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Papers In Press, published online ahead of print February 19, 2004
Physiology and Neuroscience, Medical University of South Carolina, Charleston, SC 29464
Corresponding Author: jayanthi{at}musc.edu
The effects of norepinephrine (NE) in the brain and periphery are terminated primarily by active reuptake of the catecholamine via cocaine- and amphetamine- sensitive norepinephrine transporters (NETs). Activation of protein kinase C (PKC) down-regulates NET by sequestering it from the plasma membrane although the underlying mechanism is not yet known. Previously, we showed robust expression of endogenous NETs in rat placental trophoblasts (Jayanthi et al, 2002, Br. J. Pharmacol. 135, 1927-1934). Here, we report a significant reduction in native NET function and surface expression in these cells following phorbol-ester (ß-PMA)-treatment. The ß-PMA-mediated down-regulation of NET occurs by a rapid sequestration of NETs from the plasma membrane and is calcium-independent. Reversible biotinylation experiments revealed a significant enhancement of NET endocytosis following ß-PMA-treatment. Chemical treatments and expression of dominant negative mutants of dynamin 1 and 2 failed to prevent the ß-PMA effect suggesting a clathrin-independent pathway. In contrast, treatment with the cholesterol-disrupting agent filipin, that blocks caveolae/lipid-raft mediated internalization, completely blocked the ß-PMA-mediated NET-sequestration. Discontinuous sucrose density-gradient centrifugation revealed NET in the lipid-raft fractions. Following ß-PMA-treatment, there was reduced NET levels in the lipid-raft fractions suggesting that cholesterol-rich lipid-rafts mediate PKC-triggered NET-internalization. Metabolic labeling and immunoprecipitation studies revealed that NET phosphorylation is stimulated several fold by PKC-activation and protein- phosphatase 1/2A inhibition. Together, these findings, demonstrate for the first time, that in trophoblasts, (i) PKC activation regulates NET function and surface expression by an enhanced internalization process that is lipid-raft mediated and (ii) PKC and protein-phosphatase(s) modulation regulates NET phosphorylation.
J. Biol. Chem, 10.1074/jbc.M311172200
Submitted on October 10, 2003
Revised on January 29, 2004
Accepted on February 19, 2004
Regulated internalization and phosphorylation of the native norepinephrine transporter in response to phorbol esters: Evidence for localization in lipid rafts and lipid raft mediated internalization
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