Abstract 153: Using Enzyme-Catalyzed Proximity Labeling to Define the Interactome of the Insulin-like Growth Factor-1 Receptor

Abstract

Introduction: Manipulation of I nsulin-like G rowth F actor- 1 R eceptor (IGF1R) signaling has been proposed to be an important therapeutic for the treatment of heart failure and myocardial infarction. However, activation of this receptor has, under certain conditions, been shown to result in deleterious side-effects such as pathological hypertrophy. Development of therapeutic strategies that avoid these side-effects requires a more comprehensive understanding of the proteins involved in the IGF1R signaling axis. To this end, we have made use of the enzyme-catalyzed proximity labeling technique called BioID. Objective: To comprehensively define the interactome of the activated IGF1R. Methods and Results: Briefly, BioID makes use of a biotin protein ligase called BirA* that promiscuously biotinylates neighboring proteins. The protein of interest is fused with BirA* and is then expressed in a particular cell line. Following administration of an excess amount of biotin, BirA* will biotinylate those proteins that are adjacent to or interact with the protein of interest. Interacting proteins are identified by tandem mass spectrometry (MS/MS). We have made a HEK 293T cell line that stably expresses the BirA* biotin ligase fused to the C-terminus of IGF1R. We performed BioID using this cell line (N=3) to identify a set of proteins that bind to the IGF1R following stimulation by IGF1. This set included many known interactors of the IGF1R, such as IRS2 and SHC1, thereby validating this technique. Of the many interesting novel interactors, we chose to pursue validation of sorting nexin 6 (SNX6) as some SNX proteins have been shown to be involved in receptor recycling to the plasma membrane following stimulation. We have found that SNX6 knockdown results in a dramatic diminution of IGF1-mediated ERK phosphorylation (2-fold; N=4; p <0.05). However, there was no effect on IGF1-mediated Akt phosphorylation (N=4). We are currently assessing the role of SNX6 in IGF1R-mediated function and recycling of this receptor. Conclusion: We have demonstrated the ability of the BioID technique to identify a set of proteins that can interact with the activated IGF1R. Moreover, BioID has identified a number of novel interactors that further define IGF1R signaling and function.