Grb2 Is Necessary for EGF-Stimulated EGFR Uptake 1793

Research Article 1791 Role of Grb2 in EGF-stimulated EGFR internalization

Tetsuo Yamazaki1, Kristien Zaal2, Dale Hailey2, John Presley2, Jennifer Lippincott-Schwartz2 and Lawrence E. Samelson1,* 1Laboratory of Cellular and Molecular Biology, Division of Basic Science, National Cancer Institute, Bethesda, MD 20892-4255, USA 2Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA *Author for correspondence (e-mail: [email protected])

Accepted 28 January 2002 Journal of Cell Science 115, 1791-1802 (2002) © The Company of Biologists Ltd

Summary Grb2 is an adaptor molecule that couples membrane transferrin uptake, had no effect on EGF-induced receptors such as the epidermal growth factor receptor internalization of EGFR. YFP-tagged Grb2 containing an (EGFR) to intracellular signaling pathways. To gain insight inhibitory mutation in either N- or C-SH3 domain into the trafficking pathways followed by these molecules redistributed to the plasma membrane upon EGF after activation by EGF, we visualized Grb2 and EGFR stimulation, but the macropinocytic structures containing fused to GFP spectral variants in single live cells. In Grb2-YFP and EGFR-CFP did not translocate inward and nonstimulated cells, Grb2-YFP was primarily localized appeared to remain tethered to the plasma membrane. This diffusely in the cytoplasm, whereas EGFR-CFP was found suggested that the Grb2 SH3 domain was responsible for on the plasma membrane and in endocytic structures coupling the membranes containing EGFR with localized in the perinuclear area. Within 1 minute of EGF downstream effectors involved in internalization of these stimulation, Grb2 redistributed to the plasma membrane membranes. Transferrin uptake was unaffected in the where it bound EGFR-CFP in an SH2 dependent manner. presence of all of the SH3 domain mutants, consistent with The plasma membrane then began to dynamically ruffle, the EGF-stimulated EGFR internalization pathway being and Grb2-YFP and EGFR-CFP were found to internalize clathrin-independent. These results demonstrate a role for together in large macropinocytic structures. These Grb2 in events associated with a macropinocytic structures were morphologically distinct from internalization pathway for EGFR in activated cells. conventional, clathrin-derived endosomes and did not label with transferrin, AP-2 or clathrin heavy chain. Evidence Movies available on-line that these structures did not require clathrin for internalization came from experiments showing that expression of the C-terminus of AP-180, which inhibited Key words: Grb2, EGFR, SH3 domain, Endocytosis, GFP

Introduction The EGFR has been extensively investigated over many Adaptor molecules are modular protein structures containing years, and it serves as an excellent model of a receptor that protein and lipid binding domains, and stretches of sequence mediates such diverse cellular phenomena as proliferation and to which some of these domains bind (Pawson, 1995). differentiation (Sorkin, 1998). The EGF ligand induces Adaptors are essential to the signal transduction process in dimerization of the receptor, which leads to activation of the cells, and link ligand-activated integral membrane receptors to protein tyrosine kinase activity intrinsic to the receptor. complex intracellular biochemical pathways. Less-well studied Phosphorylation of multiple tyrosine residues on the cytosolic is the role of these molecules in downregulating signaling domain of the receptor creates binding sites for interaction with events. Our studies focus on the fate of the epidermal growth enzymes such as phospholipase Cγ1 [PLCγ1 (Zhu et al., 1992)] factor receptor (EGFR) and the Grb2 adaptor molecule upon and phosphatidylinositol 3-kinase [PI3K (Hu et al., 1992)], and activation of the receptor at the cell surface, and address the adaptors Shc (Okabayashi et al., 1994; Soler et al., 1994) questions about the function of Grb2 in EGFR dynamics. and Grb2 (Buday and Downward, 1993; Lowenstein et al., Grb2 consists of an SH2 domain flanked by two SH3 1992; Okutani et al., 1994). Such interactions are critical to the domains (Lowenstein et al., 1992; Rozakis-Adcock et al., induction of those signaling events that the receptor regulates. 1992). The SH2 domain, by its interactions with Attenuation of EGFR signals has also been extensively phosphotyrosine residues in a specific sequence context, studied. One mechanism for downregulation is EGFR mediates binding to a variety of activated receptors and adaptor internalization. It has been reported that mutant EGF receptors molecules. The SH3 domains of Grb2 bind proline-rich that are defective in internalization lead to ligand-induced sequences such as those found in the Ras guanine nucleotide transformation (Wells et al., 1990), supporting the importance exchange factor, SOS (Chardin et al., 1993; Egan et al., 1993; of receptor downregulation. To date, the role for Grb2 in EGFR Li et al., 1993; Rozakis-Adcock et al., 1993). Hence, Grb2 internalization has been controversial, and biochemical and links ligand-activated receptors coupled to tyrosine kinases to immunofluorescence microscopy studies have produced the distal signaling apparatus. conflicting results (Chang et al., 1991; Chang et al., 1993; 1792 Journal of Cell Science 115 (9)

