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Emerging data indicate the existence of multiple regulatory processes supporting serotonin (5HT) transporter (SERT) capacity including regulated trafficking and catalytic activation, influenced by post-translational modifications and transporter-associated proteins. In the present study, using differential extraction and sedimentation procedures optimized for the purification of cytoskeletal and membrane-skeletal associated proteins, we analyze SERT localization in platelets. We find that most of the plasma membrane SERT is associated with the membrane skeleton. This association can be enhanced by both transporter activation and 5HT2A receptor activation. Inactivation of transport activity by phorbol ester treatment of intact platelets relocates SERT to the cytoskeleton fraction, consequently leading to transporter internalization. The translocation of SERT between these compartments is correlated with changes in the interaction with the LIM domain adaptor protein Hic-5. Co-immunoprecipitation and uptake activity studies suggest that Hic-5 is a determinant of transporter inactivation and relocation to a compartment subserving endocytic regulation. Associations of SERT with Hic-5 are evident in brain synaptosomes, suggesting the existence of parallel mechanisms operating to regulate SERT at serotonergic synapses.