Roxrud Ingrid, Raiborg Camilla, Pedersen Nina Marie, Stang Espen, Stenmark Harald
Centre for Cancer Biomedicine, University of Oslo and the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
J Cell Biol. 2008 Mar 24;180(6):1205-18. doi: 10.1083/jcb.200708115.
Down-regulation of activated and ubiquitinated growth factor (GF) receptors by endocytosis and subsequent lysosomal degradation ensures attenuation of GF signaling. The ubiquitin-binding adaptor protein Eps15 (epidermal growth factor receptor [EGFR] pathway substrate 15) functions in endocytosis of such receptors. Here, we identify an Eps15 isoform, Eps15b, and demonstrate its expression in human cells and conservation across vertebrate species. Although both Eps15 and Eps15b interact with the endosomal sorting protein Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in vitro, we find that Hrs specifically binds Eps15b in vivo (whereas adaptor protein 2 preferentially interacts with Eps15). Although Eps15 mainly localizes to clathrin-coated pits at the plasma membrane, Eps15b localizes to Hrs-positive microdomains on endosomes. Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect. Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR. These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex.
通过内吞作用及随后的溶酶体降解来下调活化的和泛素化的生长因子（GF）受体，可确保GF信号传导的减弱。泛素结合衔接蛋白Eps15（表皮生长因子受体[EGFR]途径底物15）在这类受体的内吞作用中发挥作用。在此，我们鉴定出一种Eps15异构体Eps15b，并证明其在人类细胞中的表达以及在脊椎动物物种中的保守性。尽管Eps15和Eps15b在体外均与内体分选蛋白Hrs（肝细胞生长因子调节的酪氨酸激酶底物）相互作用，但我们发现Hrs在体内特异性结合Eps15b（而衔接蛋白2优先与Eps15相互作用）。尽管Eps15主要定位于质膜上的网格蛋白包被小窝，但Eps15b定位于内体上Hrs阳性微区。与Hrs过表达类似，Eps15b过表达会抑制配体介导的EGFR降解，而Eps15则无此作用。同样，Eps15b而非Eps15的缺失会延迟EGFR的降解并促进其回收。这些结果表明，Eps15b是Eps15的一种定位于内体的异构体，它通过与Hrs直接相互作用存在于Hrs复合物中，并且对该复合物的分选功能很重要。