Wang and Moran, 1996). The exact function of Grb2 in EGF R86K mutation was ligated to the BglII-digested pYFP/Grb2 receptor dynamics remains poorly understood. (pYFP/R86K Grb2). In this study we made use of the GFP spectral variants, YFP (3) pYFP/P49L Grb2 (encoding NSH3m-YFP). The BamHI-BglII and CFP (Ellenberg et al., 1998; Ellenberg et al., 1999), which fragment from the pGEX-3X plasmid containing human Grb2 with a we fused to Grb2 and EGFR, respectively. Because YFP and P49L mutation was ligated to the BglII-digested pYFP/Grb2 (pYFP/P49L Grb2). CFP are distinguishable from each other based on the (4) pYFP/P49L, G203R Grb2 (encoding NCSH3m-YFP). A PCR difference in their fluorescence excitation and emission product with the G203R mutation was created using pGEX-3X patterns, we were able to follow the spatio-temporal plasmid containing G203R Grb2 as a template with the oligos 5′ TAT relationship of these two chimeric molecules (Grb2-YFP and CAC AGA TCT ACA TCT 3′ and 5′ GGC GAC CGG TCC ACC EGFR-CFP) in single live cells using confocal microscopy. TCC ACC GAC GTT CCG GTT CAC GGG 3′. Then, the BglII/AgeI- We show that Grb2-YFP binds EGFR-CFP in an SH2- digested PCR product was ligated to the BglII-AgeI fragment of dependent fashion. Following activation, both internalize pYFP/P49L Grb2 (pYFP/P49L, G203R Grb2). The sequence of this together in large macropinocytic structures that are construct was verified using the Sequenase II DNA sequencing kit. temporally, morphologically and biochemically distinct from (5) pYFP/G203R Grb2 (encoding CSH3m-YFP). The NheI-BglII conventional transferrin-containing endocytic structures. fragment of pYFP/P49L, G203R Grb2 was replaced with XbaI-BglII fragment from pYFP/Grb2 to remove the P49L mutation Expression of Grb2-YFP containing mutations in either SH3 (pYFP/G203R Grb2). domain did not block recruitment of Grb2 to the plasma (6) pCFP/EGFR (encoding EGFR-CFP). The XhoI fragment from membrane upon EGF stimulation but impeded the subsequent LTR2-EGFR (kindly provided by J. Rubin, Laboratory of Cellular and inward translocation of the macropinocytic structures enriched Molecular Biology, NCI, NIH) containing the entire human EGFR in Grb2 and EGFR. These results demonstrate a role of Grb2 was ligated to XhoI-digested pBluescript II KS (pBKS-EGFR). The in events associated with EGFR internalization in activated HindIII-KpnI fragment from pBKS/EGFR was subcloned into cells. HindIII/KpnI-digested pCFP-N1 vector (pCFP/EGFR Hind-Kpn). pCFP/EGFR Hind-Kpn was digested with SalI, filled in with Klenow fragment, and then religated (pCFP/Sal BL). The junction between Materials and Methods the EGFR and CFP was created by PCR using pBKS-EGFR as a template together with the oligos 5′ GCC ACC CAT ATG TAC CAT Reagents C 3′ and 5′ GGC CCG CGG TGC TCC AAT AAA TTC ACT G 3′. Reagents used in this article were purchased as indicated: murine EGF The AccI/SacII digested PCR product was ligated to the AccI-SacII (Calbiochem); mouse anti-EGF, anti-Grb2, anti-SOS1/2, anti-Cbl and fragment of pCFP/Sal BL (pCFP/EGFR). anti-dynamin II antibody (Santa Cruz Biotechnology); sheep anti- EGFR antibody (Upstate Biotechnology); anti-GFP antibody (monoclonal and polyclonal: Clontech); TRITC-labeled rabbit anti- Transfection goat IgG and goat anti-mouse IgG antibody (Kirkegarrd & Perry COS-7 cells were transfected by electroporation using 10 µg of Laboratories Inc.); biotin-XX EGF conjugate (B-EGF) (Molecular pCFP/EGFR and 5 µg of either pYFP/Grb2 or pYFP/mutant Grb2. 10 Probes); biotinylated transferrin (B-Tfn) and streptavidin-Quantum µg of either pYFP/Grb2 or pYFP/mutant was transfected into A431 Red conjugate (Sigma). cells. Electoroporation was performed at 250 V and 500 µF for COS- 7 cells and at 310 V and 960 µF for A431 cells with a Gene Pulser (Bio-Rad). Cells Both A431 cells and COS-7 cells were grown in complete DMEM medium (DMEM containing 10% fetal bovine serum (FBS), 2 mM Confocal time-lapse imaging glutamine, and 50 µg/ml gentamicin). Transfected cells were grown in coverglass chambers (LabTek) for 24 hours in complete DMEM medium. The medium was then replaced with DMEM medium free of phenol red and supplemented with 0.5% Constructs FBS, 2 mM glutamine and 50 µg/mg gentamicin. After a further 16 Both YFP and CFP (human codon-optimized W7) GFP variants were hours of incubation, the cells were subjected to time-lapse imaging. described previously (Ellenberg et al., 1998; Ellenberg et al., 1999). For dual color GFP time-lapse imaging (Ellenberg et al., 1998; pYFP-N1 and pCFP-N1 vectors have the same backbone as pEGFP- Ellenberg et al., 1999), the chambers were mounted on a temperature- N1, except the EGFP sequence is replaced by the YFP and the CFP controlled stage of a confocal microscope (model LSM 410; Carl sequence, respectively. The construct encoding the C-terminal domain Zeiss) using the 63× objective (NA 1.4). To image YFP, the 488 nm of AP180-C (Ford et al., 2001) was a kind gift of H. McMahon, MRC laser line (Omnichrome Series 43, Omnichrome Inc.) was used with Laboratory of Molecular Biology, Cambridge. a standard dual FT 488/568 dichroic and a BP 515-560 (both Carl Plasmid constructions were conducted as follows: (1) pYFP/Grb2 Zeiss). To image CFP, the 413 nm Kr laser line (Coherent Enterprise (encoding wild-type Grb2-YFP). The BamHI-EcoRI fragment from II, Coherent Inc.) was used in conjunction with a FT440 dichroic and the pGEX-3X plasmid containing human Grb2 was ligated to the a LP 460 emission filter (both Chroma Technology Corp.). Images of BamHI-EcoRI fragment of pBluescript II KS (pBKS/Grb2). The Grb2-YFP and EGFR-CFP were captured just before addition of EGF AflIII-NspI fragment was removed from pBKS/Grb2, and then the (100 ng/ml for COS-7 and 500 ng/ml for A431, Calbiochem) directly vector was religated using the oligos 5′ TTT CCC CGC AAT TAT to the chamber, and subsequent images were taken every 30 seconds. GTC ACC CCC GTG AAC CGG AAC GTC GGT GGA GGT GGA Time-lapse sequence was recorded using a macro program that allows CCG GTA 3′ and 5′ CAT GTA CCG GTC CAC CTC CAC CGA CGT autofocusing on the coverslip surface in the reflection mode before TCC GGT TCA CGG GGG TGA CAT AAT TGC GGG GAA ACA capturing confocal fluorescence images. TG 3′ (pBKS/Grb2-4G). The SacI-AgeI fragment from pBKS/Grb2- 4G was ligated to SacI/AgeI-digested pYFP-N1 vector (pYFP/Grb2). (2) pYFP/R86K Grb2 (encoding SH2m-YFP). The BamHI-BglII Immunofluorescence staining of fixed cells fragment from the pGEX-3X plasmid containing human Grb2 with a COS-7 cells were grown in coverglass chambers (LabTek) for 24 Grb2 is necessary for EGF-stimulated EGFR uptake 1793

hours in complete DMEM medium, which was then replaced with DMEM medium free of phenol red supplemented with 0.5% FBS, 2 mM glutamine, and 50 µg/mg gentamicin. After a further 16 hours incubation, EGF stimulation (100 ng/ml) was carried out. Cells were then ﬁxed in 3.7% paraformaldehyde in PBS for 30 minutes at room temperature, washed (three times) in PBS containing 10% fetal bovine serum (PBS/FBS), permeabilized using 0.1% Triton X-100 in PBS for 4 minutes at room temperature, washed (three times), and incubated for 45 minutes in PBS/FBS for preblocking. Cells were then incubated with anti-EGFR antibody in PBS/FBS for 45 minutes at room temperature, washed, and incubated with TRITC-labeled goat anti- mouse IgG antibody for 45 minutes, followed by washing with PBS (three times). Stained samples were viewed using the 568-nm laser line of a confocal laser scanning microscope using the 63× objective (NA 1.4).

