Mechanisms in thyroid eye disease - the TSH receptor interacts directly with the IGF-1 receptor Abstract: The pathogenesis of Thyroid Eye Disease (TED) has been suggested as due to signal enhancement in orbital fibroblasts as a result of autoantibody-induced, synergistic, interaction between the TSH receptor (TSHR) and the IGF-1 receptor (IGF-1R). This interaction has been explained by a "receptor cross talk", mediated via β-arrestin binding. Here, we have examined if this interaction can be mediated via direct receptor contact using modeling and experimental approaches. First, we docked a model of the leucine rich domain (LRD) of the TSHR ectodomain (ECD) to an available cryo-EM based structure of the active-state IGF-1R dimer and demonstrated the stability of the complex using molecular dynamics (MD) simulations. We then extended the complex with the full-length TSHR and the transmembrane helices of the IGF1R and a 3000 ns simulation also showed stability of this complex. We then performed co-immunoprecipitation studies with anti-TSHR and anti-IGF-1R antibodies using cells expressing the IGF-1R and the full-length TSHR and also cells that expressed the IGF-1R and only the TSHR-ECD and, therefore, unable to bind β-arrestin. These studies showed a 360 kD complex protein in the immunoprecipitation which was present in both the full-length TSHR and the TSHR-ECD-only expressing cells evidencing a direct interaction of receptors via their ectodomains in the absence of arrestin. Co-localized staining of TSHR and IGF1R in the TSHR-ECD cells further supported this direct interaction. These data showed that the TSHR and IGF-1R can interact directly and in the absence of β-arrestin binding. Understanding these interactions is important in the pathogenesis of TED and its therapeutic intervention.