Belouzard Sandrine, Rouillé Yves
Centre National de la Recherche Scientifique, Unité Propre de Recherche 2511, Institut Pasteur de Lille, Lille Cedex, France.
EMBO J. 2006 Mar 8;25(5):932-42. doi: 10.1038/sj.emboj.7600989. Epub 2006 Feb 16.
Leptin receptors are constitutively endocytosed in a ligand-independent manner. To study their endocytosis, leptin receptors OB-Ra and OB-Rb were expressed in HeLa cells. Both receptor isoforms were ubiquitylated, internalized by clathrin-mediated endocytosis and transported to Hrs-positive endosomes after their internalization. Proteasome inhibitors inhibited OB-Ra but not OB-Rb internalization from the cell surface. OB-Ra ubiquitylation occurred on lysine residues K877 and K889 in the cytoplasmic tail, the mutation of which abolished OB-Ra internalization. Fusion of an ubiquitin molecule at the C-terminus of an OB-Ra construct defective both in ubiquitylation and endocytosis restored clathrin-dependent endocytosis of the receptor. The internalization of this constitutively mono-ubiquitylated construct was no longer sensitive to proteasome inhibitors, which inhibited OB-Ra endocytosis by blocking its ubiquitylation. Fusion of an ubiquitin molecule to a transferrin receptor deleted from its own endocytosis motif restored clathrin-mediated endocytosis. We propose that mono-ubiquitin conjugates act as internalization motifs for clathrin-dependent endocytosis of leptin receptor OB-Ra.
瘦素受体以不依赖配体的方式持续进行内吞作用。为了研究它们的内吞作用，瘦素受体OB-Ra和OB-Rb在HeLa细胞中表达。两种受体亚型均被泛素化，通过网格蛋白介导的内吞作用内化，并在其内化后转运至Hrs阳性的内体。蛋白酶体抑制剂抑制OB-Ra从细胞表面的内化，但不抑制OB-Rb。OB-Ra的泛素化发生在细胞质尾巴中的赖氨酸残基K877和K889上，其突变消除了OB-Ra的内化。在泛素化和内吞作用均有缺陷的OB-Ra构建体的C末端融合一个泛素分子，可恢复该受体的网格蛋白依赖性内吞作用。这种组成性单泛素化构建体的内化不再对蛋白酶体抑制剂敏感，蛋白酶体抑制剂通过阻断OB-Ra的泛素化来抑制其内吞作用。将一个泛素分子融合到从其自身内吞基序缺失的转铁蛋白受体上，可恢复网格蛋白介导的内吞作用。我们提出，单泛素缀合物作为瘦素受体OB-Ra的网格蛋白依赖性内吞作用的内化基序。
