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• W2080340766 abstract "Clathrin-coated pit (CCP) formation occurs as a result of the targeting and assembly of cytosolic coat proteins, mainly the plasma membrane clathrin-associated protein complex (AP-2) and clathrin, to the intracellular face of the plasma membrane. In the present study, the mechanisms by which Eps15, an AP-2-binding protein, is targeted to CCPs was analyzed by following the intracellular localization of Eps15 mutants fused to the green fluorescent protein. Our previous results indicated that the N-terminal Eps15homology (EH) domains are required for CCP targeting. We now show that EH domains are, however, not sufficient for targeting to CCPs. Similarly, neither the central coiled-coil nor the C-terminal AP-2 binding domains were able to address green fluorescent protein to CCPs. Thus, targeting of Eps15 to CCPs likely results from the collaboration between EH domains and another domain of the protein. An Eps15 mutant lacking the coiled-coil domain localized to CCPs showing that Eps15 dimerization is not strictly required. In contrast, Eps15 mutants lacking all AP-2 binding sites showed a dramatic decrease in plasma membrane staining, showing that AP-2 binding sites, together with EH domains, play an important role in targeting Eps15 into CCPs. Finally, the effect of the Eps15 mutants on clathrin-dependent endocytosis was tested by both immunofluorescence and flow cytometry. The results obtained showed that inhibition of transferrin uptake was observed only with mutants able to interfere with CCP assembly. Clathrin-coated pit (CCP) formation occurs as a result of the targeting and assembly of cytosolic coat proteins, mainly the plasma membrane clathrin-associated protein complex (AP-2) and clathrin, to the intracellular face of the plasma membrane. In the present study, the mechanisms by which Eps15, an AP-2-binding protein, is targeted to CCPs was analyzed by following the intracellular localization of Eps15 mutants fused to the green fluorescent protein. Our previous results indicated that the N-terminal Eps15homology (EH) domains are required for CCP targeting. We now show that EH domains are, however, not sufficient for targeting to CCPs. Similarly, neither the central coiled-coil nor the C-terminal AP-2 binding domains were able to address green fluorescent protein to CCPs. Thus, targeting of Eps15 to CCPs likely results from the collaboration between EH domains and another domain of the protein. An Eps15 mutant lacking the coiled-coil domain localized to CCPs showing that Eps15 dimerization is not strictly required. In contrast, Eps15 mutants lacking all AP-2 binding sites showed a dramatic decrease in plasma membrane staining, showing that AP-2 binding sites, together with EH domains, play an important role in targeting Eps15 into CCPs. Finally, the effect of the Eps15 mutants on clathrin-dependent endocytosis was tested by both immunofluorescence and flow cytometry. The results obtained showed that inhibition of transferrin uptake was observed only with mutants able to interfere with CCP assembly. plasma membrane clathrin-associated protein complex Golgi clathrin-associated protein complex clathrin-coated pit Eps15 homology green fluorescent protein transferrin transferrin receptor phosphate-buffered saline bovine serum albumin Clathrin-coated vesicle formation represents the initial step of the major pathway for receptor-mediated endocytosis. The known roles of AP-2,1 clathrin and dynamin in this process are as follows. AP-2 is believed to drive clathrin assembly at the plasma membrane and binds to tyrosine-based internalization signals, playing a central role in both formation and function of clathrin-coated pits (CCPs); clathrin gives an organizing framework to the pit; and the dynamin GTPase activity is required for membrane fusion events leading to coated vesicle formation (1.Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1338) Google Scholar, 2.Kirchhausen T. Bonifacino J.S. Riezman H. Curr. Opin. Cell Biol. 1997; 9: 488-495Crossref PubMed Scopus (352) Google Scholar, 3.Hirst J. Robinson M.S. Biochim. Biophys. Acta. 1998; 1404: 173-193Crossref PubMed Scopus (327) Google Scholar, 4.Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (355) Google Scholar, 5.McNiven M.A. Cell. 1998; 94: 151-154Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The mechanisms by which these soluble proteins are targeted to the plasma membrane to form an organized clathrin coat have been extensively studied. Targeting of AP-2 complexes is mediated by its α-adaptin subunit. Studies of chimeras in which equivalent domains of α-adaptin from AP-2 and γ-adaptin from AP-1 were exchanged allowed the identification of a CCP targeting signal within a 200-amino acid region of the N-terminal “head” or “trunk” domain of α-adaptin. These studies also established a role for the C-terminal “ear” domain of α-adaptin (6.Robinson M.S. J. Cell Biol. 1993; 123: 67-77Crossref