epslS and epslSR Are Essential Components of the Endocytic ...We addressed the relevance of eps 15 and epslSR to receptor-mediated internalization by testing endocytosis of the EGFR

[CANCER RESEARCH 57. 5498-5504. December 15. 19971

Advances in Brief

epslS and epslSR Are Essential Components of the Endocytic Pathway1

Roberta Carbone, Silvia FrÃ©,Gioacchin lamiÃ³lo, Francesca Belleudi, Patrizia Mancini, Pier Giuseppe Pelicci,Maria Rosaria Torrisi, and Pier Paolo Di Fiore2

Department of Experimental Oncology, European Institute of Oncology, 20141 Milan. Italy Â¡R.C.. S. F.. C. /.. P. C. P.. P. P. D. F.J; Dipartimento di Medicina Sperimentale ePatologia, UniversitÃ di Roma La Sapienyi, 00161 Rome, Italy Â¡F.Bâ€žP. M., M. R. T.Â¡:Istituto di Patologia Speciale Medica. University of Parma, 43100 Panna, Italy [P. G. P.];and Â¡Minilodi Microbiologia, University of Bari, 70124 Bari, Italy Â¡P.P. D. F. /

Abstract

epslS and epslSR are substrates of the epidermal growth factor (EGF)receptor kinase that are characterized by the presence of a protein:proteininteraction domain, the EH domain, and by their ability to bind to theclathrin adaptor protein complex adaptor protein 2. Indirect evidencesuggests that eps 15 and epslSR are involved in endocytosis. Here we showthat microinjection of antibodies against epslS and epslSR inhibits inter-

nalization of EGF and transferrin. In addition, fragments of epslS (encompassing its EH domains or the COOH-terminal region that binds to

adaptor protein 2) inhibit EGF internalization or endocytosis of Sindbisvirus. These results demonstrate that epslS and epslSR are essentialcomponents of the endocytic machinery.

Introduction

epsl5 (1) and epslSR (2) are related proteins originally identifiedas substrates for the kinase activity of the EGFR.3 They are charac

terized by the presence of three copies of a novel protein:proteininteraction domain, the EH domain (2). A number of observationshave recently linked eps 15. epslSR. and other EH-containing proteinsto coated pits-mediated internalization: (a) epslS (3, 4) and epslSR4

colocalize with markers of the plasma membrane clathrin-coated pits

and vesicles; (b) by electron microscopy. epslS is found at the rim ofbudding coated vesicles (4); (c) epslS and epslSR are constitutivelyassociated with the clathrin AP complex AP-2 (S);4 (d) a putative

160-kDa EH-containing protein is associated with the y-subunit of theGolgi adaptor complex AP-1 (6); (e) End3p, an EH-containing yeastprotein, is essential for endocytosis of the a-factor receptor (7); (f)

mutations in the EH domains of another yeast protein, Panlp, impairendocytosis (8, 9); and (#) the amino acid motif NPF (Asn-Pro-Phe)

is a ligand for the EH domain ( 10) and functions as an internalizationmotif in yeast (II). By a combined approach, using microinjectionand/or transfection of dominant negative mutants of eps 15 and mi-croinjection of anti-epslS and anti-epslSR affinity-purified Abs, we

Received 11/5/97; accepted 11/13/97.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' Supported in part by grants from Ministero dell'UniversitÃ e Ricerca Scientifica e

Tecnologica (to M. R. T.) and by grants from Associazione Italiana per la Ricerca sulCancro and from Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologicafrom Consiglio Nazionale delle Ricerche. Italy (to P. P. D. F., P. G. P.. and M. R. T.).Support from the European Community BIOMED-2 Programme (to P. P. D. F. andP. G. P.) and from the FerrerÃ² Foundation (to P. P. D. F.) is also acknowledged. G. I. isthe recipient of an Associazione Italiana per la Ricerca sul Cancro fellowship. R. C. andS. F. contributed equally to this work

" To whom requests for reprints should be addressed, at Istituto Europeo di Oncologia,

Via Ripamonti 435, 20141 Milan. Italy. Phone: 39-2-57489855; Fax: 39-2-57489851;E-mail: [email protected].

