Interaction of Shc with Adaptor Protein Adaptins*

, The role of Shc as a substrate of receptors for growth factors and cytokines is well established. To gain further insight into the function of Shc in signal transduc- tion, we used an affinity method to identify potential Shc-binding proteins. Incubation of bovine brain lysates with a glutathione S -transferase (GST)-Shc fusion protein immobilized on glutathione-Sepharose beads re- sulted in the binding of cellular proteins of (cid:59) 115, 110, and 100 kDa as well as those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides revealed that the 100-kDa protein was almost identical to (cid:98) -adaptin and that the 110- and 115-kDa proteins were almost identical to (cid:97) A -adaptin. Using immunoblot analysis, anti- (cid:97) -adap- tin antibody recognized several proteins of 100 (cid:59) 115 kDa, and anti- (cid:98) -adaptin antibody recognized a 100-kDa protein, suggesting that (cid:97) A -, (cid:97) C -, and (cid:98) -adaptins are bound to the GST-Shc fusion protein. Immunoblot analysis with anti- (cid:97) -adaptin antibody revealed that (cid:97) -adap-tin was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates, suggesting a specific interaction of Shc and adaptins in intact cells. A binding study using reverse transcrip-tion-polymerase chain reaction the the an of three (GCA), which in an in-frame alanine insertion corresponding amino acid 308 HI site by site-directed mutagen- esis the digested cDNA was inserted into the expression plasmid pGEX Biotech, Uppsala). Mutant Shc constructs were prepared by polymerase chain reaction or site-directed mutagenesis. Some C-terminal deletion mutants were obtained by restriction enzyme digestion and blunting of digested cDNA, followed by self-ligation. pGEX- Shc SH2 was prepared as reported (14). (cid:109) g/ml aprotinin. Affinity Purification and Sequencing of Shc-binding Proteins— Affin- ity resins for the isolation of Shc-binding proteins were prepared by immobilizing (cid:59) 20 (cid:109) g of GST-Shc on glutathione-Sepharose 4B beads (Pharmacia) as described (19). Similar resins were prepared using GST alone as a control for the specificity of interaction. Affinity resins were incubated for with bovine brain lysates prepared by homog-enizing brain M (cid:109) g/ml aprotinin, by sequential cen- trifugation (cid:51) and separated by SDS-polyacryl- gel electrophoresis and visualized by silver stain or Coomassie Blue stain. The regions of the gel containing the Shc-binding proteins were excised, and a digestion was performed for 16 h at 37 °C in the gel slice using endopeptidase Lys-C (Promega). 2 Peptides were separated by serial anion-exchange and reverse-phase high pressure liquid chromatography and sequenced using a modified Applied Biosys- tems 477A sequencer (20). Mutant GST-Shc Proteins—

Many receptor tyrosine kinases such as those for epidermal growth factor (EGF), 1 platelet-derived growth factor, and insulin transmit intracellular signals for cell proliferation and differentiation (1). The binding of ligands and the subsequent conformational alteration of the extracellular domain induce receptor oligomerization, which results in elevated protein-tyrosine kinase activity of the receptor and leads to increased autophosphorylation and intracellular substrate phosphorylation (1,2). Receptor autophosphorylation is an important structural feature of the association of the receptor with proteins containing Src homology 2 (SH2) domains (1,3,4). A number of proteins that contain SH2 domains have been identified, including phospholipase C-␥1, Ras GTPase-activating protein, the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, Grb2, and Shc (5). Many of these proteins also contain one or more SH3 domains, which specifically interact with proline-rich sequences, thus serving as linker molecules to bring other proteins into the signaling complex (5).