PubMed Scopus (59) Google Scholar, 7.Page L.J. Robinson M.S. J. Cell Biol. 1995; 131: 619-630Crossref PubMed Scopus (129) Google Scholar), a result confirmed by recent data showing efficient targeting of a GFP-ear construct to CCP (8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar). Furthermore, the α-adaptin N-terminal domain also contains a phosphoinositide binding site (9.Gaidarov I. Chen Q. Falck J.R. Reddy K.K. Keen J.H. J. Biol. Chem. 1996; 271: 20922-20929Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), which seems necessary for efficient incorporation of AP-2 complexes into CCPs (10.Gaidarov I. Keen J.H. Mol. Biol. Cell. 1997; 8: 92Google Scholar). Clathrin assembly at the plasma membrane requires plasma membrane-bound AP-2 complexes (11.Moore M.S. Mahaffey D.T. Brodsky F.M. Anderson R.G. Science. 1987; 236: 558-563Crossref PubMed Scopus (93) Google Scholar,12.Mahaffey D.T. Moore M.S. Brodsky F.M. Anderson R.G. J. Cell Biol. 1989; 108: 1615-1624Crossref PubMed Scopus (30) Google Scholar) and is thought to result from direct AP-2/clathrin interactions. Dynamin directly binds to salt-stripped membranes and to phospholipids containing liposomes (13.Tuma P.L. Collins C.A. J. Biol. Chem. 1995; 270: 26707-26714Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14.Sweitzer S.M. Hinshaw J.E. Cell. 1998; 93: 1021-1029Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar, 15.Takei K. Haucke V. Slepnev V. Farsad K. Salazar M. Chen H. De Camilli P. Cell. 1998; 94: 131-141Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar), suggesting that dynamin might directly interact with the plasma membrane in vivo. This hypothesis was confirmed by the fact that dynamin is homogeneously redistributed at the plasma membrane when CCP assembly is inhibited (8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar). Its localization to CCPs requires its C-terminal proline-rich domain (16.Shpetner H.S. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1996; 271: 13-16Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), which binds to the SH3 domain of amphiphysin (17.David C. McPherson P.S. Munndigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (350) Google Scholar). Recently, several new CCP-associated proteins were identified including Eps15 (18.Tebar F. Sorkina T. Sorkin A. Kirchhausen T. J. Biol. Chem. 1996; 271: 28727-28730Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), amphiphysin (19.Bauerfeind R. Takei K. De Camilli P. J. Biol. Chem. 1997; 272: 30984-30992Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 20.Wigge P. Kohler K. Vallis Y. Doyle C.A. Owen D. Hunt S.P. McMahon H.T. Mol. Biol. Cell. 1997; 8: 2003-2015Crossref PubMed Scopus (209) Google Scholar), and epsin (21.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (271) Google Scholar). The mechanisms by which these accessory coat proteins are targeted to CCPs have not yet been investigated. The Eps15 protein is constitutively and ubiquitously associated with AP-2 (22.Benmerah A. Gagnon J. Bègue B. Mégarbané B. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1995; 131: 1831-1838Crossref PubMed Scopus (151) Google Scholar). Recent data obtained both in vivo and in a perforated cell assay showed that Eps15 is required for the early steps of clathrin-dependent endocytosis (23.Carbone R. Fre S. Iannolo G. Belleudi F. Mancini P. Pelicci P.G. Torrisi M.R. Difiore P.P. Cancer Res. 1997; 57: 5498-5504PubMed Google Scholar, 24.Benmerah A. Lamaze C. Bègue B. Schmid S.L. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1998; 140: 1055-1062Crossref PubMed Scopus (300) Google Scholar). The fact that Eps15 is not found in coated vesicles (25.Cupers P. Jadhav A.P. Kirchhausen T. J. Biol. Chem. 1998; 273: 1847-1850Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) suggested a coated pit-restricted function. Results showing that the α-adaptin ear domain, the Eps15 binding site on AP-2 (26.Benmerah A. Bègue B. Dautry-Varsat A. Cerf-Bensussan N. J. Biol. Chem. 1996; 271: 12111-12116Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 27.Iannolo C. Salcini A.E. Gaidarov I. Goodman O.B. Baulida J. Carpenter G. Pelicci P.G. Di Fiore P.P. Keen J.H. Cancer Res. 1997; 57: 240-245PubMed Google Scholar), is involved in the targeting of AP-2 to CCPs (6.Robinson M.S. J. Cell Biol. 1993; 123: 67-77Crossref PubMed Scopus (59) Google Scholar, 7.Page L.J. Robinson M.S. J. Cell Biol. 1995; 131: 619-630Crossref PubMed Scopus (129) Google Scholar, 8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar) have suggested that Eps15 may be part of the AP-2 docking machinery. This hypothesis is strengthened by the fact that an Eps15 mutant lacking EH domains inhibits CCP assembly (8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar). Eps15 is a conserved protein (28.Fazioli F. Minichiello L. Matoskova B. Wong W.T. Di Fiore P.P. Mol. Cell. Biol. 1993; 13: 5814-5828Crossref PubMed Scopus (238) Google Scholar, 29.Wong W.T. Kraus M.H. Carlomagno F. Zelano A. Druck T. Croce C.M. Huebner K. Di Fiore P.P. Oncogene. 