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EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking.

Mills IG, Praefcke GJ, Vallis Y, Peter BJ, Olesen LE, Gallop JL, Butler PJ, Evans PR, McMahon HT - J. Cell Biol. (2003)

Bottom Line: Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively.Thus, potentially, two AP1 complexes can bind to one epsinR.This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.

ABSTRACT
EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

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The ENTH domain of epsinR. (A) The ENTH domain of epsinR was modeled on epsin1 ENTH with Ins(1,4,5)P3 bound. The major difference in surface electrostatic potential (red − 10 kT e−1; blue + 10 kT e−1) is in the PtdInsP binding pocket where several positively charged residues are missing in epsinR ENTH domain. (B) Ribbon diagram of the modeled epsinR ENTH domain showing the residues in the binding pocket and the succession of hydrophobic residues on the outer surface of helix zero (α0), just like in epsin1 ENTH domain. (C) Sequence homologies between epsin1 homologues and epsinR homologues. Both Drosophila (D) and C. elegans (Ce) have one homologue of each, but humans have three homologues of epsin1 (1–3), and yeast (Sc, Saccharomyces cerevisiae) also has multiple homologues. Hydrophobic residues on the outer surface of helix zero are marked in yellow. Conserved (blue) and nonconserved (green) Ptd(4,5)P2 binding residues and other major differences (orange) are marked. Some of the key residues in the human epsinR sequence referred to in the paper are numbered. (D) Domain structure of epsin1 compared with epsinR. Clathrin binding motifs (pink), DPW/DLF adaptor binding motifs (purple) and regions of alternative splicing (dotted lines) are shown. The splice site at amino acid 460 is an insert of the residues QPLQNVSTVLQKPNPLYN. Below are shown all the constructs, mutations, truncations and peptides used in the paper. The ubiquitin-interacting motifs (UIMs) and the Eps15 binding motifs (NPFs) found in epsin1 are not present in epsinR. We also know that there are additional clathrin binding motifs in epsinR (at least one in the N2 domain) that we have not mapped.
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fig1: The ENTH domain of epsinR. (A) The ENTH domain of epsinR was modeled on epsin1 ENTH with Ins(1,4,5)P3 bound. The major difference in surface electrostatic potential (red − 10 kT e−1; blue + 10 kT e−1) is in the PtdInsP binding pocket where several positively charged residues are missing in epsinR ENTH domain. (B) Ribbon diagram of the modeled epsinR ENTH domain showing the residues in the binding pocket and the succession of hydrophobic residues on the outer surface of helix zero (α0), just like in epsin1 ENTH domain. (C) Sequence homologies between epsin1 homologues and epsinR homologues. Both Drosophila (D) and C. elegans (Ce) have one homologue of each, but humans have three homologues of epsin1 (1–3), and yeast (Sc, Saccharomyces cerevisiae) also has multiple homologues. Hydrophobic residues on the outer surface of helix zero are marked in yellow. Conserved (blue) and nonconserved (green) Ptd(4,5)P2 binding residues and other major differences (orange) are marked. Some of the key residues in the human epsinR sequence referred to in the paper are numbered. (D) Domain structure of epsin1 compared with epsinR. Clathrin binding motifs (pink), DPW/DLF adaptor binding motifs (purple) and regions of alternative splicing (dotted lines) are shown. The splice site at amino acid 460 is an insert of the residues QPLQNVSTVLQKPNPLYN. Below are shown all the constructs, mutations, truncations and peptides used in the paper. The ubiquitin-interacting motifs (UIMs) and the Eps15 binding motifs (NPFs) found in epsin1 are not present in epsinR. We also know that there are additional clathrin binding motifs in epsinR (at least one in the N2 domain) that we have not mapped.

Mentions: Epsins were originally identified as Eps15-interacting proteins, but the family is now better described as being ENTH domain (phosphatidylinositol phosphate [PtdInsP] binding) proteins with clathrin/adaptor binding sequences. Epsins 1 and 2 are brain-enriched (Rosenthal et al., 1999), whereas epsin3 is expressed in wounded epithelia (Spradling et al., 2001). Previously, we identified another epsin in the database (Ford et al., 2002) that is more distantly related to epsins 1–3 in that it does not have any Eps15-binding motifs (NPFs), and so is not a classical “epsin,” and thus, we named it epsinR (for epsin-related protein). The ENTH domain of epsinR is conserved, although the lipid specificity is predicted to be different (Ford et al., 2002). The clathrin/adaptor binding domain is also conserved, although the motifs present are distinctly different (Fig. 1 D). EpsinR does not have the ubiquitin interacting motifs present in epsins 1–3 that bind to ubiquitin and are essential for the monoubiquitination of the protein (Oldham et al., 2002; Polo et al., 2002). EpsinR homologues are also present in other species, including Drosophila and C. elegans (Fig. 1 C), and have no NPF motifs, but have multiple DxF motifs in place of the multiple DPWs in the epsin1 homologues. In this paper, we investigate the function of mammalian epsinR in vesicle budding events.


EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking.

