THE JO~AL OF BIO~ICAL 268, No. 26, Issue of 15, pp. 19312 ... › content › 268 › 26 ›...

THE J O ~ A L OF B I O ~ I C A L CHENISTRY 8 1993 by The American Society for Biochemistry and Molecular Biology, Inc

vel. 268, No. 26, Issue of September 15, pp. 19312-19320, 1993 Printed in U.S.A.

Ligand-induced Internalization of the Epidermal Growth Factor Receptor Is Mediated by Multiple Endoiytic Codes Analogous to the Qrosine Motif Found in Constitutively Internalized Receptors*

(Received for publication, April 8, 1993, and in revised form, June 1, 1993)

Chia-Ping ChangSP, Cheri S. Lazar$, Brenda J. Walshn, Masato Komuroll, James F. Collawn** $$, Leslie A. Kuhnll II, John A. Tainerll 0, Ian S. Trowbridge**, Marilyn G. Farquhadl, Michael G. RosenfeldSO 98, H. Steven Wilefl m, and Gordon N. Gill$ From the $Department of Medicine, the (Division of Cellular and Molecular Medicine, and the §Howard Hughes Medical Institute, University of California-Sun Diego, La Jolla, California 92093, the **Department of Cancer Biology, The Salk Institute for Biological Studies, Sun Diego, California 92186, 11 IDepartment of Molecular Biology, Scripps Research Znstitute, La Jolla, California 92037, and the VDepartment of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132

Ligand-induced internalization of epidermal growth factor (EGF) receptors via a high affinity saturable pathway requires sequences located in the carboxyl terminus distal to the tyrosine kinase domain. Three regions were found to contain endocytic motifs as defined by their ability to restore internalization function to EGF receptors truncated at the distal border of the kinase domain at residue 958. Deletional analysis identified the sequence -QQGFF as essential for function of the region encompassing residues 993-1022. QQGFF and the deduced sequence of the region encompassing residues 973-991 (s7sFYRAL) could effectively replace the endogenous endocytic code of the transferrin receptor WTRF). FYRAL and YTRF were less active than QQGFF when substituted into region 993-1022 of the EGF receptor, but a synthetic sequence (NNAYF), predicted to have structural features of a tight turn, effectively replaced QQGFF for EGF receptor internalization. Whereas EGF receptor sequences functioned effectively in the transferrin receptor, function of these sequences in the EGF receptor was strictly dependent on intrinsic tyrosine kinase activity as demonstrated kinetically and by immunofluorescence using semithin cryosections. Ligand- dependent endocytosis and down-regulation of the EGF receptor thus require multiple sequence motifs that are exchangeable between ligand-dependent and -independent receptors, but that require intrinsic tyrosine kinase activity for function in the context of the EGF receptor.

The function of membrane receptors is dependent on correct trafficking between cellular compartments. The low density lipoprotein (LDL)I and transferrin (M receptors, whose pri-

Grant CB3P and Council for Tobacco Research U. S. A., Inc. Grant 1622 * This work was supported in part by the American Cancer Society

(to G. N. G.), National Institutes of Health Grants CA34789 (to I. S. T.) and DK39949 (to M. G. R,), and National Science Foundation Grants BCS9111940 (to H. S. W.) and DIR8822385 (to J. A. T.). The costs of publication of this article were defrayed in part by the payment of page

in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. charges. This article must therefore be hereby marked “uduertisement”

$$ Arthritis Foundation Investigator. Present address: Dept. of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294.

$9 Investigator of the Howard Hughes Medical Institute. Ill Supported by a National Institutes of Health research career de-

The abbreviations used are: LDL, low density lipoprotein; Tf, trans- velopment award.

mary function is transport of nutritional molecules into the cell, are internalized by virtue of their constitutive binding to clath- rin-coated pits (1). Endocytosis of this class of receptors is independent of ligand binding, but depends on specific cytoplasmic motifs that contain an aromatic residue residing in a tight turn structure (2-5). Because the behavior of both occupied and unoccupied receptors is similar, ligand binding does not alter cell-surface receptor concentration or intracellular distribution. Mutations in specific &-amino acid sequences abolish the high constitutive internalization rates observed in wild- type receptors, and the resulting low endocytic rates appear due to random entrapment in coated pits (3-6). “he 4-6-amino acid endocytic sequence codes can be interchanged between the Tf receptor and the cation-independent mannose 6-phosphate receptor, indicating that their internalization occurs through a similar mechanism (7, 8).

