Litterst Claudia, Georgakopoulos Anastasios, Shioi Junichi, Ghersi Enrico, Wisniewski Thomas, Wang Rong, Ludwig Andreas, Robakis Nikolaos K
Department of Psychiatry and Neuroscience, Mount Sinai School of Medicine New York University, New York, NY 10029, USA, and Institute for Molecular Cardiovascular Research, University Hospital Reinisch-West Faelische Technische Hochschule, Aachen, Germany.
J Biol Chem. 2007 Jun 1;282(22):16155-63. doi: 10.1074/jbc.M611449200. Epub 2007 Apr 10.
Binding of EphB receptors to ephrinB ligands on the surface of adjacent cells initiates signaling cascades that regulate angiogenesis, axonal guidance, and neuronal plasticity. These functions require processing of EphB receptors and removal of EphB-ephrinB complexes from the cell surface, but the mechanisms involved are poorly understood. Here we show that the ectodomain of EphB2 receptor is released to extracellular space following cleavage after EphB2 residue 543. The remaining membrane-associated fragment is cleaved by the presenilin-dependent gamma-secretase activity after EphB2 residue 569 releasing an intracellular peptide that contains the cytoplasmic domain of EphB2. This cleavage is inhibited by presenilin 1 familial Alzheimer disease mutations. Processing of EphB2 receptor depends on specific treatments: ephrinB ligand-induced processing requires endocytosis, and the ectodomain cleavage is sensitive to peptide inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal but insensitive to metalloproteinase inhibitor GM6001. The ligand-induced processing takes place in endosomes and involves the rapid degradation of the extracellular EphB2. EphrinB ligand stimulates ubiquitination of EphB2 receptor. Calcium influx- and N-methyl-d-aspartic acid-induced processing of EphB2 is inhibited by GM6001 and ADAM10 inhibitors but not by N-benzyloxycarbonyl-Val-Leu-leucinal. This processing requires no endocytosis and promotes rapid shedding of extracellular EphB2, indicating that it takes place at the plasma membrane. Our data identify novel cleavages and modifications of EphB2 receptor and indicate that specific conditions determine the proteolytic systems and subcellular sites involved in the processing of this receptor.
EphB受体与相邻细胞表面的ephrinB配体结合，启动调节血管生成、轴突导向和神经元可塑性的信号级联反应。这些功能需要对EphB受体进行加工处理，并从细胞表面去除EphB-ephrinB复合物，但其中涉及的机制尚不清楚。在这里，我们表明EphB2受体的胞外域在EphB2第543位残基处裂解后释放到细胞外空间。剩余的膜相关片段在EphB2第569位残基后被早老素依赖的γ-分泌酶活性裂解，释放出一个包含EphB2胞质域的细胞内肽段。这种裂解被早老素1家族性阿尔茨海默病突变所抑制。EphB2受体的加工处理取决于特定的处理方式：ephrinB配体诱导的加工处理需要内吞作用，胞外域裂解对肽抑制剂N-苄氧羰基-Val-Leu-亮氨醛敏感，但对金属蛋白酶抑制剂GM6001不敏感。配体诱导的加工处理发生在内体中，并涉及细胞外EphB2的快速降解。EphrinB配体刺激EphB2受体的泛素化。钙内流和N-甲基-D-天冬氨酸诱导的EphB2加工处理被GM6001和ADAM10抑制剂抑制，但不被N-苄氧羰基-Val-Leu-亮氨醛抑制。这种加工处理不需要内吞作用，并促进细胞外EphB2的快速脱落，表明它发生在质膜上。我们的数据确定了EphB2受体的新裂解和修饰，并表明特定条件决定了参与该受体加工处理的蛋白水解系统和亚细胞位点。
