From the Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065 and.
Department of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Center for Alzheimer Research, Karolinska Institutet, 141 57 Huddinge, Sweden.
J Biol Chem. 2017 Dec 29;292(52):21623-21630. doi: 10.1074/jbc.M116.758961. Epub 2017 Nov 3.
How an optimal level of human dopamine D4 receptor (hD4R) is maintained in synaptic membranes is not known. We show here that hD4R is ubiquitinated in primary neurons. We go on to show that ubiquitin is attached to hD4R through isopeptide and ester bonds. When lysine (Lys) residues of the hD4R are substituted with arginine (Arg) residues, cellular hD4R protein levels increase. A synergistic effect on hD4R levels is noted when cytoplasmic serine (Ser) and threonine (Thr) residues are mutated. Chloroquine, an inhibitor of lysosomal degradation, did not have an effect on hD4R protein levels. However, treatment with bortezomib, an inhibitor of the 20S proteasome, caused a dose-dependent increase in hD4R protein levels. The effect of bortezomib was attenuated in the receptor variants that lacked Lys or Ser/Thr residues, and the hD4R mutant that lacked 17 cytoplasmic Lys, Ser, and Thr residues was nearly insensitive to bortezomib treatment. We conclude that both isopeptide and ester bond ubiquitination regulate proteasomal degradation of hD4R.
目前尚不清楚突触膜中多巴胺 D4 受体（hD4R）的最佳水平是如何维持的。我们在这里表明 hD4R 在原代神经元中发生泛素化。我们接着表明，通过异肽键和酯键将泛素连接到 hD4R 上。当 hD4R 的赖氨酸（Lys）残基被精氨酸（Arg）残基取代时，细胞内 hD4R 蛋白水平增加。当细胞质丝氨酸（Ser）和苏氨酸（Thr）残基发生突变时，对 hD4R 水平有协同作用。氯喹是溶酶体降解的抑制剂，对 hD4R 蛋白水平没有影响。然而，蛋白酶体 20S 抑制剂硼替佐米的处理导致 hD4R 蛋白水平呈剂量依赖性增加。在缺乏 Lys 或 Ser/Thr 残基的受体变体中，硼替佐米的作用减弱，并且缺乏 17 个细胞质 Lys、Ser 和 Thr 残基的 hD4R 突变体对硼替佐米处理几乎不敏感。我们的结论是，异肽键和酯键泛素化都调节 hD4R 的蛋白酶体降解。