Cell-surface tyrosine kinase receptors that activate signal transduction pathways display a different pattern of internalization. Ligand binding activates their intrinsic tyrosine kinase activity and results in redistribution of diffuse cell-surface receptors to coated pits, enhanced rates of endocytosis, and down- regulation from the surface (9, 10). Occupancy-induced internalization of epidermal growth factor (EGF) and insulin receptors requires both intrinsic protein-tyrosine kinase activity and specific sequence motifs (9-12). Two internalization codes have been identified in exon 16 located in the juxtamem- brane region of the cytoplasmic domain of the insulin receptor (131, and peptides corresponding to both sequences adopt a tight turn structure in solution (5, 14). Deletional analysis of the EGF receptor has shown that sequences required for occupancy-induced internalization are located in the carboxyl terminus distal to the core tyrosine kinase domain bounded at an exon-intron junction (9). Sequences required for maximum rates of ligand-induced endocytosis have been proposed to re- side in more than one portion of the 229-amino acid regulatory carboxyl terminus (9, 15).

In this study, we have identified two sequence codes in the regulatory carboxyl terminus of the EGF receptor that are involved in ligand-induced endocytosis. These sequences can substitute for the endogenous sequence code of the Tfreceptor, and conversely, the Tf receptor endocytic code can replace an endogenous code in the EGF receptor. Although both EGF and Tf receptor internalization sequences function in the context of the Tf receptor that lacks intrinsic enzymatic activity, function

ferrin; EGF, epidermal growth factor; IC, internalization component.

19312

Endocytic Codes in EGF Receptor 19313

of these sequences in the context of the EGF receptor is strictly dependent on ligand binding and intrinsic tyrosine kinase activity. Because these sequences function in the context of EGF receptors that lack self-phosphorylation sites, the kinase requirement appears due to tyrosine phosphorylation of a cellular component involved in endocytosis.

MATERIALS AND METHODS Construction of Mutant Receptors-For construction of mutant EGF

receptors, the vectors expressing holoreceptor and c'991 and c'958 f993-1022 mutants were used (15). In the nomenclature used, c' identifies the carboxyl-terminal amino acid, and f identifies the amino acid residues fused to the carboxyl terminus. Deletion mutants c'958 flO24- 1186 and c'958 flOOl-1022 were prepared by the strategy used to pre- pare mutant c'958 f993-1022 EGF receptors with 2 amino acids (Val and Asp) created at the fusion point. To obtain a truncation receptor c'958 f993-1000, a termination codon was introduced a t in c'958 f993-1022 EGF receptor cDNA. The triple VMXA mutant was constructed by oligonucleotide-directed mutagenesis with sequences 5'-

CAGTGTTGAGGGCCTCCATGACGCCCACTGCA-3', a n d 5 ' - AGTCCTGCTGGGCGTCCATGACGTCCAGGCTA-3' (16). Similarly, the AldAsn substitution was generated with the sequence 5'-

3' between residues 1010 and 1022. The codons for the amino acids QQGFF between residues 995 and 1000 were replaced by the codons for the amino acid internalization motif YTRFS of the transferrin receptor ( 5 , 7) by using a 45-base pair oligonucleotide with the sequence 5'-

AC-3'. Oligonucleotides used to replace QQGFF with FYRAL and with N N A Y F h a d t h e f o l l o w i n g s e q u e n c e s : f o r F Y R A L , 5 ' -

A C 0 3 ' ; a n d f o r N N A Y F , 5 " T G A G G G G C T G C T G A A - ATATGCGTTG'MTGGGATGAGGTCGAC-3'. All the mutated regions were verified by sequencing (17). Mutagenized EGF receptor cDNAfrag- ments were reconstituted in the eukaryotic expression vector pX (18).