Shc protein consists of three overlapping polypeptides of ϳ46, 52, and 66 kDa. Shc proteins of 46 and 52 kDa encoded by a 3.4-kilobase mRNA are ubiquitously expressed, whereas a 66-kDa Shc protein is likely to be encoded by a distinct shc transcript and is absent in some hematopoietic cells (6). Shc is composed of a single SH2 domain at the C terminus, an adjacent glycine/proline-rich region that is 50% homologous to human ␣1-collagen, and a distinct N-terminal phosphotyrosinebinding domain (PTB domain) (6,7). Although Shc lacks apparent catalytic activity, overexpression of Shc protein induces malignant transformation in 3T3 cells (6) and neurite outgrowth in PC12 cells (8). Shc has been shown to be involved in Ras activation by a number of receptors for growth factors as well as cytokines (9 -11). Shc is phosphorylated on tyrosine upon stimulation of these receptors (8) and subsequently interacts with Grb2, which forms a complex with Sos, a Ras guanine nucleotide exchange protein (12). Shc is also a good substrate for Src family kinases, and in v-Src-and v-Fps-expressing cells, Shc is constitutively tyrosine-phosphorylated and forms a complex with Grb2 (13). Thus, tyrosine 317 within the collagen homologous region of Shc is suggested to be the binding site of Grb2 (6), and Shc interacts with tyrosine-phosphorylated growth factor receptors such as EGF receptors, TrkA, and ErbB-2 (14 -16). Recent reports have shown that the N-terminal PTB domain as well as the SH2 domain interact with tyrosine-phosphorylated growth factor receptors (7,17,18), suggesting the assembly of different signaling complexes. Thus, Shc seems to have multiple functions in signal transduction. The role of the collagen homologous domain remains to be defined. To gain further insight into the function of Shc in signal transduction, we tried to identify Shc-binding proteins using glutathione S-transferase (GST)-Shc fusion proteins.

MATERIALS AND METHODS
Tissue Culture-Human glioblastoma A172 and SV40-transformed African green monkey kidney COS cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Human epidermoid carcinoma KB cells were maintained in Eagle's minimum essential medium supplemented with 10% fetal calf serum and nonessential amino acids, and rat pheochromocytoma PC12 cells were in RPMI 1640 medium supplemented with 10% horse serum and 5% fetal calf serum. KB and PC12 cells were provided by the Japanese Cancer Research Resources Bank, Foundation for Promotion of Cancer Research (Tokyo). A172 and COS cells were obtained from the American Type Culture Collection (Rockville, MD).
Bacterial Expression of Shc Mutants as GST Fusion Proteins-A full-length human cDNA clone of Shc was isolated by reverse transcription-polymerase chain reaction using total RNA extracted from A172 cells, the sense primer 5Ј-CGGAGAATTCATGAGGCCCTGGACATG-AACAAGC-3Ј, and the antisense primer 5Ј-AAGAGAATTCTAGGGCA-GATCACAGTTTCCGC-3Ј. The cDNA was cloned into M13 to verify the sequence. Sequencing revealed an insertion of three bases (GCA), which resulted in an in-frame alanine insertion corresponding to amino acid 308 as reported (7). A BamHI site was created by site-directed mutagenesis using the primer 5Ј-CACTCAGCTGGATCCTGTCCAGGG-3Ј, and digested cDNA was inserted into the bacterial expression plasmid pGEX (Pharmacia Biotech, Uppsala). Mutant Shc constructs were prepared by polymerase chain reaction or site-directed mutagenesis. Some C-terminal deletion mutants were obtained by restriction enzyme digestion and blunting of digested cDNA, followed by self-ligation. pGEX-Shc SH2 was prepared as reported (14).
Bacterial cultures expressing pGEX-Shc were grown in Luria-Bertani medium containing 50 g/ml ampicillin, and the expression of fusion protein was induced by adding isopropyl-␤-D-thiogalactopyranoside to a final concentration of 0.25 mM. After a 4-h incubation at 37°C or a 16-h incubation at 25°C, induced bacteria were lysed by sonication in 50 mM Tris-HCl (pH 7.4) containing 1% Triton X-100, 1% Tween 20, 2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 10 g/ml aprotinin.
Affinity Purification and Sequencing of Shc-binding Proteins-Affinity resins for the isolation of Shc-binding proteins were prepared by immobilizing ϳ20 g of GST-Shc on glutathione-Sepharose 4B beads (Pharmacia) as described (19). Similar resins were prepared using GST alone as a control for the specificity of interaction. Affinity resins were incubated for 2 h at 4°C with bovine brain lysates prepared by homogenizing bovine brain in 10 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml aprotinin, followed by sequential centrifugation at 15,000 ϫ g for 60 min and at 40,000 ϫ g for 30 min at 4°C. After extensive washing, proteins bound to the resins were released either by boiling in SDS sample buffer or by elution with 1 M Tris-HCl (pH 7.4) containing 0.5% Triton X-100, separated by SDS-polyacrylamide gel electrophoresis (PAGE), and visualized by silver stain or Coomassie Blue stain. The regions of the gel containing the Shc-binding proteins were excised, and a digestion was performed for 16 h at 37°C in the gel slice using endopeptidase Lys-C (Promega). 2 Peptides were separated by serial anion-exchange and reverse-phase high pressure liquid chromatography and sequenced using a modified Applied Biosystems 477A sequencer (20).