Uptake assays Transfected cells were incubated as described above in complete DMEM medium, then in DMEM medium containing 0.5% FBS before assays. Cells were incubated with transferrin-TRITC conjugate (50 µg/ml) (Molecular Probes) at 37°C for 40 minutes. Following three washes with phenol-red-free DMEM medium containing 0.5% FBS, uptake of transferrin-TRITC was monitored in the absence of EGF by time-lapse imaging. For Fig. 1. Chimeric molecules containing Grb2 or EGFR. uptake assays in the presence of (A) YFP-tagged Grb2. Mutations in each chimeric EGF, cells were incubated with molecule are indicated. GGGG; four-glycine linker. (B) C- transferrin-TRITC at 4°C for 40 terminally tagged EGFR. (C) Immunoblot of Grb2-YFP. minutes, washed with ice-cold Lysates of COS-7 cells transfected with either wild-type phenol-red-free DMEM containing Grb2 or mutant Grb2 fused to YFP were analyzed by 0.5% FBS, then incubated with the western blotting (lane 1, YFP alone; lane 2, wild-type same medium containing EGF (100 Grb2-YFP; lane 3, SH2m-YFP; lane 4, NSH3m-YFP; lane ng/ml) at 37°C. Incorporation of 5, CSH3m-YFP; lane 6, NCSH3m-YFP). The blot was transferrin-TRITC was monitored probed with anti-GFP antibody and reprobed with anti- using confocal microscopy. Grb2 antibody. (D) Western blot of EGFR-CFP. Lysates of COS-7 cells expressing either CFP alone or EGFR-CFP were analyzed by western blotting. The blot was probed with anti-GFP antibody (lanes 1,2) and reprobed with anti-EGFR antibody (lanes 3,4). Arrows point to EGFR-CFP (upper band) and Sequestration assay endogenous EGFR (lower band). Molecular weight marker proteins (kDa) are indicated at the left. Internalization of either EGFR or (e) Grb2-YFP associates with EGFR in an EGF-dependent manner. Wild-type Grb2-YFP was TfnR was measured by avidin immunoprecipitated by anti-GFP antibody from the lysate of A431 cells expressing wild-type Grb2- inaccessibility (Vieira et al., 1996) YFP either before or after EGF stimulation. The blot was probed with anti-GFP antibody (bottom) or using a FACScan (Becton- anti-EGFR antibody (top). (F) Grb2-YFP constitutively associates with SOS, Cbl and dynamin II. Dickinson). Transfected COS-7 GFP immunoprecipitates from lysates of A431 cells transfected with either YFP alone (lane 1) or cells were dissociated for 5 minutes wild-type Grb2-YFP (lane 2) were immunoblotted with either anti-SOS, anti-Cbl, anti-dynamin II or at 37°C in PBS containing 5 mM anti-GFP antibody. EDTA, washed and resuspended at 1794 Journal of Cell Science 115 (9)

Fig. 2. EGF-induced redistribution of Grb2-YFP to the cell periphery in A431 cells. A431 cells were transfected with YFP fused to either wild-type Grb2, the SH2 domain mutant or the SH3 domain mutants of Grb2, as follows. (A,B) Wild- type Grb2-YFP, (C,D) SH2m- YFP, (E,F) NSH3m-YFP, (G,H) CSH3m-YFP and (I,J) NCSH3m-YFP. Images of live A431 cells in each column were taken prior to (A,C,E,G,I) and 1 minute after EGF stimulation (B,D,F,H,J) using confocal microscopy. Bars, 5 µm.

2×106 cells/ml in DMEM containing 0.5% BSA and 25 mM Hepes removed by washing with buffer A at 0°C. Cells were split into (buffer A) at 0°C. Either B-EGF or B-Tfn was added to a final aliquots (2×105 cells), incubated at 37°C for the indicated times and concentration of 40 ng/ml or 10 µg/ml, respectively, and incubation then returned to icy water. 5 µl of streptavidin-Quantum Red was was continued for 15 minutes at 0°C. Unbound B-EGF or B-Tfn was added to 20 µl aliquots of cells resuspended in PBS containing 0.1% BSA and 0.1% sodium azide (staining buffer). Following washing with staining buffer, data was collected on a FACScan and analyzed using CELLQuest software (Becton-Dickinson). Avidin inaccessibility (AI) was calculated as in the equation, AI (%) = {1 – (MFt – BG) / (MF0 – BG)} × 100 , where MFt is the mean fluorescence intensity of cells stimulated for the indicated times (t), MF0 is the mean fluorescence intensity of unstimulated cells (time 0), and BG is the mean fluorescence intensity of cells incubated at 0°C with streptavidin-Quantum Red alone. To determine the effect of EGF stimulation on Tfn uptake, avidin inaccessibility was similarly measured except that cells were incubated with B-Tfn (10 µg/ml) plus EGF (100 ng/ml). Nonspecific binding of either B-EGF or B-Tfn was determined in the presence of at least 100-fold molar excess unlabeled EGF or Tfn, respectively.

Immunoprecipitation assays Unstimulated or EGF-stimulated transfected cells were lysed in Brij/Octyl glucoside lysis buffer (1% Brij 97, 0.5% octyl glucoside, 150 mM NaCl, 5 mM EDTA) on ice for 30 minutes. After centrifugation, the supernatant was collected. Lysates were subjected to immunoprecipitation with an anti-GFP antibody. Anti-GFP immunoprecipitates were then analyzed by SDS-PAGE and western blotting using relevant antibodies

Fig. 3. Internalization of EGFR-CFP with Grb2-YFP induced by EGF. Individual live COS-7 cells co- expressing wild-type Grb2-YFP and EGFR-CFP were monitored over time by confocal time-lapse imaging. Protein movement of Grb2-YFP (middle) and EGFR-CFP (left) upon EGF stimulation is shown. Arrows point to the characteristic internalized structures (see text). Grb2 is necessary for EGF-stimulated EGFR uptake 1795

Fig. 4. Colocalization of Grb2-YFP with clathrin heavy chain, AP-2 and dynamin. COS-7 cells co-expressing EGFR-CFP with wild-type Grb2-YFP were stimulated. Cells were then immunostained with antibodies directed at clathrin heavy chain (CHC), AP-2 or dynamin, followed by a TRITC-conjugated secondary antibody (middle). Arrows point to co-localized structures.

function as tyrosine phosphorylation upon EGF stimulation, association with Shc and endocytosis via an interaction with AP-2 (Carter and Sorkin, 1998). All GFP chimeric molecules in this study demonstrated the expected apparent molecular weight by western blotting using an anti-GFP antibody (52 kDa for Grb2-YFP and 207 kDa for EGFR-CFP), and were detected speciﬁcally with antibodies directed against Grb2 or EGFR (Fig. 1C,D).

