©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Shc with Adaptor Protein Adaptins (*)

(Received for publication, August 7, 1995; and in revised form, November 17, 1995)

Yoshinori Okabayashi (1)(§) Yutaka Sugimoto (1) Nicholas F. Totty (2) Justin Hsuan (2) Yoshiaki Kido (1) Kazuhiko Sakaguchi (1) Ivan Gout (2) Michael D. Waterfield (2) Masato Kasuga (1)

From the  (1)Second Department of Internal Medicine, Kobe University School of Medicine, Kobe 650, Japan and the (2)Ludwig Institute for Cancer Research, London W1P 8BT, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 transduction, 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 resulted in the binding of cellular proteins of 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 beta-adaptin and that the 110- and 115-kDa proteins were almost identical to alpha(A)-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alpha(A)-, alpha(C)-, and beta-adaptins are bound to the GST-Shc fusion protein. Immunoblot analysis with anti-alpha-adaptin antibody revealed that alpha-adaptin 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 mutant GST-Shc fusion proteins revealed that the collagen homologous region (amino acids 233-377) of Shc was required for adaptin binding. Conversely, the collagen homologous region of Shc inhibited the binding of adaptins to GST-Shc. In addition, adaptin was able to bind mutant fusion proteins containing amino acids 233-369, 233-355, 346-369, and 346-355 of Shc, but failed to bind a mutant containing amino acids 233-345, suggesting that amino acids 346-355 (RDLFDMKPFE) in the collagen homologous region of Shc are required for adaptin binding. Thus, this study indicates the specific interaction of Shc with alpha- and beta-adaptin components of plasma membrane adaptor proteins that are thought to be involved in receptor endocytosis.


INTRODUCTION

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 46, 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 alpha1-collagen, and a distinct N-terminal phosphotyrosine-binding 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, 10, 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, 15, 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 5`-CGGAGAATTCATGAGGCCCTGGACATGAACAAGC-3`, and the antisense primer 5`-AAGAGAATTCTAGGGCAGATCACAGTTTCCGC-3`. 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 5`-CACTCAGCTGGATCCTGTCCAGGG-3`, 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 µg/ml ampicillin, and the expression of fusion protein was induced by adding isopropyl-beta-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 µg/ml aprotinin.

Affinity Purification and Sequencing of Shc-binding Proteins

Affinity resins for the isolation of Shc-binding proteins were prepared by immobilizing 20 µg 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 µg/ml aprotinin, followed by sequential centrifugation at 15,000 times g for 60 min and at 40,000 times 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 µl 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 -80 °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 µM 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 µM 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 alpha- and beta-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 alpha- and beta-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 115, 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).


Figure 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 115, 110, and 100-kDa proteins with alpha- and beta-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 alpha(A)- and alpha(C)-adaptins(31) , and those of p100 are aligned with human beta-adaptin(27) . Dashes indicate identities to alpha(A)-adaptin.



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 20 µg 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 -20 °C. This was repeated until the amounts of Shc-binding proteins reached 5 µg. Affinity-purified Shc-binding proteins were then digested in the gel with lysyl endoproteinase. 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, KMERQVVLRTDK, KGLEISGTFTHRQGHFYMEMN, KLVYLYLVNYAK, and KNSFGVIPSTPLAI. Comparison with the sequences in the GenBank Data Bank revealed almost complete identity to rat and human beta-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, KTSVQFQNFSPTVVHPGDL, and KYLQVGHLLREPNAQAQMYRLTLRTK, and that of the 115-kDa protein yielded KTSVQFQNFSPTVVHPGDL. These amino acid sequences were almost identical to the mouse alpha(A)-adaptin, but not alpha(C)-adaptin, of the adaptor complex (Fig. 1B) (24) . These results indicate that alpha(A)- and beta-adaptins bind to Shc expressed as fusion protein with GST.

The Shc binding activity of adaptins was confirmed by immunoblot analysis with specific antibodies to alpha- and beta-adaptins. Anti-alpha-adaptin antibody (AC1-M11) recognizes both alpha(A)- and alpha(C)-adaptins, but not beta-adaptin. Anti-beta-adaptin antibody (B1-M6) has no cross-reactivity to alpha-adaptins(21) . Affinity-purified Shc-binding proteins were subjected to SDS-PAGE, transferred onto nitrocellulose, and blotted with antibodies to alpha- and beta-adaptins. Antibody to alpha-adaptin recognized several proteins of 100115 kDa that probably correspond to alpha-, alpha-, alpha-, and alpha-subunits (data not shown). Anti-beta-adaptin antibody recognized a single protein with an apparent molecular mass of 100 kDa (data not shown). Nonimmunized mouse globulin did not recognize these bands. These results indicate that Shc-binding proteins include the alpha(A)-, alpha(C)-, and beta-adaptins of the adaptor complex.