' The abbreviations used are: EGFR. epidermal growth factor receptor; EGF. epider

mal growth factor; AP. adaptor protein: GST. glutathione S-transferase: HA. hemoagglu-tinin epitope of the influenza virus; Ab, antibody; DAPI. 4'.6-diamidino-2-phenylindole;

CMV. cylomegalovirus; EGFP. enhanced green fluorescent protein.4 L. Coda, A. E. Salcini, S. Confalonieri, G. Pelicci, T. Sorkina. A. Sorkin. P. G.

Pelicci. and P. P. Di Fiore. EpslSR is a tyrosine kinase substrate with characteristics of adocking protein possibly involved in coated pits-mediated internalization. submitted forpublication.

directly addressed the relevance of these two molecules to the endocytic pathway. Our results demonstrate that epslS and epslSR areessential components of the endocytic machinery.

Materials and Methods

Microinjection, Virus Infection, and Transfection. NIH 3T3, CV-1, andCOS-7 cells were plated at a density of 5 X IO4 cells on round glass coverslips

that were coated with 2% gelatin (Sigma Chemical Co., St. Louis, MO).Injection pressure was set at 30-80 hPa. and the injection time was set at0.3-0.5 s. GST-EH, GST-L2. and GST-COIL fusion proteins (12) or control

GST was microinjected into the cytoplasm of the cells at a concentration of250 /xg/ml. Affinity-purified anti-epslS or anti-epslSR Abs were injected at a

concentration of 3 mg/ml. Injected cells were visualized by coinjection ofmouse IgG (Cappel: Organon Teknika Corp., West Chester, PA) or rabbit IgG(Jackson Immunoresearch, West Grove, PA) as marker proteins at a concentration of 500 fig/ml and 5 mg/ml. respectively. Cells were then processed forinternalization assays with EGF or transferrin or for Sindbis virus infection.

For EGF internalization assays, microinjected cells were washed in ice-coldbinding medium [20 mM HEPES (pH = 7.5) and 0.1% BSA] and treated with1 jug/ml tetramethylrhodamine-conjugated EGF (rhodamine-EGF; Molecular

Probes. Inc.. Eugene, OR) or with 100 ng/ml purified EGF (Upstate Biotechnology. Inc.) in DMEM at 4Â°Cfor 1 h. The EGF-containing medium was thenreplaced with warm DMEM. and cells were further incubated at 37Â°Cfor

15 min.For transferrin internalization assays, microinjected cells were incubated

with 50 /xg/ml tetramethylrhodamine-conjugated transferrin (rhodamine-Tf;Molecular Probes. Inc.) in DMEM at 37Â°Cfor 60 min.

For Sindbis infection assays, microinjected cells were infected by incubation with the Sindbis virus HR strain for l h at 37Â°Cas described previously

(13). At the end of incubation, the medium was replaced, and the infection wasallowed to proceed for 2.5-3.5 h at 37Â°Cbefore fixation.

Transfection of CV-1 and COS-7 cells was performed using the CaPO4 andthe DEAE-dextran standard methods, respectively.

Immunofluorescence. Cells were fixed with 4% paratbrmaldehyde in PBS(30 min at 25Â°C)and permeabilized with 0.1% Triton X-100 in PBS for 5 min.To identify injected cells, slides were incubated for l h at 25Â°Cwith FITC-

conjugated secondary Abs. either goat antimouse IgG (1:10; Cappel; OrganonTeknika Corp.) or goat antirabbit IgG (1:50; Jackson Immunoresearch). Cellswere then extensively washed in PBS and double-stained with polyclonal Abs

directed against Sindbis envelope glycoproteins (Ref. 13; 1:50 in PBS for l hat 25Â°C)followed by Texas Red-conjugated goat antirabbit IgG (1:50 in PBSfor 30 min at 37Â°C;Jackson Immunoresearch) or with l (Â¿g/mlmonoclonal Ab

directed against the EGFR (Ab-1; Oncogene Science) followed by Cy3-

conjugated goat antimouse IgG (1:400 in PBS). Nuclear counterstaining wasperformed by incubating coverslips for 5 min at 25Â°Cwith DAPI (1:3000;

Sigma Chemical Co.).

