Manipulating the lateral diffusion of surface-anchored EGF demonstrates that receptor clustering modulates phosphorylation levels

Abstract

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 displayed a lower ratio of phosphorylated EGFR to EGF 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 µm2) of ligand-receptor complex generated lower EGF receptor phosphorylation per ligand than smaller assemblies (< 1 µm2) in HCC1143 cells that were engaged to ligand-functionalized surfaces. We investigated the mechanism of 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.

Oligomerization of DDR1 ECD affects receptor–ligand binding

“…Numerous receptors, including EGFR (Stabley et al, 2013), integrins (Boettiger, 2012) toll-like receptor (Triantafilou et al, 2006), ErbB family (Yarden and Sliwkowski 2001) and Ephrin (Salaita et al, 2010) have been found to assemble into higher-order structures where downstream signaling events are often correlated to cluster formation. Consistent with our observations, a large heterogeneity in oligomer size has been reported for other RTKs, like the epidermal growth factor receptor (EGFR) (Abulrob et al, 2010).…”

“…Consistent with our observations, a large heterogeneity in oligomer size has been reported for other RTKs, like the epidermal growth factor receptor (EGFR) (Abulrob et al, 2010). A recent study using a nano-patterned supported lipid bilayer technique to control EGFR clustering levels in living cells found that large-scale clustering of EGFR dampens its phosphorylation and that the cell endocytosis machinery contributes to this clustering behavior (Stabley et al, 2013). …”

The extracellular matrix as a key regulator of intracellular signalling networks

“…ECM stiffness is known to promote tumour progression by causing integrins to cluster within focal adhesions (Paszek et al, ; Levental et al, ), and direct interactions between these RTKs and integrins are thought to facilitate this localized enrichment (Wang et al, ; Sieg et al, ; Cabodi et al, ). This spatial clustering of RTK activity on stiff substrates alters the balance between ligand concentration and receptor auto‐phosphorylation, which ultimately amplifies ligand‐induced RTK signalling (Ichinose et al, ; Levental et al, ; Stabley et al, ). Additionally, the localization of RTKs within focal adhesions on stiff substrates promotes signalling through FAK, which increases cell migration (Sieg et al, ), while FAK is also required for RTK‐mediated oncogenic transformation (Benlimame et al, ).…”

Leader cell PLCγ1 activation during keratinocyte collective migration is induced by EGFR localization and clustering

“…Cells treated with soluble EGF showed some areas of potential receptor clusters, while cells on immobilized EGF had large areas of dense EGFR signal (Figure a). Previous studies have defined receptor clusters as ranging from 0.07 to 3 μm 2 ; therefore, we classified any signal with an area less than 0.1 μm 2 as an isolated receptor and quantified the sizes of all clusters (Figure b). Few EGFR clusters were observed in cells in the bulk regardless of treatment condition (Supporting Information Figure 2); in addition, cells in the control condition had no observable receptor clusters at the wound edge.…”