Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Sci Adv. 2024 Nov 22;10(47):eadq9793. doi: 10.1126/sciadv.adq9793.
Monoamine transporters function in neuronal membranes to control extracellular concentrations of their substrates. Cell-surface expression of transporters is regulated by substrates and intracellular signaling, but the underlying mechanisms remain unclear. Here, we found that substrates of the dopamine transporter (DAT), amphetamine and dopamine, synergize with protein kinase C (PKC)-dependent DAT ubiquitination to markedly elevate clathrin-mediated endocytosis of DAT, which is accompanied by DAT movement out of plasma membrane protrusions with a negative curvature. Disruption of the outward-open (OO) DAT conformation or its stabilization in the inward-open (IO) conformation recapitulates substrate effects on DAT endocytosis. Amphetamine strongly increases PKC-dependent endocytosis of norepinephrine transporter (NET) but not of serotonin transporter (SERT), correlating with a substantially weaker ubiquitination of SERT compared to NET. We propose a "shape-transition" model whereby shifting from convex-shaped OO conformers to IO conformers minimizes retention of transporters in negatively curved membranes, which facilitates their PKC-dependent ubiquitination and recruitment to positively invaginated clathrin-coated membranes, driving robust transporter endocytosis.
单胺转运体在神经元膜中发挥作用，以控制其底物的细胞外浓度。转运体的细胞表面表达受底物和细胞内信号调节，但潜在的机制尚不清楚。在这里，我们发现多巴胺转运体（DAT）的底物安非他命和多巴胺与蛋白激酶 C（PKC）依赖性 DAT 泛素化协同作用，显著增加 DAT 的网格蛋白介导内吞作用，这伴随着 DAT 从具有负曲率的质膜突出部移出。破坏外向开放（OO）DAT 构象或其向内开放（IO）构象的稳定可再现底物对 DAT 内吞作用的影响。安非他命强烈增加去甲肾上腺素转运体（NET）但不是 5-羟色胺转运体（SERT）的 PKC 依赖性内吞作用，与 SERT 的泛素化明显弱于 NET 相关。我们提出了一个“形状转换”模型，即从凸形 OO 构象体转变为 IO 构象体，可最大限度地减少转运体在负曲率膜中的保留，从而促进其 PKC 依赖性泛素化，并募集到正向凹陷的网格蛋白包被的膜中，从而驱动转运体的强大内吞作用。