Results and Discussion

We addressed the relevance of eps 15 and epslSR to receptor-

mediated internalization by testing endocytosis of the EGFR undervarious conditions designed to block the action of these two molecules. In CV-1 cells (Fig. 1) and COS-7 cells (data not shown), EGF

and EGFR were efficiently internalized after exposure of intact cellsto EGF. CV-1 cells treated with EGF at 4Â°Cdid not internalize either

5498

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ept.15 AND cpslSR IN ENDOCYTOS1S

Fig. 1. Endocytosis of EGF and EGFR inCV-l cells. Cells were incubated with rhodamine-EGF (red; A. C, D. and F) or EOF (B and Â£)at 4Â°Cfor 60 min (A, R. and O and shiftedto 37Â°Cfor IS min (D, E, and F). In panels C and F. cells were acid-washed for 6 min with ice-cold 0.2 M acetic acid (pH 2.8) and 0.5 M NaCl lo remove surface-hound KGI;. The

EGFR (B and E) was visualized by staining with the monoclonal Ah Ab-1. followed by CyS-conjugated (red) goat antimouse IgCi. In all panels, nuclei counterstained with IJAPI arevisible in blue. All of the images were obtained using single fluorochrome filter sets for either rhodamine. fluorescein. or DAPI; recorded by a charge-coupled device camera (JVC.

Japan): and merged using a computer analysis program.

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cpslS AND epslSR IN ENDOCYTOS1S

Fig. 2. Inhibition of endocytosis of EOF and transferrin in CV-I cells by Abs to eps]5 and epsI5R. Cells were microinjected with polyclonal Abs specific for epsl5 (A and D) orepslSR (B and E) or with control rabbit IgG (C and Fi and then incubated with rhodamine-EGF (red'. A, B, and C) or rhodamine-transferrin (red', D. E, and F). Injected cells wereidentified using FITC-conjugaled (green} goat anlirabbit IgG. Cells injected with anti-epsl5 (A and DÃ¬and anti-epsl5R (B and E} Abs do not show the typical punctate staining (yellow)

of either internalized EOF or transferrin. which is visible instead in cells injected with control IgG (C and F).

the ligand (Fig. IA) or the receptor (Fig. IÃŸ),as witnessed by the receptor (Fig. If) underwent endocytosis. as evidenced by the ap-presence of a diffuse membrane-associated staining on immunofluo- pearance of a punctate/vesicular pattern of staining, indicating therescence. However, on shifting of the cells to 37Â°C.a temperature presence of the ligand/receptor complex in the various vesicles and

permissive for internalization, both the ligand (Fig. ID) and the organdÃes of the endocytic pathway. Acid treatment of intact cells,

5500



epslS AND epslSR IN ENDOCYTOSIS

1 200 400 600 800

EH Coil C-Terminalamino acid

position

EpslS

650 700

1.2

750â€”¿�iamino acid

position

Fig. 3. Inhibition of EGF intemalization inCV-1 cells by overexpression of the L2 region ofepslS. A, schematic of epslS showing the L2region (amino acids 623-750). B, C, and D, CV-1cells were transfected wilh either CMV-HA-L2(B), CMV-HA full-length epslS (O. or CMV-

EGFP (DÃ¬as control. Cells were then incubated at4Â°Cwith rhodamine-EGF and allowed to internalize at 37Â°C,as described in "Materials andMethods." Transfected cells were visualized ei

ther with an anti-HA Ab and a FITC-conjugated

(green) goat antimouse IgG (fl and Q or byspontaneous fluorescence of the EGFP (green inD). Internalized rhodamine-EGF is visible in red

in nontransfected cells and in yellow in transfected cells. Inhibition of intemalization by L2was present in more than 70% of the transfectedcells (B).

designed to remove surface-bound ligand, resulted in the completedisappearance of the membrane staining at 4Â°C(Fig. 1C), whileleaving unaltered the punctate/vesicular pattern at 37Â°C(Fig. IF).

We then microinjected Abs specific forepslS (1,2) orepsl5R4into

CV-1 (Fig. 2) and COS-7 cells (data not shown) to test their effects onreceptor-mediated intemalization. Both Abs were purified by affinity

chromatography onto the immobilized antigens and have been previously shown not to cross-react with other cellular proteins (1, 2). Inaddition, the affinity-purified anti-epsl5 Ab does not cross-react withepslSR and vice versa.4 Injection of the anti-epsl5 Ab into CV-1 cells

dramatically impaired EGF intemalization (Fig. 2A). Similar resultswere obtained after microinjection of the anti-epsl5R Ab (Fig. 2ÃŸ).