1994; 9: 1591-1597PubMed Google Scholar) organized in three structural domains. The N-terminal domain (DI) contains three repeats of ∼70 amino acids homologous to each other and to equivalent domains found in proteins from humans, yeast, and nematodes. These domains were called EH (30.Wong W.T. Schumacher C. Salcini A.E. Romano A. Castagnino P. Pelicci P.G. Di Fiore P.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9530-9534Crossref PubMed Scopus (135) Google Scholar), for Eps15 Homology, and defined a novel protein-protein interacting module (31.Di Fiore P.P. Pelicci P.G. Sorkin A. Trends Biochem. Sci. 1997; 22: 411-413Abstract Full Text PDF PubMed Scopus (79) Google Scholar) that recognizes NPF-based motifs (32.Salcini A.E. Confalonieri S. Doria M. Santolini E. Tassi E. Minenkova O. Cesareni G. Pelicci P.G. Difiore P.P. Genes Dev. 1997; 11: 2239-2249Crossref PubMed Scopus (286) Google Scholar). The central domain (DII) contains heptad repeats required for coiled-coil structures and is involved in the dimerization of Eps15 (33.Tebar F. Confalonieri S. Carter R.E. Di Fiore P.P. Sorkin A. J. Biol. Chem. 1997; 272: 15413-15418Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 34.Cupers P. Terhaar E. Boll W. Kirchhausen T. J. Biol. Chem. 1997; 272: 33430-33434Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The C-terminal domain (DIII) contains the AP-2 binding region (26.Benmerah A. Bègue B. Dautry-Varsat A. Cerf-Bensussan N. J. Biol. Chem. 1996; 271: 12111-12116Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), which spans ∼120 amino acids (24.Benmerah A. Lamaze C. Bègue B. Schmid S.L. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1998; 140: 1055-1062Crossref PubMed Scopus (300) Google Scholar) and contains three independent AP-2 binding sites (27.Iannolo C. Salcini A.E. Gaidarov I. Goodman O.B. Baulida J. Carpenter G. Pelicci P.G. Di Fiore P.P. Keen J.H. Cancer Res. 1997; 57: 240-245PubMed Google Scholar). Therefore, as Eps15 does not contain any known plasma membrane targeting signals, its localization to CCPs is likely to be mediated by protein-protein interactions through its structural domains. The aim of this study was to identify these signals. With this goal in mind, we generated mutant forms of Eps15 and followed their intracellular localization. Their effect on clathrin-dependent endocytosis was further analyzed and compared with their capacity or inability to localize in CCP. HeLa cells (ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mml-glutamine, penicillin, and streptomycin (Life Technologies, Inc.). The mouse monoclonal antibodies AP.6 (anti-AP-2 (35.Chin D.J. Straubinger R.M. Acton S. Nathke I. Brodsky F.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9289-9293Crossref PubMed Scopus (80) Google Scholar)) and OKT9 (anti-human transferrin receptor (Tf-R)) were obtained from ATCC. Texas Red-conjugated goat anti-mouse immunoglobulins were obtained from Molecular Probes (Eugene, OR), Cy5-conjugated goat anti-mouse immunoglobulins were from Amersham Pharmacia Biotech, and phycoerythrin-conjugated F(ab′)2 fragment goat anti-mouse IgG was from Immunotech (Marseille, France). The cDNA of human Eps15 subcloned in pBluescript® II KS (Stratagene, La Jolla, CA) was obtained in the laboratory (22.Benmerah A. Gagnon J. Bègue B. Mégarbané B. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1995; 131: 1831-1838Crossref PubMed Scopus (151) Google Scholar) and used as a template to generate the different cDNA fragments used in this study. The Eps15 constructs encoding each structural domain subcloned in the PGEX5.1 vector (Amersham Pharmacia Biotech) and the EH deleted mutant EΔ95/295 were described previously (8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar, 24.Benmerah A. Lamaze C. Bègue B. Schmid S.L. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1998; 140: 1055-1062Crossref PubMed Scopus (300) Google Scholar, 26.Benmerah A. Bègue B. Dautry-Varsat A. Cerf-Bensussan N. J. Biol. Chem. 1996; 271: 12111-12116Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The deleted mutants were obtained by polymerase chain reactions and introduced within Eps15 subcloned between the BamHI/XhoI sites of the PGEX5.1 vector using appropriate restriction sites. Briefly, deletion of the central coiled-coil domain (EΔCC) was obtained by deleting a 635-base pair region corresponding to nucleotides 960–1605 of human Eps15, and the mutated DNA fragment was introduced within the Eps15 sequence using the HindIII (825) and ClaI (1937) sites. Mutants of the C-terminal domain were obtained by introducing stop codons in the corresponding lower primers at position 1856 (EΔA/P/C), 2298 (EΔP/C), or 2544 (EΔC). The mutated polymerase chain reaction fragments were then introduced within the Eps15 sequence using the BglII (1337) and XhoI sites. To generate a Eps15 mutant lacking only AP-2 binding sites (EΔAP-2), a 354-base pair region (1856 to 2298) was deleted as described previously (24.Benmerah A. Lamaze C. Bègue B. Schmid S.L. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1998; 140: 1055-1062Crossref PubMed Scopus (300) Google Scholar). All of the different constructs were excised from the PGEX5.1 vector using the BamHI/XhoI sites, purified on agarose gel, and then subcloned into theBglII/SalI sites of the EGFP-C2 vector (CLONTECH). Restriction enzymes and T4 DNA ligase were from Amersham Pharmacia Biotech. All of the constructs were checked by nucleotide sequencing (Thermosequenase, Amersham Pharmacia Biotech). Sequences of the different primers used to generate the Eps15 mutants are available on request. Subconfluent HeLa cells were used for transient expression of the different constructs. Transfections were performed using the CalPhos Maximizer transfection kit fromCLONTECH. For immunofluorescence studies, transfected HeLa cells were grown on coverslips and used the day after transfection. The cells were washed in PBS and fixed in 3.7% paraformaldehyde and 0.03 msucrose for 30 min at 4 °C. The cells were then washed once in PBS and, after quenching for 10 min in 50 mm NH4Cl in PBS, washed again in PBS supplemented with 1 mg/ml bovine serum albumin (BSA). The cells were then incubated with the AP.6 antibody in permeabilization buffer (PBS with 1 mg/ml BSA and 0.05% saponin) for 45 min at room temperature. After two washes in the permeabilization buffer, the presence of antibodies was revealed by incubating the cells for 45 min at room temperature in permeabilization buffer containing labeled secondary antibody. After two washes in permeabilization buffer and one in PBS, the cells were mounted on microscope slides in 100 mg/ml Mowiol (Calbiochem), 25% glycerol (v/v), and 100 mmTris-HCl, pH 8.5. To wash out cytosolic GFP constructs, the cells were briefly permeabilized before fixation with a 2–5-min incubation with 0.03% saponin in cytosolic buffer (100 mm potassium acetate, 1 mm MgCl2, 20 mm Hepes) at 4 °C. The cells were then washed twice in cytosolic buffer and processed for immunofluorescence as described above. For surface staining, transfected cells were incubated with antibodies in PBS, 1 mg/ml BSA at 4 °C. Human transferrin (Tf) was conjugated to Cy3 dye using the CyDye FluoroLink reactive dye kit from Amersham Pharmacia Biotech following the manufacturer's instructions. Endocytosis of Cy3-conjugated Tf was performed on subconfluent HeLa cells grown on coverslips 1 day after transfection. The cells were first incubated for 20 min at 37 °C in Dulbecco's modified Eagle's medium and 20 mm Hepes, pH 7.2, to eliminate receptor-bound Tf and then incubated in Dulbecco's modified Eagle's medium, 20 mm Hepes, pH 7.2, and 1 mg/ml BSA containing 100 nm Cy3-conjugated Tf. After incubation at 37 °C for 15 min, the cells were rapidly cooled to 4 °C, washed twice in cold PBS, and then fixed as described above. The samples were examined under an epifluorescence microscope (Zeiss, Oberkochen, Germany) attached to a cooled CCD camera (Photometrics, Tucson, AZ). Cell surface Tf-Rs were quantified by flow cytometry. HeLa cells were detached from culture plates 1 day after transfection using a Costar cell lifter. Cells were washed twice in PBS, 1 mg/ml BSA and then incubated at 4 °C for 45 min with anti-Tf-R antibody OKT9 at saturating concentrations in PBS 1 mg/ml BSA. The cells were then washed twice in PBS 1 mg/ml BSA and incubated at 4 °C for 45 min in PBS 1 mg/ml BSA with a phycoerythrin-conjugated goat anti-mouse IgG. The cells were then washed twice in PBS 1 mg/ml BSA, and the levels of expression of both GFP and Tf-R on the cell surface were assessed using a FACSCAN (Becton Dickinson, San Jose, CA). Data were analyzed using the CELLQuest program (Becton Dickinson). Endogenous Eps15 is constitutively found in CCPs (18.Tebar F. Sorkina T. Sorkin A. Kirchhausen T. J. Biol. Chem. 1996; 271: 28727-28730Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 36.van Delft S. Schumacher C. Hage W. Verkleij A.J. Henegouwen P. J. Cell Biol. 1997; 136: 811-821Crossref PubMed Scopus (112) Google Scholar). Addition of the GFP to its N terminus does not modify its intracellular localization (8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar, 24.Benmerah A. Lamaze C. Bègue B. Schmid S.L. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1998; 140: 1055-1062Crossref PubMed Scopus (300) Google Scholar), with the resulting GFP-Eps15 construct showing a punctate staining at the plasma membrane that colocalizes with AP-2 (Fig. 2, a, b, and insets). In this study, the GFP fusion system was used to map the domains of Eps15 involved in its targeting to CCPs. The different mutants derived from Eps15 (see Fig. 6) were fused to GFP, the resulting constructs were transiently transfected into HeLa cells, and their intracellular distribution was compared with that of AP-2.Figure 6Structural organization of Eps15 mutants and summary of the results. A, P, andC stand for AP-2 binding, proline-rich, and C-terminal regions, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The results obtained in our previous study showed that EH domains are required for CCP targeting of Ep15. Indeed, an Eps15 construct lacking the second and third EH domains showed a diffuse cytosolic staining (8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar). The putative role of EH domains as a sufficient coated pit targeting signal was first tested. As shown in Fig.1 a, a GFP construct encoding EH domains (DI) presented a diffuse intracellular staining, suggesting that the DI construct was not targeted to CCP. This was confirmed by its lack of colocalization with AP-2 at the plasma membrane (Fig. 1,a, b, and insets), showing that EH domains were unable to specifically target GFP to CCPs. From these and previous results (8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar) it can be concluded that EH domains are required but not sufficient for CCP targeting. The central coiled-coil and C-terminal AP-2 binding domains, DII and DIII constructs, respectively, were then tested for their capacity to bring GFP to CCPs. The DII construct was diffusely distributed in cytosol and did not colocalize with AP-2 at the plasma membrane (Fig.1, c, d, and insets). A similar cytosolic staining was observed with the DIII construct (Fig.1 e). However, expression of the DIII construct, previously shown to inhibit clathrin-dependent endocytosis (24.Benmerah A. Lamaze C. Bègue B. Schmid S.L. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1998; 140: 1055-1062Crossref PubMed Scopus (300) Google Scholar), induced a mislocalization of AP-2 complexes. Cells expressing DIII showed less AP-2 dots at the plasma membrane and an increase in cytosolic staining compared with neighboring untransfected cells (Fig.1 f, indicated by arrows). These results are reminiscent of those obtained with the EΔ95/295 mutant (8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar) and indicated that expression of the DIII construct also inhibits clathrin-coated pit assembly. Nevertheless, the DIII construct did not colocalize with the AP-2 dots still present at the plasma membrane (Fig. 1, e, f, and insets), showing that the AP-2 binding domain is not a sufficient coated pit targeting signal. The lack of plasma membrane punctate staining observed for each structural domain was not due to an excess of cytosolic GFP, because brief permeabilization of the transfected cells before fixation effectively washed out cytosolic GFP constructs but did not reveal any specific plasma membrane-associated staining (data not shown). Altogether, these results show that none of the three structural domains of Eps15 is sufficient for CCP targeting. The fact that EH domains are necessary but not sufficient for coated pit targeting suggests the involvement of another domain(s) in this process. We first investigated the role of the C-terminal domain. Indeed, the C-terminal domain of Eps15 contains both AP-2 binding sites and a proline-rich region. The proline-rich region is found between amino acids 768 and 849 and contains a PALPPK (768/774) sequence that binds to SH3 domain-containing proteins (37.Schumacher C. Knudsen B.S. Ohuchi T. Di Fiore P.P. Glassman R.H. Hanafusa H. J. Biol. Chem. 1995; 270: 15341-15347Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 38.Matsuda M. Ota S. Tanimura R. Nakamura H. Matuoka K. Takenawa K. Kurata T. J. Biol. Chem. 1996; 271: 14468-14472Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Interestingly, a proline-rich domain is responsible for the targeting of dynamin to CCPs (16.Shpetner H.S. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1996; 271: 13-16Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) through interaction with the SH3 domain of amphiphysin (39.Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (403) Google Scholar). Such a mechanism could therefore also be involved in Eps15 targeting. Eps15 constructs lacking either the 849–896 (EΔC; data not shown) or the 763–896 region (EΔP/C; Fig.2 c) showed a bright plasma membrane punctate staining. Furthermore, the plasma membrane punctate staining completely colocalized with AP-2 (Fig. 2, c,d, and insets), showing that EΔP/C is targeted to CCPs. Thus, the proline-rich region of Eps15, responsible for binding to SH3 domain containing proteins, is not necessary for its targeting to CCPs. The role of AP-2 binding sites was investigated using two different constructs; EΔA/P/C lacking the entire C-terminal region after amino acid 620 and EΔAP-2 lacking only the AP-2 binding sites (621–738 region). As shown in Fig. 2, these two constructs showed a very faint plasma membrane staining (Fig. 2, e and g). This very faint staining still presented some colocalization with AP-2 (Fig.2, e-h and insets), showing that the constructs could be found in CCPs. However, the plasma membrane staining was highly reduced compared with that observed for both wild type Eps15 (Fig. 2 a) and for all of the constructs containing both EH domains and AP-2 binding sites (Fig. 2 c and Fig. 3,a and c), showing that AP-2 binding sites play an important role in CCP targeting of Eps15. Noticeably, in contrast to endogenous Eps15 2A. Benmerah, unpublished observation. and transfected GFP-Eps15 (Fig. 2 a), some constructs (including EΔP/C (Fig. 2 c) and EΔA/P/C (Fig. 2 e)) were found in the nucleus. Interestingly, the Eps15-related protein, Eps15r, is found both in plasma membrane CCPs and in the nucleus (40.Coda L. Salcini A.E. Confalonieri S. Pelicci G. Sorkina T. Sorkin A. Pelicci P.G. Difiore P.P. J. Biol. Chem. 1998; 273: 3003-3012Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Thus, Eps15 mutants may reveal a possible nucleocytoplasmic traffic for Eps15. 