Wild-type Grb2 tagged with YFP functions similarly to Grb2 Grb2 is known to associate with a variety of receptors upon receptor ligation via interactions of its SH2 domain and phosphotyrosine residues. In addition, the SH3 domains of Grb2 mediate Grb2 binding to critical signaling molecules such as SOS (Chardin et al., 1993; Egan et al., 1993; Li et al., 1993; Rozakis-Adcock et al., 1993), Cbl (Buday et al., 1996; Hanazono et al., 1996; Meisner et al., 1995), WASP (Cory et al., 1996; She et al., 1997), Results MEKK1 (Pomerance et al., 1998) and dynamin (Ando et al., GFP chimeric proteins that contain Grb2 or EGFR 1994; Barylko et al., 1998; Gout et al., 1993; Herskovits et al., Extensive biochemical evidence demonstrates that Grb2 has a 1993; Lin et al., 1997; Vidal et al., 1998; Wang and Moran, cytosolic distribution in resting cells. Upon activation of 1996). certain receptor tyrosine kinases or ligation of receptors that To test whether Grb2-YFP behaves like Grb2, we are coupled to tyrosine kinases, Grb2 translocates to the performed immunoprecipitation assays using cell lysates from plasma membrane to bind target molecules. The fate of Grb2 A431 cells transfected with wild-type Grb2-YFP. EGFR was following these events is less well characterized. In order to coimmunoprecipitated with Grb2-YFP upon EGF stimulation follow this translocation visually and to observe the entire (Fig. 1E), suggesting that Grb2-YFP becomes inducibly course of Grb2 molecular dynamics, we created Grb2-YFP bound to EGFR as does endogenous Grb2. Additionally, anti- and EGFR-CFP. GFP immunoprecipitates from A431 cells expressing wild- In addition to preparing a chimera of wild-type Grb2 with type Grb2-YFP (Fig. 1F, lane 2) contained SOS, Cbl and YFP (Grb2-YFP, Fig. 1A), we also added YFP to the C- dynamin without EGF stimulation, showing the constitutive terminus of several Grb2 mutants (SH2m-, NSH3m-, CSH3m- associations of Grb2-YFP with these critical molecules. Based and NCSH3m-YFP) in order to investigate the contribution of on its binding speciﬁcity, Grb2-YFP behaves similarly to the individual SH2 and SH3 domains to the dynamics of Grb2. Grb2. The R86K (arginine 86 to lysine) mutation in the SH2 domain is known to disrupt SH2 domain-phosphotyrosine interactions (Clark et al., 1992). The mutations P49L (proline 49 to leucine) SH2-domain-dependent translocation of Grb2-YFP to and G203R (glycine 203 to arginine) are in the N- and C- the plasma membrane terminal SH3 domains, respectively. These mutants of Grb2 To demonstrate by imaging that EGF stimulation induces the correspond to loss-of-function phenotypes in Caenorhabditis redistribution of Grb2 to the plasma membrane, we expressed elegans (Clark et al., 1992) and are known to block SH3- Grb2-YFP, as well as Grb2 mutant-YFP fusion proteins in mediated interactions (Egan et al., 1993). For all Grb2-YFP A431 cells. A431 human epidermoid cells were used as they fusion proteins, four glycine residues were placed as a linker bear abundant EGFR (Gamou et al., 1984; Haigler et al., 1979; between Grb2 and YFP to increase ﬂexibility in that region. Krupp et al., 1982). Grb2-YFP was diffusely cytosolic in the We fused CFP to the EGFR C-terminus (EGFR-CFP, Fig. resting state (Fig. 2A). Translocation towards the cell periphery 1B). A similar chimera, where EGFR was tagged with the began immediately after EGF was added. Redistribution of enhanced GFP (EGFP) at the C-terminus, was already reported Grb2-YFP to the plasma membrane was completed within a to behave like endogenous EGFR, exhibiting such expected minute of EGF stimulation (Fig. 2B). Concurrently, the amount 1796 Journal of Cell Science 115 (9)

A observations support the biochemical evidence for EGF- induced translocation of Grb2 from the cytosol to the plasma membrane where Grb2-EGFR interactions occur. Note that a signiﬁcant amount of Grb2 accumulates in the nucleus after EGF stimulation as previously reported (Romero et al., 1998). We expressed SH2m-YFP in A431 cells to conﬁrm that translocation to the membrane was dependent on the SH2 domain interactions with phosphorylated EGFR. As expected, no redistribution of SH2m-YFP was seen upon EGF stimulation (Fig. 2C,D), indicating that translocation of YFP-tagged Grb2 to the plasma membrane was dependent on an intact Grb2 SH2 domain. Grb2 SH3 domain mutants tagged with YFP (NSH3m-, CSH3m- and NCSH3m-YFP) translocated to the plasma membrane with similar kinetics to that of wild-type Grb2-YFP in response to EGF (Fig. 2F,H,J). Thus, EGF-induced redistribution of Grb2-YFP to the plasma membrane was not only SH2 domain dependent, but did not require intact SH3 domains. B

1 00 EGFR-CFP recruits Grb2-YFP to the plasma membrane and is internalized with Grb2-YFP To address the spatio-temporal relationship between 8 0 Grb2 and EGFR upon receptor activation with EGF, we co-expressed both wild-type Grb2-YFP (Grb2-YFP) and EGFR-CFP. To avoid any complication because of the large amount of endogenous EGFR on A431 cells (1- 6 0 2×106/cell) (Gamou et al., 1984; Haigler et al., 1979;

al) Krupp et al., 1982), Grb2-YFP and EGFR-CFP were co- t o

f t non-transfec(-EGF) expressed in COS-7 cells, which have low levels of o 4 0 endogenous EGFR. In unstimulated cells, EGFR-CFP Grb2 (-EGF) B-Tfn sequestered (% localized both to the plasma membrane and to punctate NSH3m (-EGF) structures scattered in the periphery and the perinuclear non-transfec(+EGF) region, whereas Grb2-YFP was diffusely cytosolic (Fig. 2 0 3, pre-EGF stimulation). A signiﬁcant pool of Grb2-YFP Grb2 (+EGF) redistributed to the plasma membrane within a minute of NSH3m (+EGF) EGF stimulation (Fig. 3, 1 minute), where it co-localized with EGFR-CFP. Note also that Grb2-YFP also 0 0 2 4 6 8 1 0 accumulated transiently in the nucleus, as found in A431 Time (min) cells. EGF-induced redistribution of Grb2-YFP in COS- 7 did not occur unless EGFR-CFP was co-expressed Fig. 5. Transferrin localization and dynamics in the presence of Grb2-YFP or (data not shown), which suggested that the NSH3m-YFP. (A) Transferrin uptake by COS-7 cells co-expressing EGFR- phosphorylation of endogenous EGFR was not sufficient CFP with either wild-type Grb2-YFP (top) or NSH3m-YFP (bottom) was to make translocation of Grb2-YFP detectable in this monitored by confocal time-lapse imaging. Images were collected 20 min context. Importantly, Grb2-YFP was not recruited to the after EGF stimulation following 40-min incubation with transferrin-TRITC. internal structures containing EGFR-CFP upon EGF (B) Uptake and sequestration of biotinylated Tfn (B-Tfn) in COS-7 cells was stimulation (Fig. 3, 1 minute and 4 minute). Given that measured by avidin inaccessibility (See Materials and Methods). Assays were performed in the absence (open symbols) or presence of EGF (closed redistribution of Grb2-YFP to the plasma membrane was symbols). Data shown are representative of three independent experiments. clearly observed upon EGF stimulation either in A431 cells expressing large amounts of endogenous EGFR, or in COS-7 cells co-expressing EGFR-CFP, it appears that of Grb2-YFP in the cytoplasm decreased 1 minute after EGF recruitment of Grb2-YFP to the plasma membrane in EGF- stimulation, leaving a central lucency. We also tested whether stimulated cells is mediated by EGFR. an N-terminally tagged Grb2 construct, YFP-Grb2, Immediately following Grb2-YFP translocation to the redistributed to the plasma membrane in the same fashion. plasma membrane upon EGF stimulation, the cells began to YFP-Grb2 translocation upon EGF stimulation was almost exhibit ruffling activity at their plasma membrane (Fig. 3). identical to C-terminally tagged Grb2 (data not shown), Spherical and sack-like structures reminiscent of indicating that the redistribution of the Grb2 chimeras with macropinosomes that contained Grb2-YFP and EGFR-CFP GFP can be attributed to the Grb2 sequence itself. These then started to appear at and detach from the plasma membrane. Grb2 is necessary for EGF-stimulated EGFR uptake 1797

Fig. 6. Internalization of EGFR-CFP is not blocked by the inhibition of clathrin-mediated uptake. COS-7 cells were co-transfected with EGFR-CFP and the C-terminal domain of AP180 and serum starved. In nontransfected cells, transferrin localizes to the perinuclear region. In co-transfected cells, the C-terminal domain of AP180 inhibits clathrin-mediated uptake and transferrin fails to localize (left). When stimulated with EGF, co-transfected cells still take up EGFR-CFP in large structures (right). Images from a time- lapse series are presented. At time 0, EGF was added to a ﬁnal concentration of 100 ng/ml. Confocal images were captured every 30 seconds. Arrows indicated a region of interest initially on the plasma membrane. The region is internalized and moves to the perinuclear region where it deforms the nuclear envelope.

The accompanying movie shows high resolution images and endocytic pathway (Fig. 5A, top row). When we stained cells video sequences of these structures, which moved towards the expressing Grb2-YFP with antibodies to clathrin heavy chain perinuclear region over time with both Grb2-YFP and EGFR- (CHC), which assembles with clathrin light chain to form CFP remaining associated (see http://jcs.biologists.org/ clathrin lattices on the cytosolic surfaces of membranes (Marsh supplemental). The perinuclear pool of EGFR-CFP observed and McMahon, 1999), very little, if any, labeling of the Grb2- prior to activation remained distinct from the large YFP-containing spherical structures was observed (Fig. 4, macropincytotic structures containing Grb2-YFP and EGFR- CHC). Antibody staining of the adaptor protein AP2, which CFP, indicating that only Grb2-YFP was recruited to and helps recruit cargo into clathrin-coated pits (Marsh and remained associated with membranes containing activated McMahon, 1999), revealed that this protein also was not EGFR. enriched on the Grb2-YFP-containing structures (Fig. 4, AP- 2). However, signiﬁcant labeling of the Grb2-YFP-containing structures with dynamin antibodies was observed (Fig. 4, Grb2-YFP-containing structures that internalize in Dynamin). TRITC-Transferrin, used to monitor clathrin- response to EGF contain dynamin but not clathrin, mediated endocytosis, showed a pattern of uptake that was transferrin or AP2 similar to that observed in unstimulated cells and that did not To further characterize the large spherical structures containing co-localize with Grb2-YFP (Fig. 5A). Moreover, the rate of Grb2-YFP and EGFR-CFP that arose off the plasma membrane uptake of biotinylated transferrin (Tfn) in the activated cells during EGF stimulation, we performed double-labeling was indistinguishable from unstimulated cells (Fig. 5B). These experiments using antibodies to proteins known to participate data suggest that EGF-induced, Grb2-YFP-containing in clathrin-mediated receptor internalization, including structures are distinct from conventional clathrin-derived clathrin, AP2 and dynamin (Schmid et al., 1998) (Fig. 4). We endocytic intermediates, and internalize plasma membrane also compared the distribution of the spherical structures to that receptors, such as EGFR, in a pathway that operates in parallel of Tf, which is internalized into the clathrin-mediated to the clathrin-mediated uptake pathway. 1798 Journal of Cell Science 115 (9) Inhibition of clathrin-mediated uptake by AP-180 from the plasma membrane (Ford et al., 2001). To assess the overexpression does not prevent EGFR internalization in extent of inhibition of the clathrin-mediated pathway by AP180 response to EGF stimulation of starved cells overexpression, transfected cells were incubated with To rule out a role of clathrin in the uptake of EGFR-CFP in rhodamine-labeled transferrin. As shown in Fig. 6, transferrin response to EGF, we co-expressed EGFR-CFP and the C- failed to localize to the perinuclear region in cells terminal domain of AP180 in COS cells. AP180 is an adaptor overexpressing AP180. When serum-starved cells were protein that plays an important role in clathrin-mediated stimulated with EGF, EGFR-CFP still internalized into large endocytosis (Ford et al., 2001). When overexpressed in cells, macropinocytic structures in cells overexpressing AP180. The it has been shown to block clathrin-mediated uptake of proteins data thus indicate that EGFR internalization into macropinocytic structures in response to EGF after serum starvation is not mediated by clathrin.

Grb2 SH3 domain mutants prevent inward translocation of endocytic structures that have sequestered EGF receptors in EGF- stimulated cells We next sought to clarify the role of Grb2 in the events associated with the EGF-stimulated EGFR uptake pathway. We co-expressed EGFR-CFP in COS-7 cells with SH3 mutants of Grb2 to test whether disruption of the structural integrity of Grb2 SH3 domains affected the dynamics of EGFR internalization. Prior to EGF stimulation, cells expressing an NSH3 mutant of Grb2 tagged with YFP (NSH3m-YFP) showed that the chimera was diffusely cytosolic, whereas EGFR-CFP was localized on the plasma membrane and in perinuclear structures (Fig. 7, 0′). Within 1 minute of EGF stimulation, NSH3m-YFP molecules translocated to the plasma membrane (Fig. 7, 1′), similar to that observed for Grb2- YFP molecules shown above. However, the subsequent fate of NSH3m-YFP and EGFR- CFP at the plasma membrane was different from that observed for wild-type Grb2-YFP and EGFR-CFP (Fig. 7, 20′, 30′). NSH3m-YFP and EGFR-CFP remained peripherally localized instead of moving into the cytoplasm (Fig. 7). They remained closely apposed to the plasma membrane for signiﬁcant periods of time after addition of EGF, with the large spherical structures containing NSH3m-YFP and EGFR- CFP unable to translocate inwards. These structures were relatively static and often appeared in confocal sections to be tethered by thin membrane tubules with the plasma membrane (Fig. 7, inset). A blockade of the inward translocation of peripheral structures containing EGFR-CFP was similarly observed in EGF-activated cells overexpressing either CSH3m- or NCSH3m-YFP. Movie versions of the blockade induced by NSH3m-, CSH3m- or NCSH3m-YFP can be found at http:// jcs.biologists.org/supplemental. Fig. 7. Requirement of the intact N-terminal SH3 domain of Grb2 for EGFR internalization. Images of live COS-7 cells co-expressing NSH3m-YFP and EGFR- The EGF-induced, spherical structures CFP were collected using confocal microscopy; NSH3m-YFP (left) and EGFR-CFP containing EGFR-CFP in NSH3m-YFP- (middle). Shown at the bottom is a higher magniﬁcation view of the boxed region. expressing cells could be labeled with dynamin Arrows point to large spherical structures associated with the plasma membrane (see antibodies, but not with antibodies to CHC or AP- text). Bar, 5 µm. 2 (Fig. 8). Additionally, TRITC-Tfn that was Grb2 is necessary for EGF-stimulated EGFR uptake 1799

