Abstract
The insulin-like growth factor 1 receptor (IGF1R) is a receptor tyrosine kinase with critical roles in various biological processes. Recent results from clinical trials targeting IGF1R indicate that IGF1R signaling pathways are more complex than previously thought. Moreover, it has become increasingly clear that the function of many proteins can be understood only in the context of a network of interactions. To that end, we sought to profile IGF1R-protein interactions with the proximity-labeling technique BioID. We applied BioID by generating a HEK293A cell line that stably expressed the BirA* biotin ligase fused to the IGF1R. Following stimulation by IGF1, biotinylated proteins were analyzed by MS. This screen identified both known and previously unknown interactors of IGF1R. One of the novel interactors was sorting nexin 6 (SNX6), a protein that forms part of the retromer complex, which is involved in intracellular protein sorting. Using co-immunoprecipitation, we confirmed that IGF1R and SNX6 physically interact. SNX6 knockdown resulted in a dramatic diminution of IGF1-mediated ERK1/2 phosphorylation, but did not affect IGF1R internalization. Bioluminescence resonance energy transfer experiments indicated that the SNX6 knockdown perturbed the association between IGF1R and the key adaptor proteins insulin receptor substrate 1 (IRS1) and SHC adaptor protein 1 (SHC1). Intriguingly, even in the absence of stimuli, SNX6 overexpression significantly increased Akt phosphorylation. Our study confirms the utility of proximity-labeling methods, such as BioID, to screen for interactors of cell-surface receptors and has uncovered a role of one of these interactors, SNX6, in the IGF1R signaling cascade.
Highlights
The insulin-like growth factor 1 receptor (IGF1R) is a receptor tyrosine kinase with critical roles in various biological processes
sorting nexin 6 (SNX6) is a novel regulator of IGF1R signaling we have discovered that the protein SNX6 plays an important role in regulating IGF1R signaling
Temporal proteomic analysis has revealed that IGF1 treatment over time promotes the production of proteins associated with vesicle trafficking, glycolysis, and cell stress, functions that have been associated with cancer cell proliferation [28]
Summary
The insulin-like growth factor 1 receptor (IGF1R) is a receptor tyrosine kinase with critical roles in various biological processes. Following stimulation by IGF1, biotinylated proteins were analyzed by MS This screen identified both known and previously unknown interactors of IGF1R. Our study confirms the utility of proximity-labeling methods, such as BioID, to screen for interactors of cell-surface receptors and has uncovered a role of one of these interactors, SNX6, in the IGF1R signaling cascade. The challenge of identifying novel protein interactions has been addressed by proximity-dependent labeling. Proximity-dependent labeling techniques such as BioID have been making a major impact on our understanding of protein interaction networks (14, 18 –24). Despite their obvious strengths, no proximity-labeling technique has been employed to identify novel interactions with an RTK such as the IGF1R
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