CONFORMATIONAL-CHANGES OF THE INSULIN-RECEPTOR UPON INSULIN BINDING AND ACTIVATION AS MONITORED BY FLUORESCENCE SPECTROSCOPY

We have characterized the changes in intrinsic fluorescence that the i nsulin receptor undergoes upon ligand binding and autophosphorylation. The binding of insulin to its receptor results in an increase in the receptor's fluorescence intensity, emission energy and anisotropy. We monitored the time course of the anisotropy change, and these data, co upled with studies monitoring the energy transfer from insulin recepto r tryptophan donors to a fluorescent-labeled insulin, allowed us to co nclude that the change in anisotropy is due to a conformational change in the receptor induced by hormone binding. Since insulin association is very fast, the time course also allowed us to estimate the slower rate of formation of this conformationally-altered state. The time cou rse of receptor autophosphorylation was measured under similar conditi ons and was found to be similar to the ligand-induced anisotropy time course. The simultaneous use of two fluorescent-labeled insulin analog s also allowed us to assess the maximum distance between the two hormo nes bound to the receptor. Addition of ATP produces a large, seemingly instantaneous increase in anisotropy. Our observation that ATP binds to the insulin receptor in the presence and absence of insulin support s the idea that the conformational change produced by insulin binding increases the rate of autophosphorylation rather than increases ATP af finity. A suggested model for these changes is presented.