Defining Distinct Actions of Insulin and IGF1 Receptors and Their Extra and Intracellular Domains at the Level of the Phosphoproteome

Insulin and IGF-1 regulate a wide variety of biological processes through insulin and IGF-1 receptors (IR and IGF1R). These receptors are highly homologous and share many overlapping downstream signaling pathways. To determine differences in signaling between IR and IGF1R that could account for their unique physiological actions, we performed global phosphoproteomic analysis of preadipocytes lacking both endogenous receptors then reconstituted with IR, IGF1R, and chimeric IR_IGF1R and IGF1R_IR receptors. Over 26,000 phosphosites were quantified. Hierarchical clustering revealed 3208 significant differentially regulated phosphosites between IR and IGF1R. These included major differences of several large networks following ligand stimulation. Thus, many phosphosites associated with mTOR signaling were preferentially up-regulated by IR, whereas many phosphosites associated with Rho-GTPases and cell cycle were uniquely up-regulated by IGF1R. Interestingly, there were also many differences in the phosphoproteome between IR and IGF1R in the basal state indicating important differences in signaling by the unoccupied IR and IGF1R. These related to membrane trafficking, chromatin remodeling, Rho-GTPase signaling and cell cycle. When these data were compared to the phosphoproteome of cells expressing IR and IGF1R chimeric receptors, i.e., receptors with the extracellular domain (ECD) of IR fused to the intracellular domain (ICD) of IGF1R and vice versa, we found that many of the differences between IR and IGF1R in the ligand-stimulated state depended on differences in the ICD of IR vs. IGF1R, whereas many differences in the basal state depended on the ECD. Thus, despite high homology, IR and IGF1R regulate different networks of proteins in both the basal and stimulated states. Understanding these pathway differences provides the unique opportunity to differentially modulate insulin and IGF-1 signaling at the post-receptor level. Disclosure H. Nagao: None. W. Cai: None. T.M. Batista: None. H. Pan: None. J. Dreyfuss: None. C. Kahn: Advisory Panel; Self; ERX Pharmaceuticals, Kaleido Biosciences. Consultant; Self; AntriaBio, Flagship Pioneering, Sana-Cobalt.