<p>Insulin receptor (IR) is a tyrosine kinase involved in vital cellular signaling processes related to glucose metabolism and cell growth. Detailed understanding of molecular mechanisms of IR signaling is important for improved treatments of diabetes and related disorders. Although many aspects of IR signaling have over the past years been elucidated, much of the available information comes from studies made in vitro, using protein preparations. However, observations performed directly in living cells, tissues and organisms are needed for proper understanding of IR signaling. To address this need, we decided to investigate GP2(1)-eGFP-CD59, a recently identified FLIP biosensor of IR conformation that takes advantage of the powerful yet underutilized technique of linear dichroism fluorescence microscopy. Our approach allowed us to detect, in living cells, changes in the IR ectodomain in response to IR agonists and inhibitors. Surprisingly, we observed rearrangements in the ectodomain even upon administering an IR kinase inhibitor. Our findings suggest that the state of the intracellular kinase domain plays a role in the stability/conformation of the ectodomain, and urge caution in using solely the ectodomain in structural studies. Furthermore, our results open doors to sensitive imaging of various aspects of IR function in living cells, tissues, and ultimately whole organisms, under conditions close to natural.</p>