Gonzalez-Villalobos Romer, Klassen R Bryan, Allen Patricia L, Navar L G, Hammond Timothy G
Tulane Hypertension and Renal Center of Excellence and Department of Physiology, Tulane University School of Medicine, New Orleans, LA 70112, USA.
Am J Physiol Renal Physiol. 2005 Feb;288(2):F420-7. doi: 10.1152/ajprenal.00243.2004. Epub 2004 Oct 5.
Megalin is an abundant membrane protein heavily involved in receptor-mediated endocytosis. The major functions of megalin in vivo remain incompletely defined as megalin typically faces specialized milieus such as glomerular filtrate, airways, epididymal fluid, thyroid colloid, and yolk sac fluid, which lack many of its known ligands. In the course of studies on ANG II internalization, we were surprised when only part of the uptake of labeled ANG II into immortalized yolk sac cells (BN-16 cells) was blocked by specific peptide inhibitors and direct competitors of the angiotensin type 1 receptor. This led us to test if megalin was a receptor for ANG II. Four lines of direct evidence demonstrate that megalin and, to a lesser extent, its chaperone protein cubilin are receptors for ANG II. First, in BN-16 cells anti-megalin and anti-cubilin antisera interfere with ANG II uptake. Second, also in BN-16 cells, pure ANG II competes for uptake of a known megalin ligand. Third, in proximal tubule cell brush-border membrane vesicles extracted from mice, anti-megalin antisera interfere with ANG II binding. Fourth, purified megalin binds ANG II directly in surface plasmon resonance experiments. The finding that megalin is a receptor for ANG II suggests a major new function for the megalin pathway in vivo. These results also indicate that ANG II internalization in some tissues is megalin dependent and that megalin may play a role in regulating proximal tubule ANG II levels.
巨膜蛋白是一种丰富的膜蛋白，在受体介导的内吞作用中起重要作用。由于巨膜蛋白通常面对的是特殊的微环境，如肾小球滤液、气道、附睾液、甲状腺胶体和卵黄囊液，这些微环境中缺乏许多已知的配体，因此其在体内的主要功能仍未完全明确。在研究血管紧张素II（ANG II）内化的过程中，我们惊讶地发现，在永生化卵黄囊细胞（BN - 16细胞）中，只有部分标记的ANG II摄取被血管紧张素1型受体的特异性肽抑制剂和直接竞争剂所阻断。这促使我们测试巨膜蛋白是否是ANG II的受体。四条直接证据表明，巨膜蛋白及其伴侣蛋白 cubilin 在较小程度上是ANG II的受体。首先，在BN - 16细胞中，抗巨膜蛋白和抗cubilin抗血清会干扰ANG II的摄取。其次，同样在BN - 16细胞中，纯ANG II会竞争已知巨膜蛋白配体的摄取。第三，在从小鼠提取的近端肾小管细胞刷状缘膜囊泡中，抗巨膜蛋白抗血清会干扰ANG II的结合。第四，在表面等离子体共振实验中，纯化的巨膜蛋白直接结合ANG II。巨膜蛋白是ANG II受体这一发现提示了巨膜蛋白途径在体内的一个主要新功能。这些结果还表明，某些组织中ANG II的内化依赖于巨膜蛋白，并且巨膜蛋白可能在调节近端肾小管ANG II水平中发挥作用。
