Kinin B2 receptor-mediated bradykinin internalization and metalloendopeptidase EP24.15-dependent intracellular bradykinin degradation
Kinin B2 receptor-mediated bradykinin internalization and metalloendopeptidase EP24.15-dependent intracellular bradykinin degradation
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DOI：
10.1124/jpet.108.136911
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发表时间：
2008-07-01
影响因子：
3.5
通讯作者：
Leeb-Lundberg, L. M. Fredrik
中科院分区：
文献类型：
--
作者：
Sanden, Caroline;Enquist, Johan;Leeb-Lundberg, L. M. Fredrik
激肽是细胞外产生的有效促炎肽，并通过受体介导的内吞作用被细胞外肽酶和细胞内肽酶快速降解。在这项研究中，我们开发了单独或一起表达激肽B-2受体（B2 R）和金属内肽酶硫代寡肽酶（EC 3.4.24.15; EP 24.15）的模型细胞系统，以解决1）这些蛋白质之间的细胞和功能关系，以及2）通过B2 R内化缓激肽（BK）后EP 24.15参与BK代谢。B2 R几乎只定位于质膜，而EP 24.15定位于细胞内和细胞表面，并分泌在培养基中。胞内EP 24.15存在于整个细胞中，包括胞质和颗粒，具有较少的核定位，并且与内质网标记物钙连接蛋白或高尔基体标记物GM 130没有共定位。使用免疫荧光显微镜没有观察到B2 R和EP 24.15的直接共定位。然而，这两种蛋白质共免疫沉淀特异性，和EP 24.15衰减最大B2 R反应性，而不影响BK的效力，刺激磷酸肌醇水解和细胞内Ca 2+动员。细胞表面结合的BK在过表达EP 24.15的细胞中保持完整，但在B2 R介导的内吞作用后以EP 24.15依赖性方式在细胞内降解。这些结果表明，EP 24.15起到负调节B2 R反应性的作用，并且它在通过该受体特异性内化的BK的降解中充当细胞内肽酶。
Kinins are potent proinflammatory peptides that are produced extracellularly and are rapidly degraded by extracellular peptidases and by intracellular peptidases accessed by kinins via receptor-mediated endocytosis. In this study, we developed model cell systems expressing the kinin B-2 receptor (B2R) and the metalloendopeptidase thimet oligopeptidase (EC 3.4.24.15; EP24.15) either individually or together to address 1) the cellular and functional relationship between these proteins and 2) the participation of EP24.15 in the metabolism of bradykinin (BK) after BK internalization via B2R. B2R was localized almost exclusively in the plasma membrane, whereas EP24.15 was localized both intracellularly and on the cell surface and secreted in the media. Intracellular EP24.15 was present throughout the cell, both cytosolic and particulate, with less nuclear localization and no colocalization with either the endoplasmic reticulum marker calnexin or the Golgi marker GM130. No direct colocalization of B2R and EP24.15 was observed using immunofluorescence microscopy. However, the two proteins coimmunoprecipitated specifically, and EP24.15 attenuated maximal B2R responsiveness without influencing the potency of BK to stimulate phosphoinositide hydrolysis and intracellular Ca2+ mobilization. Cell surface-bound BK remained intact in cells overexpressing EP24.15 but was degraded intracellularly in an EP24.15-dependent manner upon B2R-mediated endocytosis. These results show that EP24.15 acts to negatively regulate B2R responsiveness, and it serves as an intracellular peptidase in the degradation of BK specifically internalized via this receptor.