Fig. 8. Colocalization of NSH3m-YFP with clathrin heavy chain, AP-2 and dynamin. COS-7 cells co- expressing EGFR-CFP with NSH3m-YFP were stimulated. Cells were then immunostained with antibodies directed at clathrin heavy chain (CHC), AP-2 or dynamin, followed by a TRITC-conjugated secondary antibody. Arrows point to co-localized structures.

confocal images in the Z-axis showed that they were always closely associated with the plasma membrane (data not shown). By contrast, without permeabilization, these structures (Fig. 9C, arrows) could not be labeled with the antibody (Fig. 9D), indicating that EGFRs in them were not accessible to this antibody. This suggested that, in the presence of Grb2 SH3 mutants, EGFR and Grb2 are sequestered into domains of the plasma membrane that are no longer exposed to the outside. Consistent with this, we found that biotinylated EGF could be internalized into these cells (measured by avidin inaccessibility) at a rate similar to the rate in cells expressing wild-type Grb2-YFP (Fig. 9E). These results indicate that, in cells expressing Grb2 SH3 domain mutants, EGF- stimulation results in uptake of EGFR into endocytic structures that remain closely associated with the plasma membrane and that are taken up from the plasma membrane did not label these inaccessible to the extracellular environment. structures, but was found associated with other endocytic structures instead (Fig. 5A). Flow cytometric assays performed using biotinylated Tfn further revealed that the rate of Discussion internalization of Tfn was not affected in cells expressing the The adaptor protein Grb2 has been extensively analyzed Grb2 SH3 domain mutants (Fig. 5B). These data indicate that biochemically and is known to link surface receptors to over-expression of SH3 domain mutants of Grb2 has a downstream signaling molecules. Upon receptor stimulation, profound effect on the inward movement of plasma membrane- Grb2 translocates from the cytosol to the cell surface where it derived structures containing EGFR without perturbing binds, via its SH2 domain, receptors at specific sites of tyrosine clathrin-mediated uptake of Tfn. phosphorylation. Once bound to receptors, Grb2 is thought to use its SH3 domains to link ligand-activated receptors to the distal signaling apparatus. Whether Grb2 plays an additional EGF receptors internalized in EGF-stimulated cells role in receptor internalization, which attenuates receptor expressing Grb2 SH3 domain mutants are inaccessible signaling, has been controversial. to the cell exterior For the EGFR, which is known to interact with Grb2, early To further investigate the nature of the inhibitory effect on reports suggested that Grb2 might not regulate EGFR EGF-stimulated EGFR trafficking caused by expression of internalization, since internalization of EGFR mutants lacking Grb2 SH3 domain mutants, we asked whether the spherical the Grb2-binding domain still occurred but at a slower rate than structures containing EGFR-CFP that localized near the normal (Chang et al., 1991; Chang et al., 1993). However, plasma membrane under these conditions represented fully Wang and Moran showed that EGF-induced EGFR endocytosis budded membrane vesicles whose contents were inaccessible was blocked in MDCK cells following microinjection of Grb2 to the cell exterior, or represented vesicles still continuous with containing the G203R C-SH3 domain mutation (Wang and the plasma membrane whose contents were accessible to the Moran, 1996), suggesting that Grb2 functions in EGF-induced extracellular space. To distinguish between these possibilities, EGFR endocytosis. Further support to this view was provided EGF-stimulated COS-7 cells expressing EGFR-CFP and by Sorkin et al., who showed that in EGF-stimulated cells NSH3m-YFP were either permeabilized or left EGFR-CFP and Grb2-YFP co-localize in endosomes and can unpermeabilized following fixation, and were then undergo fluorescence energy transfer (FRET), an indicator of immunostained with an antibody directed against the protein-protein interactions (Sorkin et al., 2000). However, the extracellular domain of EGFR. In permeabilized COS-7 cells, specific role Grb2 plays in EGF-stimulated EGFR most of the spherical structures associated with the plasma internalization (e.g. receptor sorting, vesicle budding/pinching membrane were detected by the antibody (Fig. 9B). Some of or vesicle transport) has remained unclear. the spherical structures appeared to be detached from the In the present study we clarify the role of Grb2 in EGFR plasma membrane, but 3D reconstruction based on a series of internalization by directly visualizing EGFR and Grb2 1800 Journal of Cell Science 115 (9) dynamics in EGF-stimulated cells expressing wild-type or redistribution required an intact Grb2 SH2 domain consistent mutant SH2/SH3 domains of Grb2. Grb2-YFP redistributed to with previous biochemical data (Lowenstein et al., 1992). the cell periphery immediately after EGF stimulation. This Mutation of either or both Grb2 SH3 domains had no effect on the dynamics of translocation to the plasma membrane. However, time-lapse confocal microscopy revealed that the SH3 mutants prevented the stimulation-dependent inward translocation of EGFR-containing endocytic structures that was observed in wild-type Grb2-expressing cells. Therefore, the SH3 domains of Grb2 appear to orchestrate the machinery necessary for translocating endocytic membranes carrying EGFR from the periphery to the interior of the cell. Several properties of the endocytic membranes carrying EGFR-CFP and Grb2- YFP in EGF-stimulated cells suggested that they were distinct from clathrin-derived endocytic structures. First, their appearance was coupled to the translocation of Grb2-YFP to the plasma membrane and the subsequent ruffling activity of these membranes, which often seemed to give rise directly to the large, EGFR-CFP- and Grb2-YFP-containing endosomes. Second, the surfaces of these endocytic membranes were depleted of molecules associated with clathrin-coated pits, including clathrin heavy chain and AP-2 (Marsh and McMahon, 1999). Third, they did not contain internalized transferrin, which is a marker for the clathrin-derived endocytic E system. Finally, the macropinocytic structures carrying EGFR-CFP into the cell were not 1 00 inhibited when clathrin-mediated uptake was blocked by overexpression of AP180, a clathrin adaptor molecule. Because we did not 8 0 characterize the fate of EGFR-CFP and Grb2- YFP for periods after EGF stimulation longer than 60 minutes, it is possible that these molecules eventually are delivered into the 6 0 conventional endocytic pathway leading to lysosomes. Our time-lapse images early after al) t stimulation with EGF, however, demonstrate o

t 4 0

of that EGFR-CFP and Grb2-YFP at the plasma

non-transfec Seqestered B-EGF (% membrane are internalized into large structures Grb2 that do not resemble in size or composition the

