Molecular mechanism of regulation of growth factor receptor sorting at endosomes

在生长因子与其细胞表面受体结合后，生长因子/受体复合物被内化并转运至早期内体。在早期的内体中分选后，复合物被进一步运输到溶酶体并降解。泛素与受体的结合作为转运至溶酶体的分选信号。在这项研究中，我们研究了去泛素化酶UBPY和ALG 2在调节内体生长因子受体分选中的作用，并获得了以下结果。（1）UBPY过表达可降低EGF受体（EGFR）的泛素化水平，延缓EGF刺激后EGFR的降解。Hrs的过表达引起内源性UBPY在夸张的内体上的积累。无催化活性的UBPY突变体明显定位于内体，当细胞用EGF刺激时，它与EGFR重叠。通过RNA干扰消除内源性UBPY导致EGF激活的EGFR的泛素化升高和加速降解。这些结果表明，UBPY通过使内体上的EGFR去泛素化来负调节EGFR降解速率。(2)At在核内体中，配体激活的受体被掺入到从其界膜向内出芽的核内体的腔囊泡中。含有这种腔囊泡的内体被称为多泡体（MVB）。我们研究了ALG 2在腔囊泡形成中的作用。通过RNA干扰消耗内源性ALG 2导致磷脂LBPA水平的降低，磷脂LBPA特异性地定位于腔囊泡的膜。通过电子显微镜分析也观察到这种减少。这些结果表明ALG 2在MVB腔囊泡的形成中起重要作用。

Upon binding of growth factors to their cell surface receptors, the growth factor/receptor complexes are internalized and transported to early endosomes. After sorted at early endosomes, the complexes are further transported to lysosome and degraded. Conjugation of ubiquitin to the receptors serves as a sorting signal for transport to lysosomes. In this study, we examined the role of the deubiquitinating enzyme UBPY and ALG2 in regulation of growth factor receptor sorting at endosomes, and the following results were obtained.(1)Overexpression of UBPY reduced the ubiquitination level of EGF receptor (EGFR), and delayed its degradation in EGF-stimulated cells. Overexpression of Hrs caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. These results suggest that UBPY negatively regulates the rate of EGFR degradation by deubiquitinating EGFR on endosomes.(2)At endosomes, ligand-activated receptors are incorporated into luminal vesicles of endosomes that bud inward from its limiting membrane. Endosomes that contain such luminal vesicles are called the multivesicular body (MVB). We examined the role of ALG2 in the formation of the luminal vesicles. Depletion of endogenous ALG2 by RNA interference resulted in the reduction of the level of phospholipid LBPA, which is specifically localized to the membrane of the luminal vesicles. This reduction was also observed by an electron microscopy analysis. These results suggest thatALG2 plays an important role in the formation of the luminal vesicles in MVB.