Binding to Mutant GST-Shc Proteins-Various GST-Shc fusion protein constructs or GST alone was bound to glutathione-Sepharose beads. The beads were then washed with 20 mM Hepes (pH 7.4) containing 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, and 1 mM sodium orthovanadate. The amounts of GST fusion proteins used were normalized by Coomassie Blue stain. Affinity resins were incubated with bovine brain lysates for 2 h at 4°C. After extensive washing, the beads were boiled in sample buffer, and the released proteins were analyzed by SDS-PAGE. For the binding inhibition assay, the collagen homologous region of Shc was prepared as follows. GST-Shc protein containing amino acids 233-369 was immobilized on a glutathione-Sepharose column and incubated with 5 units of thrombin in 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 5 mM MgCl 2 , 2.5 mM CaCl 2 , and 1 mM dithiothreitol for 16 h at 4°C. Then, 20 l of p-aminobenzamidine beads (Sigma) was added to the supernatants and incubated for 30 min at 4°C. The collagen homologous region of Shc was purified from the resulting supernatant using a Centricon 10 apparatus (Amicon, Inc.). Brain lysates were first incubated with the collagen homologous region of Shc for 2 h at 4°C, then added to GST-Shc immobilized on beads, and further incubated for 2 h at 4°C. After washing, samples were processed as described above.
Immunoblot Analysis-Confluent cells were serum-starved for 12 h and then stimulated with mouse EGF (Takara Shuzo, Kyoto, Japan). Cells were immediately frozen in liquid nitrogen and stored at Ϫ80°C until lysis. Cells were lysed in 0.5 M Tris-HCl (pH 7.4) containing 0.5% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, and 20 M leupeptin. Clarified lysates were adjusted to 50 mM Tris-HCl (pH 7.4) containing 0.5% Triton X-100, 100 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, and 20 M leupeptin (IP buffer). Lysates were precleared by incubating with nonimmunized rabbit serum coupled to protein A-Sepharose (Pharmacia) for 60 min at 4°C and then incubated with anti-Shc antibody (14) coupled to protein A-Sepharose for 90 min at 4°C. Immunoprecipitates were washed with IP buffer and then boiled in SDS sample buffer. Samples were then subjected to SDS-PAGE and transferred onto nitrocellulose. Blots were blocked in 5% skim milk in Tris-buffered saline and then probed with antibodies to ␣and ␤-adaptins (AC1-M11 and B1-M6, respectively) (21). These antibodies were kindly provided by Dr. Margaret S. Robinson (Department of Clinical Biochemistry, University of Cambridge, Cambridge, United Kingdom). Bound antibodies were detected with horseradish peroxidase-conjugated antibodies to mouse immunoglobulin G (Promega) using the ECL detection system (Amersham International, Buckinghamshire, United Kingdom).
Glutathione-Sepharose beads preloaded with GST-Shc fusion protein were incubated with bovine lysates for 2 h at 4°C. After extensive washing, proteins were released from the beads by boiling in SDS sample buffer. Samples were subjected to SDS-PAGE, transferred onto nitrocellulose, and blotted with antibodies to ␣and ␤-adaptins as described above.

RESULTS
Identification of GST-Shc-binding Proteins-To investigate the unidentified role of Shc in signal transduction from receptors for growth factors and cytokines, we used affinity methods to identify potential Shc-binding proteins. An affinity matrix was prepared by immobilization of a GST-Shc-(4 -473) fusion protein on glutathione-Sepharose beads. Incubation of bovine brain lysates with immobilized GST-Shc-(4 -473) fusion protein resulted in the binding of several proteins when analyzed by SDS-PAGE and visualized by silver stain. Among these proteins, proteins with apparent molecular masses of ϳ115, 110, and 100 kDa were most prominent (Fig. 1A). Increasing the amount of brain lysate resulted in increasing association of these proteins (data not shown). These proteins did not associate with GST protein (Fig. 1A).