Conversely, the injection of control purified IgGs did not alter theendocytic process (Fig. 2C). Similar results were obtained in COS-7cells (data not shown). Interestingly, injection of either anti-epsl5(Fig. 2D) or anti-epsl5R (Fig. 2E), but not of control IgGs (Fig 2F),

also blocked intemalization of transferrin, thus suggesting that epslSand epslSR are involved in some general step of the endocyticprocess.

epslS and epslSR share a modular structure (Fig. 3A) composed of

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epslS AND epslSR IN ENDOCYTOSIS

Fig. 4. Inhibition of Sindbis virus endocytosis and infection in NIH 3T3 cells microinjected with GST-fusion proteins. Cells were microinjected with the GST-EH (a and b), GST-L2(c and i/). and GST-COIL (c and/) fusion proteins or with GST alone (# and it) and then incubated with Sindbis virus. Endocytosis of the virus and the subsequent infection was allowedto proceed for 3.5 h. Injected cells were identified using FITC-conjugated goat antimouse IgG (left panels). Double-staining with anti-Sindbis spike Abs, followed by TexasRed-conjugated goat antirabbit Igu (right panels) was performed to detect viral protein synthesis in infected cells. Cells microinjected with GST-EH (asterisk in h) or with GST-L2(asterisks in d) are not stained by anti-Sindbis Ab and therefore seem uninfecled. Cells injected with GST-COIL (asterisks in/) or GST (asterisks in h) are stained by anti-Sindbis spike

Abs. assessing a viral infection process indistinguishable from that of uninjected cells among the injected cells (h, d, f, and h).

5502



epsIS AND epslSR IN ENDOCYTOSIS

a NH2-terminal portion, which contains three EH domains (EH region), a central putative coiled-coil region composed of several contiguous heptads (COIL region), and a COOH-terminal domain displaying multiple copies of an aspartate-proline-phenylalanine motif, aproline-rich region capable of interacting with the SH3 domain of theCrk proto-oncogene (14), and a region responsible for binding to AP-2(12, 15). The minimal binding region of epslS to AP-2 was previouslyidentified as a â€”¿�130amino acid region (L2 domain; Ref. 12) con

taining three small determinants (A, n, and c in Fig. 3A; Ref. 12) of~10 amino acids, each of which participates in AP-2 binding (Fig.

3/1). We engineered a CMV promoter-based eukaryotic vector expressing the L2 region of epslS as a HA-tag fusion protein (CMV-HA-L2 vector). As shown in Fig. 3ÃŸ,CV-1 cells transfected with theCMV-HA-L2 vector exhibited a dramatic reduction of the intemal-ization phenotype. As a control, we expressed a CMV-HA-epsl5vector encoding the entire HA-tagged epslS protein or a vectorencoding the EGFP under the control of the CMV promoter (CMV-EGFP; Fig. 3, C and D), neither of which detectably affected inter-nalization. Similar results were obtained in COS-7 cells (data not

shown).The role of different domains of epslS in the endocytic pathway

was further evaluated by testing the entry of Sindbis virus in cellsmicroinjected with individual domains of epslS, engineered as fusionproteins with GST (GST-EH, GST-COIL, and GST-L2). A similar

approach has been previously used to analyze the involvement of coatproteins in the control of endocytic steps, using vesicular stomatitisvirus (Ref. 16). Both Sindbis and vesicular stomatitis virus are enveloped viruses known to enter the cells by clathrin-mediated endocyto-

sis and to require endosomal acidic pH for fusion and infection (for areview, see Ref. 17). NIH 3T3 cells were microinjected with purifiedGST-EH, GST-L2, and GST-COIL, and injected cells were identified

by coinjection of mouse IgG as marker protein. After microinjection,cells were infected with Sindbis virus. Efficient virus endocytosis andsubsequent infection were then assessed by detection of viral spikeproteins on intracellular sites along the biosynthetic exocytic pathwayof the infected cells. Cells injected with GST-EH (Fig. 4. A and B) andGST-L2 (Fig. 4. C and D) did not appear to be infected because of thelack of reactivity with anti-Sindbis spike Abs. On the contrary, in cellsinjected with GST-COIL (Fig. 4, E and F) or with GST alone (Fig. 4,

G and //), the presence of intracellular viral glycoproteins indicated anunperturbed infection process. Thus, EH and L2 domains of epslS,but not the COIL domain, were able to inhibit viral endocytosis, mostlikely by competing with the endogenous epslS.

