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An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor.

Roxrud I, Raiborg C, Pedersen NM, Stang E, Stenmark H - J. Cell Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biomedicine, University of Oslo and the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

ABSTRACT
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.

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Eps15b is an isoform of Eps15. (A) The EPS15 gene contains 25 annotated exons. Eps15 is produced from exons 1–25, whereas the Eps15b isoform originates from a transcriptional initiation in intron 12 continuing through exons 13–25. (B) The Eps15 protein contains several functional protein domains: three EH domains, a coiled-coil region, a DPF repeat domain, and two consecutive UIMs. The Eps15b isoform lacks the three EH domains and contains a short unique N-terminal region. The remaining part of the protein is identical to Eps15 and contains major parts of the coiled coil region in addition to the DPF repeat domain and the UIM motives. (C) Multiple sequence alignment of the predicted protein sequences for the putative Eps15b homologues in Homo sapiens (BX647676), Macaca fascicularis (AB172746), Mus musculus (BAC38796), Gallus gallus (CR407454), and Bos taurus (DV911715) compared with Eps15 from H. sapiens. The sequences in blue and red represent the N-terminal 32 Eps15b-specific sequences (conserved residues are shown in blue and unconserved residues in red), whereas the sequence in black indicates where Eps15b aligns with the Eps15 sequence. The asterisks indicate fully conserved amino acid residues in the Eps15b-specific region.
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fig2: Eps15b is an isoform of Eps15. (A) The EPS15 gene contains 25 annotated exons. Eps15 is produced from exons 1–25, whereas the Eps15b isoform originates from a transcriptional initiation in intron 12 continuing through exons 13–25. (B) The Eps15 protein contains several functional protein domains: three EH domains, a coiled-coil region, a DPF repeat domain, and two consecutive UIMs. The Eps15b isoform lacks the three EH domains and contains a short unique N-terminal region. The remaining part of the protein is identical to Eps15 and contains major parts of the coiled coil region in addition to the DPF repeat domain and the UIM motives. (C) Multiple sequence alignment of the predicted protein sequences for the putative Eps15b homologues in Homo sapiens (BX647676), Macaca fascicularis (AB172746), Mus musculus (BAC38796), Gallus gallus (CR407454), and Bos taurus (DV911715) compared with Eps15 from H. sapiens. The sequences in blue and red represent the N-terminal 32 Eps15b-specific sequences (conserved residues are shown in blue and unconserved residues in red), whereas the sequence in black indicates where Eps15b aligns with the Eps15 sequence. The asterisks indicate fully conserved amino acid residues in the Eps15b-specific region.

Mentions: The finding that Eps15 proteins localize to two distinct membranes begged the question of whether the form of Eps15 residing in bilayered clathrin coats on endosomes might be distinct from the canonical clathrin-coated pit-associated form. Indeed, in their initial characterization of Eps15, Fazioli et al. (1993) identified several protein species recognized by Eps15 antibodies and introduced the idea of possible isoforms of Eps15. Through searches in public databases, we identified a putative isoform of Eps15 on the basis of a full-length cDNA sequence (GenBank/EMBL/DDBJ accession no. BX647676; isolated from a human colon endothelial primary cell culture) and named it Eps15b (Fig. 2 A). The EPS15B sequence comprises exons 13–25 of the EPS15 gene and upstream 245 additional nucleotides. These 245 nucleotides align with the 3′ end of intron 12 according to the genomic sequence of EPS15 and we consequently defined this region of intron 12 as an alternative transcriptional initiation exon. These 245 nucleotides form the 5′ end of the EPS15B isoform and suggest that an alternative promoter and transcriptional start site (TSS) exists in intron 12. The DataBase of Human Transcription Start Sites (Suzuki et al., 2004), containing large-scale information on TSSs based on experimentally determined TSSs of human genes, identifies several TSSs in the ±30 nucleotide vicinity of the EPS15B start site, which suggests that this region is an active site of transcriptional initiation.