To determine the identity of the binding proteins, these proteins were purified in quantities sufficient for protein sequencing by high resolution methods (20). Bovine brain lysates (1 ml) were incubated with ϳ20 g of GST-Shc immobilized on glutathione-Sepharose. After washing, proteins bound to the resin were subjected to SDS-PAGE and visualized by Coomassie stain. The bands of Shc-binding proteins were excised and stored at Ϫ20°C. This was repeated until the amounts of Shc-binding proteins reached ϳ5 g. Affinity-purified Shcbinding proteins were then digested in the gel with lysyl endo-2 J. J. Hsuan, unpublished data.
FIG. 1. Identification of GST-Shc-binding proteins from bovine brain lysates and amino acid sequencing of these proteins. A, proteins bound to GST-Shc-(4 -473) fusion protein. Bovine brain lysates were incubated with GST or GST-Shc-(4 -473) coupled to glutathione-Sepharose beads. The beads were washed extensively, and associated proteins were released by boiling in SDS sample buffer. Released proteins were resolved by SDS-PAGE on a 6% gel and visualized by silver stain. B, alignment of tryptic peptides of ϳ115, 110, and 100-kDa proteins with ␣and ␤-adaptin sequences. Peptide sequences obtained from lysyl endoproteinase digestion (FR) are indicated in boldface above the appropriate sequence of adaptins. Tryptic peptides of p115 and p110 are aligned with mouse ␣ A -and ␣ C -adaptins (31), and those of p100 are aligned with human ␤-adaptin (27). Dashes indicate identities to ␣ A -adaptin.
proteinase. Peptides were resolved by high pressure liquid chromatography and subjected to protein sequence determination. Amino acid sequencing of the fragments of the 100-kDa protein yielded the sequences KEYATEVDVDFVRK, KMER-QVVLRTDK, KGLEISGTFTHRQGHFYMEMN, KLVYLYLV-NYAK, and KNSFGVIPSTPLAI. Comparison with the sequences in the GenBank TM Data Bank revealed almost complete identity to rat and human ␤-adaptins, a component of the plasma membrane adaptor complex of coated vesicles (Fig. 1B) (22,23). Amino acid sequencing of the fragments of the 110-kDa protein yielded the sequences KSEFRQNLGRMYLFYGNK, KTS-VQFQNFSPTVVHPGDL, and KYLQVGHLLREPNAQAQM-YRLTLRTK, and that of the 115-kDa protein yielded KTS-VQFQNFSPTVVHPGDL. These amino acid sequences were almost identical to the mouse ␣ A -adaptin, but not ␣ C -adaptin, of the adaptor complex (Fig. 1B) (24). These results indicate that ␣ A -and ␤-adaptins bind to Shc expressed as fusion protein with GST.
Association of Adaptins with Shc in Intact Cells-We next investigated the association of adaptins with Shc in intact cells. PC12, KB, and COS cells unstimulated or stimulated with 10 nM EGF for 2 min were solubilized in 0.5 M Tris-HCl (pH 7.4) containing 0.5% Triton X-100 and protease inhibitors. Anti-Shc immunoprecipitates were subjected to SDS-PAGE, transferred onto nitrocellulose, and then immunoblotted with anti-␣-adaptin antibody. In these cells, ␣-adaptin was identified in anti-Shc immunoprecipitates (Fig. 2). Antibody to ␣-adaptin (AC1-M11) recognized both ␣ A -and ␣ C -adaptins. Both ␣ A -and ␣ Cadaptins were predominant in PC12 cells, whereas ␣ C -adaptin was predominant in KB and COS cells. To see the total amount of cellular adaptins relative to that recovered in anti-Shc immunoprecipitates, lysates equal to 2% of that used for immunoprecipitation were electrophoresed and blotted with anti-␣adaptin antibody. Approximately 1-5% of cellular adaptins were coimmunoprecipitated with Shc. More ␣-adaptin was coimmunoprecipitated with Shc in KB and COS cells than in PC12 cells. However, the amount of ␣-adaptin coimmunoprecipitated with Shc was not changed upon stimulation with EGF, suggesting that the association of adaptin with Shc is constitutive.
