The neuropeptide Y (NPY) plays numerous biological roles that are mediated by a family of G-protein-coupled receptors. Among the latter, the NPY Y subtype receptor undergoes a rapid desensitization following agonist exposure. This desensitization was suggested to result from a rapid clathrin-dependent internalization of Y and its recycling at the plasma membrane via sorting/early endosomes (SE/EE) and recycling endosomes (RE). Herein, to validate and quantitatively consolidate the mechanism of NPY internalization, we quantitatively investigated the NPY-induced internalization of the Y receptor by direct stochastic optical reconstruction microscopy (dSTORM), a super-resolution imaging technique that can resolve EE and SE, which are below the resolution limit of conventional optical microscopes. Using Cy5-labeled NPY, we could monitor with time the internalization and recycling of NPY on HEK293 cells stably expressing eGFP-labeled Y receptors. Furthermore, by discriminating the SE/EE from the larger RE by their sizes and monitoring these two populations as a function of time, we could firmly consolidate the kinetic model describing the internalization mechanism of the Y receptors as the basis for their rapid desensitization following agonist exposure.
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