An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor.

Roxrud I, Raiborg C, Pedersen NM, Stang E, Stenmark H - J. Cell Biol. (2008)

Eps15b is an isoform of Eps15. (A) The EPS15 gene contains 25 annotated exons. Eps15 is produced from exons 1–25, whereas the Eps15b isoform originates from a transcriptional initiation in intron 12 continuing through exons 13–25. (B) The Eps15 protein contains several functional protein domains: three EH domains, a coiled-coil region, a DPF repeat domain, and two consecutive UIMs. The Eps15b isoform lacks the three EH domains and contains a short unique N-terminal region. The remaining part of the protein is identical to Eps15 and contains major parts of the coiled coil region in addition to the DPF repeat domain and the UIM motives. (C) Multiple sequence alignment of the predicted protein sequences for the putative Eps15b homologues in Homo sapiens (BX647676), Macaca fascicularis (AB172746), Mus musculus (BAC38796), Gallus gallus (CR407454), and Bos taurus (DV911715) compared with Eps15 from H. sapiens. The sequences in blue and red represent the N-terminal 32 Eps15b-specific sequences (conserved residues are shown in blue and unconserved residues in red), whereas the sequence in black indicates where Eps15b aligns with the Eps15 sequence. The asterisks indicate fully conserved amino acid residues in the Eps15b-specific region.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2373575&req=5

fig2: Eps15b is an isoform of Eps15. (A) The EPS15 gene contains 25 annotated exons. Eps15 is produced from exons 1–25, whereas the Eps15b isoform originates from a transcriptional initiation in intron 12 continuing through exons 13–25. (B) The Eps15 protein contains several functional protein domains: three EH domains, a coiled-coil region, a DPF repeat domain, and two consecutive UIMs. The Eps15b isoform lacks the three EH domains and contains a short unique N-terminal region. The remaining part of the protein is identical to Eps15 and contains major parts of the coiled coil region in addition to the DPF repeat domain and the UIM motives. (C) Multiple sequence alignment of the predicted protein sequences for the putative Eps15b homologues in Homo sapiens (BX647676), Macaca fascicularis (AB172746), Mus musculus (BAC38796), Gallus gallus (CR407454), and Bos taurus (DV911715) compared with Eps15 from H. sapiens. The sequences in blue and red represent the N-terminal 32 Eps15b-specific sequences (conserved residues are shown in blue and unconserved residues in red), whereas the sequence in black indicates where Eps15b aligns with the Eps15 sequence. The asterisks indicate fully conserved amino acid residues in the Eps15b-specific region.
Mentions: The finding that Eps15 proteins localize to two distinct membranes begged the question of whether the form of Eps15 residing in bilayered clathrin coats on endosomes might be distinct from the canonical clathrin-coated pit-associated form. Indeed, in their initial characterization of Eps15, Fazioli et al. (1993) identified several protein species recognized by Eps15 antibodies and introduced the idea of possible isoforms of Eps15. Through searches in public databases, we identified a putative isoform of Eps15 on the basis of a full-length cDNA sequence (GenBank/EMBL/DDBJ accession no. BX647676; isolated from a human colon endothelial primary cell culture) and named it Eps15b (Fig. 2 A). The EPS15B sequence comprises exons 13–25 of the EPS15 gene and upstream 245 additional nucleotides. These 245 nucleotides align with the 3′ end of intron 12 according to the genomic sequence of EPS15 and we consequently defined this region of intron 12 as an alternative transcriptional initiation exon. These 245 nucleotides form the 5′ end of the EPS15B isoform and suggest that an alternative promoter and transcriptional start site (TSS) exists in intron 12. The DataBase of Human Transcription Start Sites (Suzuki et al., 2004), containing large-scale information on TSSs based on experimentally determined TSSs of human genes, identifies several TSSs in the ±30 nucleotide vicinity of the EPS15B start site, which suggests that this region is an active site of transcriptional initiation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biomedicine, University of Oslo and the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus