Insulin-dependent and -independent dynamics of insulin receptor trafficking in muscle cells

Insulin resistance contributes to type 2 diabetes and can be driven by hyperinsulinemia. Insulin receptor (INSR) internalization and cell-surface dynamics at rest and during insulin exposure are incompletely understood in muscle cells. Using surface labelling 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 IGF1R, but also identified underappreciated INSR-binding factors such as ANXA2. AlphaFold-Multimer analysis of these INSR-binding proteins 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 2D super-resolution imaging revealed that high levels of insulin (20 nM) increased INSR colocalization with CAV1 but decreased its colocalization with CLTC. Single particle tracking confirmed the colocalization of cell-surface INSR with both over-expressed CAV1-mRFP and CLTC-mRFP. INSR tracks that colocalized with CAV1 exhibited longer radii and lifetimes, regardless of insulin exposure, compared to non-colocalized tracks, whereas insulin further increased the lifetime of INSR/CLTC colocalized tracks. Overall, these data suggest that muscle cells utilize both CAV1 and CLTC-dependent pathways for INSR dynamics and internalization.