The sum of our results indicates that epslS and epslSR are essentialcomponents of the endocytic pathway. Receptor-mediated endocyto

sis is known to be a saturable pathway (18); however, epslS is notlikely to represent the limiting step in this process. Overexpression offull-length epslS did not increase internalization of the EGFR (Fig.3C)- In addition, we performed internalization kinetics of I25l-labeled

EGF in CV-1 and COS-7 cells engineered to overexpress epslS up to20-fold and detected no significant increase in internalization (datanot shown). However, interference with epslS or epslSR functions,obtained with specific Abs or dominant negative mutants, inhibitedinternalization of EGF, transferrin, and Sindbis virus. Thus, epslS andepslSR are likely to act at some universal step in the endocyticprocess.

The L2 protein, which contains the region of epsl5 that is necessaryand sufficient for binding to AP-2. inhibited the internalization of both

EGFR and the Sindbis virus. A simple explanation for the relevanceof the epsl5/AP-2 interaction to the internalization process is en

hanced recruitment of receptors, such as EGFR. to forming clathrin/AP-2-containing coats through physical interaction with epslS. In

deed, evidence has been recently reported for coimmunoprecipitation

of the EGFR with epsl5, although it has not yet been establishedwhether this association is direct or mediated through other molecules(3). Alternatively, epslS might act as part of the molecular machinerydirecting AP-2 to the plasma membrane. In this scenario, AP-2 might

serve as the adaptor recruiting receptors into forming coats, as suggested by the ability of AP-2 to directly bind to activated EGFR (19).

Our results also point to an important role of the EH domains ofepslS in endocytosis. Such a role had been proposed previously (10).based on converging evidence obtained in yeast and mammals, butwas not directly demonstrated. Here we show that a GST-EH protein

acts as a dominant negative on Sindbis virus infection, most likely byinterfering with viral endocytosis. The molecular bases for EH involvement in the endocytic process are likely to be found in itsprotein:protein interaction ability. We have recently identified theamino acid triplet NPF (Asn-Pro-Phe) as a ligand for EH domains(10). Thus, NPF-containing proteins are candidates as EH partners inthe internalization machinery. In SaccharomyCfi cerevisiae, NPF-

based sequences have been shown to represent internalization motifsin a STE2p/Kex2p chimera and in STE3p. the a-factor receptor (11).In this instance, a direct interaction between EH-containing proteins

(like End3p or Panlp) and the internalizing receptor can be envisioned. However, this cannot be the case for epslS (or epsl5R),because the EGFR and the transferrin receptor do not display NPFmotifs. Thus, the search for NPF-containing binding partners of epslS

and epslSR will likely lead to the identification of other importantcomponents of the endocytic machinery.

It is to be noted that, in repeated experiments, we were not able todetect a clear-cut effect of injection of GST-EH on EGFR internal

ization (data not shown). The discrepancy between the data obtainedwith EGFR internalization and the data obtained with the Sindbissystem is not readily explained. It is possible that the immunofluo-

rescence approach that we used to study EGFR internalization onlyallows for qualitative analysis, leaving moderate alterations in theinternalization rate undetectable. In this framework, the Sindbis system might allow for higher sensitivity, necessitating only inhibition ofa few internalization events (a multiplicity of infection of 50 wasused). However, the possibility must also be considered that theinhibition by EH domains is exerted at a step in the endocytic pathwayother than internalization, as, for example, in the transport from earlyendosomes to the more acidic late endosomes, a step required for viralfusion and infection ( 16). In this scenario, epslS might be involved inthe endocytic process at multiple stages, an intriguing possibility thatwarrants further investigation.

Acknowledgments

We thank Dr. Stefano Bonatti for providing Sindbis virus and Abs and Ilio

Piras for excellent photographic work.

References

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epsl5 AND epslSR IN ENDOCYTOSIS

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1997;57:5498-5504. Cancer Res Roberta Carbone, Silvia Fré, Gioacchin Iannolo, et al. Pathwayeps15 and eps15R Are Essential Components of the Endocytic

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epslS and epslSR Are Essential Components of the Endocytic ...We addressed the relevance of eps 15 and epslSR to receptor-mediated internalization by testing endocytosis of the EGFR