2 0 NSH3m endocytic structures derived from clathrin- coated pits. CSH3m Insight into the role of Grb2 in the nonclathrin uptake pathway followed by 0 EGFR-CFP and Grb2-YFP was gained from 0 10 20 30 Time (min) experiments expressing GFP-tagged Grb2 mutants where the structural integrity of the Fig. 9. EGFR internalization is blocked by NSH3m-YFP at the step of membrane Grb2 SH3 domain was disrupted. When these fission. EGF-stimulated COS-7 cells co-expressing NSH3m-YFP and EGFR-CFP were mutants were expressed in cells, they fixed then either permeabilized (A,B) or left unpermeabilized (C,D). Cells were translocated normally to the plasma membrane immunostained with an antibody against the extracellular domain of EGFR, followed by a TRITC-conjugated secondary antibody (B,D). Bar, 5 µm. (E) EGFR in response to EGF stimulation and then internalization in COS-7 cells overexpressing the indicated YFP chimeric molecules clustered with EGFR-CFP within large, was measured by determining avidin inaccessibility. Assays were performed as endocytic structures. However, these structures described in Materials and Methods. Data shown are representative of three did not subsequently translocate into the independent experiments. interior of the cell, in contrast to what is Grb2 is necessary for EGF-stimulated EGFR uptake 1801 observed in wild-type Grb2-expressing cells. Thus, the SH3 H. L. and Albanesi, J. P. (1998). Synergistic activation of dynamin GTPase domains of Grb2 are necessary for inward translocation of by Grb2 and phosphoinositides. J. Biol. Chem. 273, 3791-3797. Grb2- and EGFR-containing endocytic structures, but not for Buday, L. and Downward, J. (1993). Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, stimulus-induced Grb2 recruitment to membranes and the and Sos nucleotide exchange factor. Cell 73, 611-620. clustering/uptake of EGFR into large endocytic structures. Buday, L., Khwaja, A., Sipeki, S., Farago, A. and Downward, J. (1996). Despite the inhibition of movement of EGFR-containing Interactions of Cbl with two adapter proteins, Grb2 and Crk, upon T cell endocytic structures by the Grb2 SH3 domain mutants, Tfn was activation. J. Biol. Chem. 271, 6159-6163. Carter, R. E. and Sorkin, A. (1998). Endocytosis of functional epidermal internalized normally with or without EGF stimulation in the growth factor receptor-green fluorescent protein chimera. J. Biol. Chem. presence of these Grb2 mutations. This finding supports the 273, 35000-35007. idea that the endocytosis machinery for EGFR differs from that Chang, C. P., Kao, J. P., Lazar, C. S., Walsh, B. J., Wells, A., Wiley, H. S., used by the TfnR, consistent with the previous evidence Gill, G. N. and Rosenfeld, M. G. (1991). Ligand-induced internalization (Warren et al., 1997; Warren et al., 1998). and increased cell calcium are mediated via distinct structural elements in the carboxyl terminus of the epidermal growth factor receptor. J. Biol. Chem. Flow cytometric analysis measuring avidin inaccessibility 266, 23467-23470. showed that, in the presence of Grb2 SH3 mutations, EGF- Chang, C. P., Lazar, C. S., Walsh, B. J., Komuro, M., Collawn, J. F., Kuhn, induced membrane vesicles could sequester internalized L. A., Tainer, J. A., Trowbridge, I. S., Farquhar, M. G., Rosenfeld, M. contents, but the vesicles were unable to detach from the G. et al. (1993). Ligand-induced internalization of the epidermal growth factor receptor is mediated by multiple endocytic codes analogous to the plasma membrane and move into the cytoplasm. Therefore, an tyrosine motif found in constitutively internalized receptors. J. Biol. Chem. important focus of future work will be in determining which 268, 19312-19320. binding partner(s) of the Grb2 SH3 domain and their Chardin, P., Camonis, J. H., Gale, N. W., van Aelst, L., Schlessinger, J., downstream signaling pathways are involved in vesicle release Wigler, M. H. and Bar-Sagi, D. (1993). Human Sos1: a guanine nucleotide and translocation into the cell center. Dynamin is thought to exchange factor for Ras that binds to GRB2. Science 260, 1338-1343. Clark, S. G., Stern, M. J. and Horvitz, H. R. (1992). C. elegans cell- mediate the process of pinching off vesicles from the signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature membrane (Schmid et al., 1998). In addition, the GTPase 356, 340-344. activity of dynamin is reported to be enhanced by its interaction Cory, G. O., MacCarthy-Morrogh, L., Banin, S., Gout, I., Brickell, P. M., with the Grb2 SH3 domains (Barylko et al., 1998; Gout et al., Levinsky, R. J., Kinnon, C. and Lovering, R. C. (1996). Evidence that the Wiskott-Aldrich syndrome protein may be involved in lymphoid cell 1993; Herskovits et al., 1993). Therefore, disruption of signaling pathways. J. Immunol. 157, 3791-3795. dynamin-Grb2 interactions by mutations in the Grb2 SH3 Egan, S. E., Giddings, B. W., Brooks, M. W., Buday, L., Sizeland, A. M. domains might be predicted to lead to the failure in targeting and Weinberg, R. A. (1993). Association of Sos Ras exchange protein with of dynamin to the cell periphery, explaining the inhibition of Grb2 is implicated in tyrosine kinase signal transduction and transformation. vesicle release. Unexpectedly, however, dynamin was found to Nature 363, 45-51. Ellenberg, J., Lippincott-Schwartz, J. and Presley, J. F. (1998). Two-color redistribute to the cell periphery upon EGF stimulation green fluorescent protein time-lapse imaging. Biotechniques 25, 838-842, regardless of the presence of the Grb2 SH3 domain mutants 844-846. (data not shown). Although it is possible that the redistributed Ellenberg, J., Lippincott-Schwartz, J. and Presley, J. F. (1999). Dual-colour dynamin GTPase activity was not enhanced sufficiently in the imaging with GFP variants. Trends Cell Biol. 9, 52-56. Ford, M., Pearse, B., Higgins, M., Vallis, Y., Owen, D., Gibson, A., presence of these mutants, it seems more likely that some other Hopkins, C., Evans, P. and McMahon, H. (2001). Simulataneous binding protein binding to the Grb2 SH3 domains is responsible for the of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices release of these vesicles from the plasma membrane and their on membranes. Science 291, 1051-1055. ability to translocate inward to the cell center. Gamou, S., Kim, Y. S. and Shimizu, N. (1984). Different responses to EGF In summary, we have examined the dynamics of Grb2 and in two human carcinoma cell lines, A431 and UCVA-1, possessing high numbers of EGF receptors. Mol. Cell Endocrinol. 37, 205-213. EGFR fused to GFP spectral variants in single live cells, and Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, P., Truong, have demonstrated that interactions through Grb2 SH3 O., Totty, N. F., Hsuan, J., Booker, G. W. et al. (1993). The GTPase domains regulate EGFR internalization. We further show that dynamin binds to and is activated by a subset of SH3 domains. Cell 75, 25- Grb2 is responsible for coupling EGFR-containing membranes 36. Haigler, H. T., McKanna, J. A. and Cohen, S. (1979). Direct visualization with downstream effectors involved in the internalization of of the binding and internalization of a ferritin conjugate of epidermal growth these membranes through a macropinocytic pathway. Grb2 has factor in human carcinoma cells A-431. J. Cell Biol. 81, 382-395. previously been thought to serve solely as an adapter linking Hanazono, Y., Odai, H., Sasaki, K., Iwamatsu, A., Yazaki, Y. and Hirai, receptors on the plasma membrane to downstream signaling H. (1996). Proto-oncogene products Vav and c-Cbl are involved in the signal pathways. Our results shed light on the additional function of transduction through Grb2/Ash in hematopoietic cells. Acta Haematol. 95, 236-242. Grb2 as a regulator of activation-induced, clathrin-independent Herskovits, J. S., Shpetner, H. S., Burgess, C. C. and Vallee, R. B. (1993). endocytosis. Microtubules and Src homology 3 domains stimulate the dynamin GTPase via its C-terminal domain. Proc. Natl. Acad. Sci. USA 90, 11468-11472. T.Y. was a fellow of the Japan Society for the Promotion of Science. Hu, P., Margolis, B., Skolnik, E. Y., Lammers, R., Ullrich, A. and We thank N. Altan, J. Ellenberg and K. Hirschberg for insightful Schlessinger, J. (1992). Interaction of phosphatidylinositol 3-kinase- discussions and V. Kapoor for technical help. associated p85 with epidermal growth factor and platelet-derived growth factor receptors. Mol. Cell. Biol. 12, 981-990. Krupp, M. N., Connolly, D. T. and Lane, M. D. (1982). Synthesis, turnover, and down-regulation of epidermal growth factor receptors in human A431 References epidermoid carcinoma requirements. Mol. Biol. Cell 4, 715-727. Ando, A., Yonezawa, K., Gout, I., Nakata, T., Ueda, H., Hara, K., Li, N., Batzer, A., Daly, R., Yajnik, V., Skolnik, E., Chardin, P., Bar-Sagi, Kitamura, Y., Noda, Y., Takenawa, T., Hirokawa, N. et al. (1994). A D., Margolis, B. and Schlessinger, J. (1993). Guanine-nucleotide-releasing complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras receptor substrate-1 after insulin treatment. EMBO J. 13, 3033-3038. signalling. Nature 363, 85-88. Barylko, B., Binns, D., Lin, K. M., Atkinson, M. A., Jameson, D. M., Yin, Lin, H. C., Barylko, B., Achiriloaie, M. and Albanesi, J. P. (1997). 1802 Journal of Cell Science 115 (9)