3V. Poupon, N. Cerf-Bensussan, A. Dautry-Varsat, and A. Benmerah, manuscript in preparation. Finally, the role of the central dimerization domain in coated pit localization was also tested. The EΔCC construct lacking the coiled-coil domain colocalized with AP-2 at the plasma membrane (Fig.3, a, b, andinsets), showing that the coiled-coil domain is not required for CCP targeting of Eps15. However, we repeatedly noticed that the CCP staining observed for EΔCC (Fig. 3 a) was fainter than for the full-length Eps15 (Fig. 2 a). Compared with EΔP/C (Fig.2 c), which only lacks the C-terminal and proline-rich regions, weaker CCP staining was also observed for the EΔCC+P/C (Fig.3 c) lacking the coiled-coil domain, the C-terminal, and the proline-rich regions. Altogether, these results suggest that the coiled-coil domain does not provide specific targeting information but rather helps to enhance the number of Eps15 molecules present in a given CCP. We next checked the effect of all of the Eps15 mutants (described above) on clathrin-dependent endocytosis. These mutants can be classified in two groups. Group I includes DIII and EΔ95/295 that affect CCP assembly, and group II includes all of the other mutants that do not affect CCP assembly (Fig. 6). Indeed, in the latter group, clathrin-dependent endocytosis could be affected at a later step, i.e. CCP invagination and/or coated vesicle formation. The effect of these mutants on Tf uptake was therefore tested. Expression of group II mutants did not inhibit internalization of Cy3-conjugated Tf (Fig. 4 B and data not shown) whereas a strong inhibition was observed with group I mutants (Fig. 4 A and data not shown). We next verified that the lack of inhibition of Tf uptake by group II mutants was not due to lower expression levels of group II mutants compared with group I mutants. We took advantage of the fact that inhibition of clathrin-dependent endocytosis induces an increase in the number of Tf-R at the cell surface (41.Durrbach A. Louvard D. Coudrier E. J. Cell Sci. 1996; 109: 457-465Crossref PubMed Google Scholar, 42.Lamaze C. Fujimoto L.M. Yin H.L. Schmid S.L. J. Biol. Chem. 1997; 272: 20332-20335Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 43.Rodal S.K. Skretting G. Garred O. Vilhardt F. van Deurs B. Sandvig K. Mol. Biol. Cell. 1999; 10: 961-974Crossref PubMed Scopus (825) Google Scholar, 44.Subtil A. Gaidarov I. Kobylarz K. Lampson M.A. Keen J.H. McGraw T.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6775-6780Crossref PubMed Scopus (488) Google Scholar). Indeed, as shown in Fig. 4 A, cells expressing the EΔ95/295 construct (a) presented both an inhibition of internalization of Cy3-Tf (b) and an increase of cell surface Tf-R (c) compared with neighboring untransfected cells. In contrast, no increase of cell surface Tf-R was observed in cells expressing group II mutants that did not inhibit Cy3-Tf uptake (Fig.4 B and data not shown). An increase of cell surface Tf-R was then used to follow the inhibition of clathrin-dependent endocytosis. HeLa cells transiently transfected with group I, group II, and control constructs were analyzed by flow cytometry. Cells expressing identical levels of GFP constructs were selected (Fig.5 a, region 2) and analyzed for cell surface Tf-R expression. As shown in Fig.5 b, cells expressing the DII construct presented similar levels of cell surface Tf-R compared with cells expressing the control construct D3Δ2, which do not inhibit clathrin-dependent endocytosis (24.Benmerah A. Lamaze C. Bègue B. Schmid S.L. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1998; 140: 1055-1062Crossref PubMed Scopus (300) Google Scholar). Similar results were also found for cells expressing the EΔCC construct (data not shown). Cells expressing either DI or EΔA/P/C constructs did not show increased cell surface Tf-R (Fig.5 b). Rather they expressed a lower level of cell surface Tf-R compared with that found for both DII and D3Δ2 constructs. Only cells expressing either DIII (Fig. 5 b) or EΔ95/295 (data not shown) showed a clear increase of cell surface Tf-R compared with cells expressing the control D3Δ2 construct. Therefore, the quantitative results obtained by flow cytometry confirm those obtained by Tf uptake experiments and show that, at identical expression levels, group I but not group II mutants inhibit CCP functions. The present results extend our previous study and indicate that EH domain-binding protein(s), together with the AP-2 complex, play an important role in coated pit targeting of Eps15 (summarized in Fig.6). Furthermore, they show that the inhibitory effect of Eps15 mutants correlates with inhibition of CCP assembly. Results obtained by several groups have shown that Eps15 EH domains bind to numerous proteins through NPF-based motifs (21.