The effects of norepinephrine 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, L. D., Vargas, G., and DeFelice, L. J. (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 (beta-PMA) treatment. The beta-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 beta-PMA treatment. Chemical treatments and expression of dominant negative mutants of dynamin 1 and 2 failed to prevent the beta-PMA effect, suggesting a clathrin-independent pathway. In contrast, treatment with the cholesterol-disrupting agent filipin, which blocks caveolae/lipid raft-mediated internalization, completely blocked the beta-PMA-mediated NET sequestration. Discontinuous sucrose density gradient centrifugation revealed NET in the lipid raft fractions. Following beta-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 severalfold 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.