Binding Site of Adaptins within Shc-We then investigated the specific binding site of adaptins within Shc by constructing a series of GST-Shc fusion proteins in which various domains were deleted. Deletion of the PTB or SH2 domain of Shc did not affect the binding of adaptins, whereas deletion of the collagen homologous region of Shc eliminated the binding of adaptins (Fig. 3). Conversely, GST-Shc fusion proteins retaining only either the PTB or SH2 domain eliminated the binding of adaptins, but the construct that retained only the collagen homologous region did bind adaptins (Fig. 3). We then prepared other GST-Shc fusion proteins containing various deletions of the collagen homologous region itself. Adaptins bound to GST-Shc fusion proteins containing amino acids 233-369 and 233-355, but failed to bind a mutant fusion protein containing amino acids 233-345 (Fig. 3). Furthermore, adaptins were able to bind mutant GST-Shc fusion proteins containing amino acids 346 -473, 346 -369, and 346 -355 (Fig. 3). These results

FIG. 2. Association of adaptins with Shc in intact cells. PC12
, KB, and COS cells untreated or treated with 10 nM EGF for 2 min were lysed in 0.5 M Tris-HCl (pH 7.4) containing 0.5% Triton X-100 and protease inhibitors. Clarified lysates were adjusted to 50 mM Tris-HCl (pH 7.4) containing 0.5% Triton X-100, 100 mM NaCl, and protease inhibitors and then incubated with anti-Shc antibody coupled to protein A-Sepharose. Immunoprecipitates (IP) were subjected to SDS-PAGE, transferred onto nitrocellulose, and immunoblotted (IB) with anti-␣adaptin antibody. Lysates equal to 2% of that used for immunoprecipitation were also electrophoresed and blotted with anti-␣-adaptin antibody. Arrowheads indicate ␣-adaptin. NRS, nonimmunized rabbit serum.

FIG. 3. Association of adaptins with mutant GST-Shc proteins.
Various GST-Shc fusion proteins corresponding to the indicated residues were immobilized on glutathione-Sepharose beads and incubated with bovine brain lysates for 2 h at 4°C. Associated proteins were released in SDS sample buffer, resolved by SDS-PAGE, and stained with silver. Gly/Pro, glycine/proline-rich collagen homologous domain.
indicate that amino acid sequence 346 -355 of Shc is necessary for adaptin binding.
Since ␣and ␤-adaptins are components of the plasma membrane adaptor complex of clathrin-coated vesicles and the adaptor complex also contains two other subunits of ϳ50 and 17 kDa, we then investigated whether two other subunits could be identified in affinity-purified proteins with GST-Shc-(4 -473). When affinity-purified proteins were released by boiling in SDS sample buffer or by elution with 1 M Tris-HCl (pH 7.4), proteins with lower molecular masses of ϳ50, 47,45,35,19, and 17 kDa as well as ϳ115, 110, and 100 kDa were identified (Fig. 4, B and C). Among these lower molecular mass proteins, proteins of 47, 45, and 35 kDa bound to GST (Fig. 4A), indicating that proteins of ϳ50, 19, and 17 kDa as well as those of 115, 110, and 100 kDa specifically bind to Shc. Association of these proteins with GST-Shc-(4 -473) was inhibited by the collagen homologous domain of Shc-(233-369) prepared by thrombin digestion of the GST fusion protein (Fig. 4, B and C). In contrast, Shc-(233-345) failed to inhibit the association of these proteins with GST-Shc-(4 -473) (Fig. 4D). These results support the finding that the collagen homologous region of Shc is a binding site for adaptins and also suggest that adaptins bind to Shc as a plasma membrane adaptor complex. DISCUSSION An affinity chromatography approach was used to identify Shc-binding proteins from bovine brain lysates. This resulted in the identification of three major proteins of ϳ115, 110, and 100 kDa and several bands of lower molecular mass including ϳ50 and 17 kDa. Microsequencing of 100ϳ115-kDa proteins showed them to be almost identical to the previously characterized proteins ␣ A -and ␤-adaptins. These adaptins are components of adaptor proteins, also referred to as assembly proteins or clathrin-associated proteins, which anchor the clathrin lattice on the surface of coated pits and coated vesicles (25,26). Two structurally related classes of adaptor proteins are present. Plasma membrane adaptor HA2-AP2 consists of ␣and ␤-adaptins and two smaller subunits of 50 and 17 kDa, and Golgi adaptor HA1-AP1 consists of ␥and ␤Ј-adaptins and two smaller subunits of 47 and 19 kDa (27,28). In addition, there are two isoforms of ␣-adaptin (␣ A and ␣ C ) encoded by distinct but highly homologous genes (24). Two alternative transcripts of ␣ A -adaptin have been identified: one is expressed in brain (24), and a smaller isoform is expressed ubiquitously (29). The smaller isoform