Jayanthi Lankupalle D, Samuvel Devadoss J, Ramamoorthy Sammanda
Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2004 Apr 30;279(18):19315-26. doi: 10.1074/jbc.M311172200. Epub 2004 Feb 19.
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.
去甲肾上腺素在大脑和外周的作用主要通过可卡因和苯丙胺敏感的去甲肾上腺素转运体（NETs）对儿茶酚胺的主动重摄取而终止。蛋白激酶C（PKC）的激活通过将NET从质膜中隔离来下调NET，尽管其潜在机制尚不清楚。此前，我们在大鼠胎盘滋养层细胞中发现了内源性NETs的强烈表达（Jayanthi, L. D., Vargas, G., and DeFelice, L. J. (2002) Br. J. Pharmacol. 135, 1927 - 1934）。在此，我们报告佛波酯（β - PMA）处理后这些细胞中天然NET功能和表面表达显著降低。β - PMA介导的NET下调通过NET从质膜的快速隔离发生，且不依赖钙。可逆生物素化实验显示β - PMA处理后NET内吞作用显著增强。用化学试剂处理以及表达发动蛋白1和2的显性负性突变体均未能阻止β - PMA的作用，提示这是一条不依赖网格蛋白的途径。相反，用破坏胆固醇的试剂制霉菌素处理，其可阻断小窝/脂筏介导的内化作用，完全阻断了β - PMA介导的NET隔离。不连续蔗糖密度梯度离心显示脂筏组分中有NET。β - PMA处理后，脂筏组分中的NET水平降低，提示富含胆固醇的脂筏介导PKC触发的NET内化。代谢标记和免疫沉淀研究显示，PKC激活和蛋白磷酸酶1/2A抑制可使NET磷酸化增加数倍。总之，这些发现首次证明在滋养层细胞中：（i）PKC激活通过脂筏介导的增强内化过程调节NET功能和表面表达；（ii）PKC和蛋白磷酸酶调节NET磷酸化。