Phosphatidylinositol (4,5)-bisphosphate-dependent activation of dynamins I (1993). The SH2 and SH3 domains of mammalian Grb2 couple the EGF and II lacking the proline/arginine-rich domains. J. Biol. Chem. 272, 25999- receptor to the Ras activator mSos1. Nature 363, 83-85. 26004. Schmid, S. L., McNiven, M. A. and De Camilli, P. (1998). Dynamin and its Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, partners: a progress report. Curr. Opin. Cell Biol. 10, 504-512. R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D. and Schlessinger, J. (1992). She, H. Y., Rockow, S., Tang, J., Nishimura, R., Skolnik, E. Y., Chen, M., The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine Margolis, B. and Li, W. (1997). Wiskott-Aldrich syndrome protein is kinases to ras signaling. Cell 70, 431-442. associated with the adapter protein Grb2 and the epidermal growth factor Marsh, M. and McMahon, H. T. (1999). The structural era of endocytosis. receptor in living cells. Mol. Biol. Cell 8, 1709-1721. Science 285, 215-220. Soler, C., Beguinot, L. and Carpenter, G. (1994). Individual epidermal Meisner, H., Conway, B. R., Hartley, D. and Czech, M. P. (1995). growth factor receptor autophosphorylation sites do not stringently deﬁne Interactions of Cbl with Grb2 and phosphatidylinositol 3′-kinase in activated association motifs for several SH2-containing proteins. J. Biol. Chem. 269, Jurkat cells. Mol. Cell. Biol. 15, 3571-3578. 12320-12324. Okabayashi, Y., Kido, Y., Okutani, T., Sugimoto, Y., Sakaguchi, K. and Sorkin, A. (1998). Endocytosis and intracellular sorting of receptor tyrosine Kasuga, M. (1994). Tyrosines 1148 and 1173 of activated human epidermal kinases. Frontiers Biosci. 3, d729-d738. growth factor receptors are binding sites of Shc in intact cells. J. Biol. Chem. Sorkin, A., McClure, M., Huang, F. and Carter, R. (2000). Interaction of 269, 18674-18678. EGF receptor and Gb2 in living cells visualized by ﬂuorescence resonance Okutani, T., Okabayashi, Y., Kido, Y., Sugimoto, Y., Sakaguchi, K., energy transfer (FRET) microscopy. Curr. Biol. 10, 1395-1398. Matuoka, K., Takenawa, T. and Kasuga, M. (1994). Grb2/Ash binds Vidal, M., Montiel, J. L., Cussac, D., Cornille, F., Duchesne, M., Parker, directly to tyrosines 1068 and 1086 and indirectly to tyrosine 1148 of F., Tocque, B., Roques, B. P. and Garbay, C. (1998). Differential activated human epidermal growth factor receptors in intact cells. J. Biol. interactions of the growth factor receptor-bound protein 2 N-SH3 domain Chem. 269, 31310-31314. with son of sevenless and dynamin. Potential role in the Ras-dependent Pawson, T. (1995). Protein modules and signalling networks. Nature 373, 573- signaling pathway. J. Biol. Chem. 273, 5343-5348. 580. Vieira, A. V., Lamaze, C. and Schmid, S. L. (1996). Control of EGF receptor Pomerance, M., Multon, M. C., Parker, F., Venot, C., Blondeau, J. P., signaling by clathrin-mediated endocytosis. Science 274, 2086-2089. Tocque, B. and Schweighoffer, F. (1998). Grb2 interaction with MEK- Wang, Z. and Moran, M. F. (1996). Requirement for the adapter protein kinase 1 is involved in regulation of Jun-kinase activities in response to GRB2 in EGF receptor endocytosis. Science 272, 1935-1939. epidermal growth factor. J. Biol. Chem. 273, 24301-24304. Warren, R. A., Green, F. A. and Enns, C. A. (1997). Saturation of the Romero, F., Ramos-Morales, F., Dominguez, A., Rios, R. M., endocytic pathway for the transferrin receptor does not affect the endocytosis Schweighoffer, F., Tocque, B., Pintor-Toro, J. A., Fischer, S. and of the epidermal growth factor receptor. J. Biol. Chem. 272, 2116-2121. Tortolero, M. (1998). Grb2 and its apoptotic isoform Grb3-3 associate with Warren, R. A., Green, F. A., Stenberg, P. E. and Enns, C. A. (1998). Distinct heterogeneous nuclear ribonucleoprotein C, and these interactions are saturable pathways for the endocytosis of different tyrosine motifs. J. Biol. modulated by poly(U) RNA. J. Biol. Chem. 273, 7776-7781. Chem. 273, 17056-17063. Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R., Li, Wells, A., Welsh, J. B., Lazar, C. S., Wiley, H. S., Gill, G. N. and Rosenfeld, W., Batzer, A., Thomas, S., Brugge, J., Pelicci, P. G. et al. (1992). M. G. (1990). Ligand-induced transformation by a noninternalizing Association of the Shc and Grb2/Sem5 SH2-containing proteins is epidermal growth factor receptor. Science 247, 962-964. implicated in activation of the Ras pathway by tyrosine kinases. Nature 360, Zhu, G., Decker, S. J. and Saltiel, A. R. (1992). Direct analysis of the binding 689-692. of Src-homology 2 domains of phospholipase C to the activated epidermal Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T. and Bowtell, D. growth factor receptor. Proc. Natl. Acad. Sci. USA 89, 9559-9563.