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-alpha-adaptin antibody. In these cells, alpha-adaptin was identified in anti-Shc immunoprecipitates (Fig. 2). Antibody to alpha-adaptin (AC1-M11) recognized both alpha(A)- and alpha(C)-adaptins. Both alpha(A)- and alpha(C)-adaptins were predominant in PC12 cells, whereas alpha(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-alpha-adaptin antibody. Approximately 1-5% of cellular adaptins were coimmunoprecipitated with Shc. More alpha-adaptin was coimmunoprecipitated with Shc in KB and COS cells than in PC12 cells. However, the amount of alpha-adaptin coimmunoprecipitated with Shc was not changed upon stimulation with EGF, suggesting that the association of adaptin with Shc is constitutive.


Figure 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-alpha-adaptin antibody. Lysates equal to 2% of that used for immunoprecipitation were also electrophoresed and blotted with anti-alpha-adaptin antibody. Arrowheads indicate alpha-adaptin. NRS, nonimmunized rabbit serum.



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 indicate that amino acid sequence 346-355 of Shc is necessary for adaptin binding.


Figure 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.



Since alpha- and beta-adaptins are components of the plasma membrane adaptor complex of clathrin-coated vesicles and the adaptor complex also contains two other subunits of 50 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 50, 47, 45, 35, 19, and 17 kDa as well as 115, 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 50, 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.


Figure 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.




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 115, 110, and 100 kDa and several bands of lower molecular mass including 50 and 17 kDa. Microsequencing of 100115-kDa proteins showed them to be almost identical to the previously characterized proteins alpha(A)- and beta-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 alpha- and beta-adaptins and two smaller subunits of 50 and 17 kDa, and Golgi adaptor HA1-AP1 consists of - and beta`-adaptins and two smaller subunits of 47 and 19 kDa(27, 28) . In addition, there are two isoforms of alpha-adaptin (alpha(A) and alpha(C)) encoded by distinct but highly homologous genes(24) . Two alternative transcripts of alpha(A)-adaptin have been identified: one is expressed in brain(24) , and a smaller isoform is expressed ubiquitously(29) . The smaller isoform of alpha(A)-adaptin migrates on SDS-polyacrylamide gels with a mobility similar to that of alpha(C)-adaptin, which is also expressed ubiquitously(24) . In the present study, amino acid sequencing revealed that the 100-kDa protein is beta-adaptin, whereas immunoblot analysis with antibodies to alpha- and beta-adaptins disclosed that the 100-kDa protein consists of alpha- and beta-adaptins. The anti-alpha-adaptin antibody recognizes both alpha(A)- and alpha(C)-adaptins, but not the beta-subunit(21) . Both isoforms of alpha-adaptin are present in brain, and a smaller isoform of alpha(A)- and alpha(C)-adaptins migrates very closely to beta-adaptin on SDS-PAGE. In addition, we could detect the association of alpha-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 alpha(A)-, alpha(C)-, and beta-adaptins bind to Shc. The reason why we failed to detect peptide fragments of a smaller isoform of alpha(A)- and alpha(C)-adaptins is probably due to the relatively low amount of these isoforms. Both alpha- and beta-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, 31, 32, 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 beta-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 (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 (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 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, 40, 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.


FOOTNOTES

*
This work was supported by a grant (to Y. O.) and by a grant-in-aid for cancer research (to M. K.) from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Second Dept. of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan. Tel.: 81-78-341-7451; Fax: 81-78-382-2080.

(^1)
The abbreviations used are: EGF, epidermal growth factor; SH2, Src homology 2; PTB domain, phosphotyrosine-binding domain; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.

(^2)
J. J. Hsuan, unpublished data.

(^3)
Y. Okabayashi and M. Kasuga, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Margaret S. Robinson for providing antibodies to alpha- and beta-adaptins (AC1-M11 and B1-M6) and critical reading of the manuscript, Drs. W. Boll and T. Kirchhausen for providing an immunoprecipitation protocol for adaptins, and the Foundation for Promotion of Cancer Research for providing KB and PC12 cells.


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