerization, typically thought to result from a ligand-induced conformational change in the ectodomain that exposes a loop (dimerization arm) required for receptor association. Ligand binding may also trigger allosteric changes in the cytoplasmic domain of the receptor that is crucial for signalling. Despite these insights, ensemble-averaging approaches have not determined the precise mechanism of receptor activation in situ. Using quantum-dot-based optical tracking of single molecules combined with a novel time-dependent diffusivity analysis, here we present the dimerization dynamics of individual EGFRs on living cells. Before ligand addition, EGFRs spontaneously formed finite-lifetime dimers kinetically stabilized by their dimerization arms. The dimers were primed both for ligand binding and for signalling, such that after EGF addition they rapidly showed a very slow diffusivity state that correlated with activation. Although the kinetic stability of unliganded dimers was in principle sufficient for EGF-independent activation, ligand binding was still required for signalling. Interestingly, dimers were enriched in the cell periphery in an actin- and receptor-expression-dependent fashion, resulting in a peripheral enhancement of EGF-induced signalling that may enable polarized responses to growth factors.
Chung
Inhee
I
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.
Akita
Robert
R
Vandlen
Richard
R
Toomre
Derek
D
Schlessinger
Joseph
J
Mellman
Ira
I
D000199:Actins / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D016764:Cell Polarity; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004058:Diffusion; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D066246:ErbB Receptors / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D008024:Ligands; D055503:Protein Multimerization / Q000187:drug effects; D021381:Protein Transport; D015398:Signal Transduction; D013816:Thermodynamics
D000199:Actins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008024:Ligands; D004815:Epidermal Growth Factor; D066246:ErbB Receptors