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (271) Google Scholar, 32.Salcini A.E. Confalonieri S. Doria M. Santolini E. Tassi E. Minenkova O. Cesareni G. Pelicci P.G. Difiore P.P. Genes Dev. 1997; 11: 2239-2249Crossref PubMed Scopus (286) Google Scholar). Some of the identified proteins including RAB/RABr are not found in CCPs nor involved in clathrin-dependent endocytosis and therefore can be excluded as physiological partners for Eps15 targeting. The most interesting candidate is the recently identified EH domain-binding protein, epsin. Like Eps15, epsin is a constitutive component of CCPs that is associated with AP-2 and required for clathrin-dependent endocytosis. Interestingly, overexpressed wild type epsin is localized to the cytosolic face of the plasma membrane in regions devoid of clathrin coat (21.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (271) Google Scholar). This is in agreement with a role of epsin as a plasma membrane-associated docking protein, which could therefore link Eps15 to the plasma membrane. Furthermore, Eps15 recruitment into CCPs also requires binding to AP-2. The fact that AP-2/Eps15 interaction sites (26.Benmerah A. Bègue B. Dautry-Varsat A. Cerf-Bensussan N. J. Biol. Chem. 1996; 271: 12111-12116Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) are involved in their respective targeting to CCPs (6.Robinson M.S. J. Cell Biol. 1993; 123: 67-77Crossref PubMed Scopus (59) Google Scholar, 7.Page L.J. Robinson M.S. J. Cell Biol. 1995; 131: 619-630Crossref PubMed Scopus (129) Google Scholar, 8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar) suggests that Eps15 is targeted to CCPs complexed to AP-2. The Eps15 mutants can be classified as group I mutants, which inhibit both CCP assembly and clathrin-dependent endocytosis and group II mutants, which do not (Fig. 6). The group I mutants contain the AP-2 binding sites. However, the presence of AP-2 binding sites is not sufficient for these inhibitory effects because the EΔCC construct that contains both EH domains and the AP-2 binding domain did not inhibit coated pit assembly nor clathrin-dependent endocytosis. The EΔCC construct was still targeted to CCP (Fig. 3) whereas DIII and EΔ95/295 were not (see Fig. 1 and Ref. 8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar). Therefore, the inhibitory effects of Eps15 mutants on clathrin-dependent endocytosis requires both the presence of AP-2 binding sites and a lack of CCP localization. Comparable domain requirements were also reported for dynamin. Dynamin mutants lacking the pleckstrin homology domain were recently shown to inhibit clathrin-dependent endocytosis of Tf (45.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (145) Google Scholar, 46.Lee A. Frank D.W. Marks M.S. Lemmon M.A. Curr. Biol. 1999; 9: 261-264Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 47.Vallis Y. Wigge P. Marks B. Evans P.R. McMahon H.T. Curr. Biol. 1999; 9: 257-260Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). As previously shown for GTPase-deficient mutants (48.Okamoto P.M. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1997; 272: 11629-11635Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), the C-terminal amphiphysin binding region is required for the inhibitory effect of the mutants on clathrin-dependent endocytosis (47.Vallis Y. Wigge P. Marks B. Evans P.R. McMahon H.T. Curr. Biol. 1999; 9: 257-260Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Furthermore, as observed for Eps15 with EH domains, the autonomously expressed dynamin pleckstrin homology domain does not inhibit clathrin-dependent endocytosis (49.Jost M. Simpson F. Kavran J.M. Lemmon M.A. Schmid S.L. Curr. Biol. 1998; 8: 1399-1402Abstract Full Text Full Text PDF PubMed Google Scholar, 50.Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Recently, the ear domain of α-adaptin, the Eps15 binding site on AP-2, was shown to bind to many other proteins including epsin, amphiphysin, auscilin, and AP-180 (51.Owen D.J. Vallis Y. Noble M.E. Hunter J.B. Dafforn T.R. Evans P.R. McMahon H.T. Cell. 1999; 97: 805-815Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). However, auxillin and AP-180 are only expressed in neuronal cells (52.Morris S.A. Schroder S. Plessmann U. Weber K. Ungewickell E. EMBO J. 1993; 12: 667-675Crossref PubMed Scopus (92) Google Scholar, 53.Schroder S. Morris S.A. Knorr R. Plessmann U. Weber K. Nguyen G.V. Ungewickell E. Eur. J. Biochem. 