Serotonin-, Protein Kinase C-, and Hic-5-associated Redistribution of the Platelet Serotonin Transporter

Ana Marin D Carneiro,Randy D Blakely

doi:10.1074/jbc.m603877200

Abstract

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.

Highlights

serotonin (5HT) transporter (SERT) are targets of multiple psychostimulants and likely play a critical role in the mechanisms of substance abuse (8 –10)
Most of the active plasma membrane SERT is associated with the membrane skeleton, whereas SERT appears to associate with the dense actin-cytoskeleton during inactivation and internalization
Antibodies used for immunoprecipitation as we recovered SERT equivalently when cytoskeletal pellets (CS) and membrane skeleton (MS) sample input was balanced for SERT abundance. These findings indicate that under basal conditions, the bulk of platelet SERT resides in intracellular, TS membranes, whereas the fraction of SERT that is resident at the plasma membrane is enriched in the MS fraction

Summary

Regulated Redistribution of Platelet Serotonin Transporter

Specific binding to a particular membrane skeleton/cytoskeleton may be controlled by phosphorylation [17]. Recent reports show that cell surface levels of SERT can be modified by protein kinase G (PKG) activation and p38 MAPK inactivation [36]. These initial studies suggest that the macromolecular organization of SERT protein complexes is differentially linked to specific SERT-associated membrane subdomains that in turn dictate SERT activity or mobility, and 5HT clearance capacity. We hypothesize that SERT inactivation and internalization is mediated by protein-protein associations that lead to active movement of the transporter through membrane skeleton/cytoskeleton domains. Most of the active plasma membrane SERT is associated with the membrane skeleton, whereas SERT appears to associate with the dense actin-cytoskeleton during inactivation and internalization. The interaction of Hic-5 with SERT can inactivate the transporter and facilitate SERT internalization through by promoting interactions with the actin cytoskeleton

EXPERIMENTAL PROCEDURES

RESULTS

Findings

DISCUSSION