of ␣ A -adaptin migrates on SDS-polyacrylamide gels with a mobility similar to that of ␣ C -adaptin, which is also expressed ubiquitously (24). In the present study, amino acid sequencing revealed that the 100-kDa protein is ␤-adaptin, whereas immunoblot analysis with antibodies to ␣and ␤-adaptins disclosed that the 100-kDa protein consists of ␣and ␤-adaptins. The anti-␣-adaptin antibody recognizes both ␣ A -and ␣ C -adaptins, but not the ␤-subunit (21). Both isoforms of ␣-adaptin are present in brain, and a smaller isoform of ␣ Aand ␣ C -adaptins migrates very closely to ␤-adaptin on SDS-PAGE. In addition, we could detect the association of ␣-adaptin with Shc in intact cells derived from tissues other than those of neural origin such as human epidermoid carcinoma KB cells or monkey kidney COS cells. These findings suggest that ␣ A -, ␣ C -, and ␤-adaptins bind to Shc. The reason why we failed to detect peptide fragments of a smaller isoform of ␣ A -and ␣ C -adaptins is probably due to the relatively low amount of these isoforms. Both ␣and ␤-adaptins are present in cells as an HA2-AP2 complex with two smaller subunits. The analysis on gradient gels showed the association of 50-and 17-kDa proteins with Shc. Therefore, it seems likely that the HA2-AP2 complex binds to Shc.
HA2-AP2 is implicated in functions essential for the dynamic cycle of clathrin-coated pits and vesicles. Sorting of membrane proteins and receptors into clathrin-coated vesicles is thought to require recognition of their cytoplasmic domains by adaptors. In vitro binding of adaptors to the cytoplasmic domain of transmembrane proteins such as low density lipoprotein, cation-independent mannose 6-phosphate/insulin-like growth factor II, asialoglycoprotein receptors, and lysosomal acid phosphatase has been demonstrated (30 -33). Furthermore, structural analysis of protein sequence motifs around tail residues shown to be critical for rapid internalization has indicated that the signal for clathrin-mediated internalization contains a tyrosine residue, which is exposed in a ␤-turn conformation. The amino acid sequences required for rapid internalization of transferrin, cation-independent mannose 6-phosphate/insulin-like growth factor II, cation-dependent mannose 6-phosphate, asialoglycoprotein receptors, and lysosomal acid phosphatase are YTDL, YSKV, YRGV, YQDL, and YRHV, respectively, where the tyrosine residue has been shown to be critical for rapid internalization (reviewed in Ref. 34). An in vitro study of lysosomal acid phosphatase has shown that the HA2-AP2 adaptor fails to bind the peptide derived from the cytoplasmic domain of lysosomal acid phosphatase in which the tyrosine residue has been changed to alanine (33), suggesting that the tyrosine residue is required for HA2-AP2 binding. Another group of internalization recognition sequences is seen in the cytoplasmic domains of low density lipoprotein (FDNPVY) and cation-dependent mannose 6-phosphate (FPHLAF) receptors (reviewed in Ref. 34). Mutagenesis analysis of these receptors revealed that both the phenylalanine and the tyrosine residues of the low density lipoprotein receptor and both phenylalanine residues of the cation-dependent mannose 6-phosphate receptor are required for rapid internalization (35,36), suggesting that the complete internalization signal spans a 6-amino acid region with critical aromatic residues at positions 1 and 6. However, there is no direct evidence indicating that this region is an HA2-AP2-binding site. In the present study, we have shown that amino acids 346 -355 of Shc are required for adaptin binding. The amino acid sequence of this region of Shc is RDLFDMKPFE, which contains a 6-amino acid segment with aromatic residues on both sides. It is noteworthy that the adaptin-binding site of Shc contains an amino acid sequence motif similar to that which FIG. 4. Association of proteins from bovine brain lysates with GST-Shc fusion protein and inhibition of binding by the collagen homologous region of Shc. A, GST or GST-Shc-(4 -473) fusion protein immobilized on glutathione-Sepharose beads was incubated with or without bovine brain lysates for 2 h at 4°C. After extensive washing, associated proteins were released by elution with 1 M Tris-HCl (pH 7.4). B-D, brain lysates were first incubated with or without the collagen homologous region of Shc prepared by thrombin digestion of GST-Shc-(233-369) or GST-Shc-(233-345) and then added to GST-Shc-(4 -473) immobilized on glutathione-Sepharose beads. Associated proteins were released either by boiling in SDS sample buffer (B) or by elution with 1 M Tris-HCl (pH 7.4) (C and D). Released proteins were resolved by SDS-PAGE and stained with silver.