1995; 228: 297-304Crossref PubMed Scopus (28) Google Scholar); Eps15, epsin, and amphiphysin seemed to be the main α-ear domain partners in peripheral cells. Interestingly, all of these different proteins bear DPF repeats first found in the AP-2 binding domain of Eps15 (26.Benmerah A. Bègue B. Dautry-Varsat A. Cerf-Bensussan N. J. Biol. Chem. 1996; 271: 12111-12116Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) and were shown to share with Eps15 the same α-ear binding site (51.Owen D.J. Vallis Y. Noble M.E. Hunter J.B. Dafforn T.R. Evans P.R. McMahon H.T. Cell. 1999; 97: 805-815Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). We could not formally exclude the possibility that the effects that we observed by expression of AP-2 binding sites containing Eps15 mutants on CCPs were because of a general inaccessibility of the α-ear domain. Nevertheless, the increasing number of α-ear binding proteins suggests a very important role of this AP-2 domain in clathrin coat function, an hypothesis confirmed by the fact that expression of α-ear inhibits clathrin-dependent internalization of Tf. What could be these functions? The α-ear domain could bring accessory proteins to the pit as it is thought for dynamin via amphiphysin (39.Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (403) Google Scholar). Another possibility, and both are not exclusive, is that α-ear plays an active role in the recruitment of AP-2 complexes onto the plasma membrane. This hypothesis is sustained by the fact that the ear domains of α-, γ- and δ-adaptins are the most divergent parts of assembly protein complexes (54.Robinson M.S. J. Cell Biol. 1989; 108: 833-842Crossref PubMed Scopus (108) Google Scholar, 55.Robinson M.S. J. Cell Biol. 1990; 111: 2319-2326Crossref PubMed Scopus (102) Google Scholar, 56.Simpson F. Peden A.A. Christopoulou L. Robinson M.S. J. Cell Biol. 1997; 137: 835-845Crossref PubMed Scopus (308) Google Scholar, 57.Dell'Angelica E.C. Ohno H. Ooi C.E. Rabinovich E. Roche K.W. Bonifacino J.S. EMBO J. 1997; 16: 917-928Crossref PubMed Scopus (331) Google Scholar) and could therefore discriminate between distinct docking machinery on their target membranes. The fact that an α-ear domain GFP fusion protein is efficiently targeted to CCP (8.Benmerah A. Bayrou M. Cerf-Bensussan N. Dautry-Varsat A. J. Cell Sci. 1999; 112: 1303-1311Crossref PubMed Google Scholar) shows that this domain effectively acts as a sufficient coated pit targeting signal. In this model, epsin and Eps15 seemed to be interesting potential candidates as constituents of the plasma membrane-associated AP-2 docking machinery. We thank V. Collin and V. Mallarde for helpful technical assistance and D. Ojcius for careful reading of the manuscript." @default.
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• W2080340766 cites W1550712876 @default.
• W2080340766 cites W161116775 @default.
• W2080340766 cites W1821459756 @default.
• W2080340766 cites W1965530119 @default.
• W2080340766 cites W1972812883 @default.
• W2080340766 cites W1974336925 @default.
• W2080340766 cites W1975081377 @default.
• W2080340766 cites W1977731456 @default.
• W2080340766 cites W1981593511 @default.
• W2080340766 cites W1985670865 @default.
• W2080340766 cites W1994167793 @default.
• W2080340766 cites W1996376778 @default.
• W2080340766 cites W1997878751 @default.
• W2080340766 cites W1999280423 @default.
• W2080340766 cites W2004972894 @default.
• W2080340766 cites W2011226078 @default.
• W2080340766 cites W2015799458 @default.
• W2080340766 cites W2025533401 @default.
• W2080340766 cites W2025929812 @default.
• W2080340766 cites W2029500247 @default.
• W2080340766 cites W2032554631 @default.
• W2080340766 cites W2042776102 @default.
• W2080340766 cites W2048869016 @default.
• W2080340766 cites W2056701485 @default.
• W2080340766 cites W2061462231 @default.
• W2080340766 cites W2071640907 @default.
• W2080340766 cites W2076364749 @default.
• W2080340766 cites W2078748790 @default.
• W2080340766 cites W2079286074 @default.
• W2080340766 cites W2085340755 @default.
• W2080340766 cites W2093139459 @default.
• W2080340766 cites W2093299463 @default.
• W2080340766 cites W2095939432 @default.
• W2080340766 cites W2099162346 @default.
• W2080340766 cites W2108328973 @default.
• W2080340766 cites W2110885272 @default.
• W2080340766 cites W2114022699 @default.
• W2080340766 cites W2120140317 @default.
• W2080340766 cites W2121357228 @default.
• W2080340766 cites W2134344512 @default.
• W2080340766 cites W2143205348 @default.
• W2080340766 cites W2149897005 @default.
• W2080340766 cites W2152678670 @default.
• W2080340766 cites W2152741987 @default.
• W2080340766 cites W2154335145 @default.
• W2080340766 cites W2155259994 @default.
• W2080340766 cites W2158177598 @default.
• W2080340766 cites W2162163372 @default.
• W2080340766 cites W2163777695 @default.
• W2080340766 cites W2166175048 @default.
• W2080340766 cites W4239169171 @default.
• W2080340766 cites W84752278 @default.
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