Mills IG, Praefcke GJ, Vallis Y, Peter BJ, Olesen LE, Gallop JL, Butler PJ, Evans PR, McMahon HT - J. Cell Biol. (2003)

The ENTH domain of epsinR. (A) The ENTH domain of epsinR was modeled on epsin1 ENTH with Ins(1,4,5)P3 bound. The major difference in surface electrostatic potential (red − 10 kT e−1; blue + 10 kT e−1) is in the PtdInsP binding pocket where several positively charged residues are missing in epsinR ENTH domain. (B) Ribbon diagram of the modeled epsinR ENTH domain showing the residues in the binding pocket and the succession of hydrophobic residues on the outer surface of helix zero (α0), just like in epsin1 ENTH domain. (C) Sequence homologies between epsin1 homologues and epsinR homologues. Both Drosophila (D) and C. elegans (Ce) have one homologue of each, but humans have three homologues of epsin1 (1–3), and yeast (Sc, Saccharomyces cerevisiae) also has multiple homologues. Hydrophobic residues on the outer surface of helix zero are marked in yellow. Conserved (blue) and nonconserved (green) Ptd(4,5)P2 binding residues and other major differences (orange) are marked. Some of the key residues in the human epsinR sequence referred to in the paper are numbered. (D) Domain structure of epsin1 compared with epsinR. Clathrin binding motifs (pink), DPW/DLF adaptor binding motifs (purple) and regions of alternative splicing (dotted lines) are shown. The splice site at amino acid 460 is an insert of the residues QPLQNVSTVLQKPNPLYN. Below are shown all the constructs, mutations, truncations and peptides used in the paper. The ubiquitin-interacting motifs (UIMs) and the Eps15 binding motifs (NPFs) found in epsin1 are not present in epsinR. We also know that there are additional clathrin binding motifs in epsinR (at least one in the N2 domain) that we have not mapped.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172650&req=5

fig1: The ENTH domain of epsinR. (A) The ENTH domain of epsinR was modeled on epsin1 ENTH with Ins(1,4,5)P3 bound. The major difference in surface electrostatic potential (red − 10 kT e−1; blue + 10 kT e−1) is in the PtdInsP binding pocket where several positively charged residues are missing in epsinR ENTH domain. (B) Ribbon diagram of the modeled epsinR ENTH domain showing the residues in the binding pocket and the succession of hydrophobic residues on the outer surface of helix zero (α0), just like in epsin1 ENTH domain. (C) Sequence homologies between epsin1 homologues and epsinR homologues. Both Drosophila (D) and C. elegans (Ce) have one homologue of each, but humans have three homologues of epsin1 (1–3), and yeast (Sc, Saccharomyces cerevisiae) also has multiple homologues. Hydrophobic residues on the outer surface of helix zero are marked in yellow. Conserved (blue) and nonconserved (green) Ptd(4,5)P2 binding residues and other major differences (orange) are marked. Some of the key residues in the human epsinR sequence referred to in the paper are numbered. (D) Domain structure of epsin1 compared with epsinR. Clathrin binding motifs (pink), DPW/DLF adaptor binding motifs (purple) and regions of alternative splicing (dotted lines) are shown. The splice site at amino acid 460 is an insert of the residues QPLQNVSTVLQKPNPLYN. Below are shown all the constructs, mutations, truncations and peptides used in the paper. The ubiquitin-interacting motifs (UIMs) and the Eps15 binding motifs (NPFs) found in epsin1 are not present in epsinR. We also know that there are additional clathrin binding motifs in epsinR (at least one in the N2 domain) that we have not mapped.
Mentions: Epsins were originally identified as Eps15-interacting proteins, but the family is now better described as being ENTH domain (phosphatidylinositol phosphate [PtdInsP] binding) proteins with clathrin/adaptor binding sequences. Epsins 1 and 2 are brain-enriched (Rosenthal et al., 1999), whereas epsin3 is expressed in wounded epithelia (Spradling et al., 2001). Previously, we identified another epsin in the database (Ford et al., 2002) that is more distantly related to epsins 1–3 in that it does not have any Eps15-binding motifs (NPFs), and so is not a classical “epsin,” and thus, we named it epsinR (for epsin-related protein). The ENTH domain of epsinR is conserved, although the lipid specificity is predicted to be different (Ford et al., 2002). The clathrin/adaptor binding domain is also conserved, although the motifs present are distinctly different (Fig. 1 D). EpsinR does not have the ubiquitin interacting motifs present in epsins 1–3 that bind to ubiquitin and are essential for the monoubiquitination of the protein (Oldham et al., 2002; Polo et al., 2002). EpsinR homologues are also present in other species, including Drosophila and C. elegans (Fig. 1 C), and have no NPF motifs, but have multiple DxF motifs in place of the multiple DPWs in the epsin1 homologues. In this paper, we investigate the function of mammalian epsinR in vesicle budding events.

Bottom Line: Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively.Thus, potentially, two AP1 complexes can bind to one epsinR.This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.

ABSTRACT
EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

Show MeSH
Related in: MedlinePlus