Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility.

Zhongwen Chen,Kabir Hassan Biswas,Dongmyung Oh,Jay T Groves,Ronen Zaidel-Bar

doi:10.7554/elife.67379

Abstract

Clustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly 1 µm, and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances both receptor phosphorylation and downstream signaling activity. Single-molecule imaging clearly resolves increased molecular binding dwell times at EphA2 clusters for both Grb2:SOS and NCK:N-WASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation.

Highlights

The cell membrane surface is studded with a broad array of receptor proteins that interact with numerous ligands, which can be soluble, membrane‐bound on an apposed cell surface, or associated with the extracellular matrix (Casaletto & McClatchey, 2012; Downward, 2001; Groves &Kuriyan, 2010)
Im ile ob Receptor clustering lies at the center of incorporating extracellular signals into amplified cellular responses (Bray et al, 1998; Cebecauer et al, 2010; Taylor et al, 2017)
The utility of this technology is clearly demonstrated by the observation of increased phosphorylation of clustered EphA2 compared to the non‐clustered ones

Summary

Introduction

The cell membrane surface is studded with a broad array of receptor proteins that interact with numerous ligands, which can be soluble, membrane‐bound on an apposed cell surface, or associated with the extracellular matrix (Casaletto & McClatchey, 2012; Downward, 2001; Groves &Kuriyan, 2010). Ligand binding is followed by activation of elaborate signal transduction pathways in the cell, which mediate cellular decision making. In the case of receptor tyrosine kinases (RTKs), initial receptor activation after ligand binding involves phosphorylation of specific tyrosine residues on the receptors, followed by recruitment of adaptor proteins which mediate activation of additional signaling molecules. Of cell surface receptors into clusters or organized arrays is a common feature of cell membranes (Dustin & Groves, 2012; Garcia‐Parajo et al, 2014; Janes et al, 2012; Lee et al., 2002; Mossman et al, 2005; Salaita et al, 2010), and has long been implicated as an important factor for modulating signaling activity (Bray et al, 1998; Cebecauer et al, 2010). In T cell receptor signaling system, the Linker for activation of T cells (LAT)

Methods

Results

Conclusion