Manipulating the Lateral Diffusion of Surface-Anchored EGF Demonstrates that Receptor Clustering Modulates its Phosphorylation Levels

Journal Article · Tue Jan 01 04:00:00 EST 2013 · Integrative Biology

OSTI ID:1073680

Stabley, Daniel ^[1]; Retterer, Scott T ^[2]; Marshal, Stephen ^[1]; Salaita, Khalid ^[1]

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1. Emory University
2. ORNL

Upon activation, the epidermal growth factor (EGF) receptor becomes phosphorylated and triggers a vast signaling network that has profound effects on cell growth. The EGF receptor is observed to assemble into clusters after ligand binding and tyrosine kinase autophosphorylation, but the role of these assemblies in the receptor signaling pathway remains unclear. To address this question, we measured the phosphorylation of EGFR when the EGF ligand was anchored onto laterally mobile and immobile surfaces. We found that cells generated clusters of ligand-receptor complex on mobile EGF surfaces, and generated a lower ratio of phosphorylated EGFR to EGF than when compared to immobilized EGF that is unable to cluster. This result was verified by tuning the lateral assembly of ligand-receptor complexes on the surface of living cells using patterned supported lipid bilayers. Nanoscale metal lines fabricated into the supported membrane constrained lipid diffusion and EGF receptor assembly into micron and sub-micron scale corrals. Single cell analysis indicated that clustering impacts EGF receptor activation, and larger clusters (> 1 m2) of ligand-receptor complex generated lower EGF receptor phosphorylation per ligand than smaller assemblies (< 1 m2) in HCC1143 cells that were engaged to ligand-functionalized surfaces. We investigated EGFR clustering by treating cells with compounds that disrupt the cytoskeleton (Latrunculin-B), clathrin-mediated endocytosis (Pitstop2), and inhibit EGFR activation (Gefitinib). These results help elucidate the nature of large-scale EGFR clustering, thus underscoring the general significance of receptor spatial organization in tuning function.

• Research Organization: Oak Ridge National Laboratory (ORNL); Center for Nanophase Materials Sciences
• Sponsoring Organization: SC USDOE - Office of Science (SC)
• DOE Contract Number: AC05-00OR22725
• OSTI ID: 1073680
• Journal Information: Integrative Biology, Journal Name: Integrative Biology Journal Issue: DOI:10.1 Vol. DOI:10.1
• Country of Publication: United States
• Language: English