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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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Two γ-adaptin appendage domains can bind simultaneously with high affinity to epsinR. (A) Affinity measurement by calorimetric titrations for α-, β-, and γ-appendage domains and GGA appendage domain with epsinR N3 constructs. Where the stoichiometry of the interaction was 2:1, the data showed a robust fit to a two-site model, and the KDs for both sites are shown. The bold lettering indicates the protein in the syringe. (B) Peptides from epsinR and γ-synergin binding to the γ-adaptin appendage domain. Profiles of typical calorimetric titrations and the integrated normalized data are shown on the right. A comprehensive table of calorimetric data is available in Table SI.
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fig5: Two γ-adaptin appendage domains can bind simultaneously with high affinity to epsinR. (A) Affinity measurement by calorimetric titrations for α-, β-, and γ-appendage domains and GGA appendage domain with epsinR N3 constructs. Where the stoichiometry of the interaction was 2:1, the data showed a robust fit to a two-site model, and the KDs for both sites are shown. The bold lettering indicates the protein in the syringe. (B) Peptides from epsinR and γ-synergin binding to the γ-adaptin appendage domain. Profiles of typical calorimetric titrations and the integrated normalized data are shown on the right. A comprehensive table of calorimetric data is available in Table SI.

Mentions: Given the binding of epsinR to adaptor complexes in brain and COS cell extracts, we next tested which appendage domains were responsible for the interactions (Fig. 4 C). In this experiment, Myc-tagged epsins were overexpressed in COS cells and GST-appendage domains were used to fish the proteins out. All appendage domains tested bound to both epsin1 and epsinR; however, γ-appendage has a distinct preference for epsinR. The L762E mutant of the γ-appendage (Kent et al., 2002) abolished the interaction (Fig. 4 C). This preference for the γ-appendage was confirmed by ITC where the affinity for the α-appendage was much weaker than for the β- and γ-appendages (Fig. 5). For both the β- and γ-appendages, the binding stoichiometry was 2:1, and we were thus able to fit a curve describing two binding sites to the data. These were very robust fits, giving a site with a submicromolar affinity and a further lower affinity site in each case. These experiments also show that multiple appendage domains can interact at the same time. GGAs, which have a γ-adaptin appendage homology domain would also be predicted to bind epsinR. GST-GGA appendage domains interacted with epsinR (Fig. 4 D), and the reverse experiment also showed that GST-epsinR N1 construct bound to GGAs 1 and 2 (Fig. 4 B and unpublished data). However, despite the very high conservation of critical residues between the predicted binding sites in GGAs and γ-adaptin appendages, deletion constructs point to clear differences in the binding specificities (Fig. 4 B). The affinity of GGA1 for the epsinR N3 construct was also much lower than the affinity of the γ-appendage (Fig. 5). Of all the appendage domains tested the much higher affinities of γ- and β-appendages for epsinR imply that the AP1 complex is the major interacting adaptor.


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)

Two γ-adaptin appendage domains can bind simultaneously with high affinity to epsinR. (A) Affinity measurement by calorimetric titrations for α-, β-, and γ-appendage domains and GGA appendage domain with epsinR N3 constructs. Where the stoichiometry of the interaction was 2:1, the data showed a robust fit to a two-site model, and the KDs for both sites are shown. The bold lettering indicates the protein in the syringe. (B) Peptides from epsinR and γ-synergin binding to the γ-adaptin appendage domain. Profiles of typical calorimetric titrations and the integrated normalized data are shown on the right. A comprehensive table of calorimetric data is available in Table SI.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172650&req=5

fig5: Two γ-adaptin appendage domains can bind simultaneously with high affinity to epsinR. (A) Affinity measurement by calorimetric titrations for α-, β-, and γ-appendage domains and GGA appendage domain with epsinR N3 constructs. Where the stoichiometry of the interaction was 2:1, the data showed a robust fit to a two-site model, and the KDs for both sites are shown. The bold lettering indicates the protein in the syringe. (B) Peptides from epsinR and γ-synergin binding to the γ-adaptin appendage domain. Profiles of typical calorimetric titrations and the integrated normalized data are shown on the right. A comprehensive table of calorimetric data is available in Table SI.
Mentions: Given the binding of epsinR to adaptor complexes in brain and COS cell extracts, we next tested which appendage domains were responsible for the interactions (Fig. 4 C). In this experiment, Myc-tagged epsins were overexpressed in COS cells and GST-appendage domains were used to fish the proteins out. All appendage domains tested bound to both epsin1 and epsinR; however, γ-appendage has a distinct preference for epsinR. The L762E mutant of the γ-appendage (Kent et al., 2002) abolished the interaction (Fig. 4 C). This preference for the γ-appendage was confirmed by ITC where the affinity for the α-appendage was much weaker than for the β- and γ-appendages (Fig. 5). For both the β- and γ-appendages, the binding stoichiometry was 2:1, and we were thus able to fit a curve describing two binding sites to the data. These were very robust fits, giving a site with a submicromolar affinity and a further lower affinity site in each case. These experiments also show that multiple appendage domains can interact at the same time. GGAs, which have a γ-adaptin appendage homology domain would also be predicted to bind epsinR. GST-GGA appendage domains interacted with epsinR (Fig. 4 D), and the reverse experiment also showed that GST-epsinR N1 construct bound to GGAs 1 and 2 (Fig. 4 B and unpublished data). However, despite the very high conservation of critical residues between the predicted binding sites in GGAs and γ-adaptin appendages, deletion constructs point to clear differences in the binding specificities (Fig. 4 B). The affinity of GGA1 for the epsinR N3 construct was also much lower than the affinity of the γ-appendage (Fig. 5). Of all the appendage domains tested the much higher affinities of γ- and β-appendages for epsinR imply that the AP1 complex is the major interacting adaptor.

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