Abstract
Analysis of protein–protein interactions in living cells by protein micropatterning is currently limited to the spatial arrangement of transmembrane proteins and their corresponding downstream molecules. Here, we present a robust and straightforward method for dynamic immunopatterning of cytosolic protein complexes by use of an artificial transmembrane bait construct in combination with microstructured antibody arrays on cyclic olefin polymer substrates. As a proof, the method was used to characterize Grb2-mediated signaling pathways downstream of the epidermal growth factor receptor (EGFR). Ternary protein complexes (Shc1:Grb2:SOS1 and Grb2:Gab1:PI3K) were identified, and we found that EGFR downstream signaling is based on constitutively bound (Grb2:SOS1 and Grb2:Gab1) as well as on agonist-dependent protein associations with transient interaction properties (Grb2:Shc1 and Grb2:PI3K). Spatiotemporal analysis further revealed significant differences in stability and exchange kinetics of protein interactions. Furthermore, we could show that this approach is well suited to study the efficacy and specificity of SH2 and SH3 protein domain inhibitors in a live cell context. Altogether, this method represents a significant enhancement of quantitative subcellular micropatterning approaches as an alternative to standard biochemical analyses.
Highlights
Analysis of protein−protein interactions in living cells by protein micropatterning is currently limited to the spatial arrangement of transmembrane proteins and their corresponding downstream molecules
In order to compensate for the low rigidity of the cyclic olefin polymer (COP) foil, the patterned substrate was bonded with a 384-well plastic casting (Figure 1A), resulting in a ready-to-use multiwell plate that can be further functionalized in a modular manner for subsequent analysis of protein−protein interactions (PPIs) in live cells with high experimental throughput
To proof Grb[2] activation and mediation of downstream signaling, we investigated the ability of bait-PAR-Grb[2] to corecruit further adapter proteins such as the SHC-transforming protein 1 (Shc1), which has been reported to be recognized by the Grb[2] SH2 domain,[34] similar to the epidermal growth factor receptor (EGFR)
Summary
Analysis of protein−protein interactions in living cells by protein micropatterning is currently limited to the spatial arrangement of transmembrane proteins and their corresponding downstream molecules. Others and we have developed protein micro- and nanopatterning approaches on solid substrates for the quantitative investigation of protein−protein interactions (PPIs) in the live cell.[4,6−14] The fundamental strategy of these approaches is the spatial rearrangement of a cell surface protein (“bait”, e.g., receptor) in the shape of the printed patterns within the plasma membrane (e.g., by use of antibodies or ligands) and the monitoring of the lateral distribution of a putative interaction partner (“prey”, e.g., cytosolic adapter protein) This enables the investigation of PPIs in a native environment and membrane composition, and importantly, allows for straight-forward characterization of PPIs in the living cell.
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