Insulin receptor trafficking and interactions in muscle cells

Context: Insulin action is critical for energy homeostasis and its dysfunction in muscle cells is associated with type 2 diabetes. Insulin receptor (INSR) internalization and cell-surface dynamics at rest and during insulin exposure are incompletely understood in muscle cells. Objective: We aimed to characterized the INSR dynamics and interactions in muscle. Methods: We applied inter-domain tagged INSR, microscopy, immunoprecipitation, mass spectrometry, and AlphaFold multimer to comprehensively profile INSR internalization and interactions with or without insulin stimulation. Results: Using surface labeling and live-cell imaging, we observed robust basal internalization of INSR in C2C12 myoblasts, without an effect of added insulin. Mass spectrometry using INSR knockout cells as controls identified high-confidence binding partners, including proteins associated with internalization. We confirmed known interactors, including insulin-like growth factor 1 receptor, and also identified underappreciated INSR-binding factors, such as annexin A2. AlphaFold multimer analysis predicted potential INSR-binding sites of these proteins. Protein-protein interaction network mapping suggested links between INSR and caveolin-mediated endocytosis. INSR interacted with both caveolin and clathrin heavy chain (CLTC) in mouse skeletal muscle and C2C12 myoblasts. Whole-cell 2-dimensional super-resolution imaging revealed that high levels of insulin (20 nM) increased INSR colocalization with caveolin-1 (CAV1) but decreased its colocalization with CLTC. Single-particle tracking confirmed the colocalization of cell-surface INSR with both overexpressed CAV1-mRFP (monomeric red fluorescent protein) and CLTC-mRFP. INSR tracks that colocalized with CAV1 exhibited longer radii and lifetimes, regardless of insulin exposure, compared with noncolocalized tracks, whereas insulin further increased the lifetime of INSR/CLTC-colocalized tracks. Conclusion: Overall, these data suggest that muscle cells utilize both CAV1- and CLTC-dependent pathways for INSR mobilization and internalization.