has been shown to be required for rapid internalization of membrane proteins.
Recent progress in protein-protein interaction studies has revealed the specificity of binding by certain modular domains. SH2 domains specifically interact with phosphorylated tyrosines, and SH3 domains with proline-rich sequences (5). For example, with respect to EGF receptors, the SH2-containing proteins phospholipase C-␥1, Shc, and Grb2 directly bind to phosphorylated tyrosine residues of EGF receptors (14,37,38). Shc becomes tyrosine-phosphorylated after EGF receptor activation, and the SH2 domain of Grb2 binds to EGF receptors indirectly via the phosphorylated tyrosine 317 of Shc. Grb2 possesses two SH3 domains, which bind to proline-rich sequences of Sos. Recently, the SH3 domains of Grb2 and phospholipase C-␥1 were found to bind proteins other than Sos, such as dynamin, synapsin I, and 145-kDa protein (39 -41). Dynamin has extensive similarity to the product of the Drosophila gene shibire (42) and possesses intrinsic GTPase activity. It has been demonstrated that point mutations in the GTP-binding domain of human dynamin specifically inhibit early events in receptor-mediated endocytosis (43). In fact, mammalian dynamin was recently shown to have a role in clathrin-coated vesicle function (44,45). In NIH/3T3 cells, phospholipase C-␥1 associates with dynamin, and dynamin associates with platelet-derived growth factor receptors in a platelet-derived growth factor-dependent manner (41), suggesting a role for dynamin in ligand-induced receptor endocytosis. In addition, adaptins associate with the C-terminal tail of EGF receptors in an EGF-dependent manner (46), 3 although adaptins constitutively associate with Shc. Thus, it is interesting that components implicated in receptor endocytosis, dynamin and HA2-AP2, associate with substrates of receptors for growth factors and cytokines.
Recently, novel types of association of Shc with other proteins have been reported. Shc was shown to associate with the PEST tyrosine phosphatase (47). Two serine residues at positions 5 and 29 in the N-terminal 45-amino acid region of 52-kDa Shc are suggested to be a binding site for the PEST tyrosine phosphatase (47). Shc has also been shown to bind a tyrosine-phosphorylated protein of 145 kDa in Balb/3T3 cells and L6 myoblasts via the PTB domain (7). Using mutant GST-Shc fusion proteins, the N-terminal region of Shc-(46 -232) was shown to be responsible for the binding of tyrosine-phosphorylated p145. In addition, Shc-(46 -209) binds to tyrosine-phosphorylated growth factor receptors such as EGF receptors and TrkA (17). In the present study, we have shown that the collagen homologous region of Shc (amino acids 346 -355) is implicated in the association with the HA2-AP2 complex. From our data, it is unclear which subunit of the HA2-AP2 complex binds to Shc. In addition, the role of a Shc-HA2-AP2 complex in signal transduction remains unclear. One possibility is that Shc may play a role in ligand-induced receptor internalization since HA2-AP2 complexes are implicated in receptor endocytosis. However, it is possible that this complex plays some other role in signal transduction. It will be important to answer this issue.