For construction of mutant Tf receptors, a CZaI fragment containing the entire coding region of the human Tf receptor was cloned into the phagemid pBluescript SK (Stratagene, La Jolla, CAI (7). Oligonucle- otide site-directed mutagenesis was performed with pBluescript SK phagemid templates of the wild-type Tf receptor by the method of Kunkel (16) using the Muta-gene mutagenesis kit (Bio-Rad) as described previously (4). Mutants were selected by restriction mapping, and ClaI fragments encoding the mutant receptors were then excised and cloned into the retroviral expression vector BH-RCAS (19). The mutations were verified by dideoxynucleotide sequencing (17,20) of the BH-RCAS constructs using the Sequenase kit (U. S. Biochemical Corp.) according to the manufacturer's directions.

Preparation of Cells Expressing Mutant Receptors-Mutant EGF receptors were transfected into mouse B82 L cells that lacked endogenous EGF receptor mRNA and protein using calcium phosphate precipitation (18,21). Stable clonal lines were selected with 400 nM methotrexate and used for subsequent analysis. Human Tf receptors were expressed in chicken embryo fibroblasts, which were obtained from SPAFAS, Inc. (Norwich, CN) as primary embryo fibroblasts (C/O, Line 22) and grown in Dulbecco's modified Eagle's medium supplemented with 1% (v/v) chicken serum, 1% (v/v) defined calf serum (HyClone Laboratories, Lo- gan, UT), 2% (v/v) tryptose phosphate broth (Difco). Chicken embryo fibroblasts were transfected with 30 pg of retroviral construct DNA/ 10-cm tissue culture plate of 40% confluent cells using the Polybrenel dimethyl sulfoxide method (22). Stable expression of wild-type and mutant human Tf receptors was achieved using a helper-independent retroviral vector (BH-RCAS) derived from Rous sarcoma virus (19). 1-2 weeks after transfection, the chicken embryo fibroblasts stably expressed wild-type and mutant receptors on their cell surface as a result of infection by recombinant virus. Cell-surface expression was confirmed by '251-labeled human Tf binding a t 37 "C under steady-state conditions.

Internalization of EGF Receptors-Mouse EGF was purified from submaxillary glands (23) and iodinated with lZ5I (Amersham Corp.) using IODO-BEAD (Pierce Chemical Co.) according to the manufacturer's recommendation. Free iodine was separated from the radiolabeled ligands using an 0.8 x 20-cm column of Sephadex G-10 equilibrated with phosphate-buffered saline. The specific activity of '251-labeled EGF was generally between 600 and 1800 cpxdfmol. Cells grown to conflu- ence in 35-mm dishes were switched from growth medium to serum-free

GGCTGATTGTGGGCGACCATGACCTGCACAGAG-3' , 5"

GGTGGAA'I"l'GTTTGCG""I'GCTGCCAGG'RTGCCAGGAGGGGAGT-

CGTGGAGGGGCTGCTGCTGAACCGGGTATATGGGATGAGGTCG-

CGTGGAGGGGCTGCTCAGGGCACGGTAGAATGGGATGAGGTCG-

Dulbecco's modified Eagle's medium containing 20 mM HEPES (pH 7.4) and 0.1% bovine serum albumin and no bicarbonate (DHB medium) 18 h before experiments. Binding experiments were initiated by changing to DHB medium containing the indicated concentrations of labeled ligand. The addition of ligand and the rinsing of cells were done with a semiautomatic apparatus (24). Binding was terminated by rapidly rinsing six times with 2 ml of ice-cold WHIPS buffer (20 mM HEPES (pH 7.4), 140 mM NaC1, 5 mM KCl, 0.5 mM MgCl2, 1 mM CaCI,, 1 mg/ml polyvinylpyrrolidone). The relative amounts of ligand associated with the cell surface and interior were determined by acid stripping at 0 "C using 50 mM glycine HCI, 100 mM NaC1, 2 mg/ml polyvinylpyrrolidone, 2 M urea (pH 3.0). Stripping efficiencies were generally 98%. Nonspecific binding was determined by measuring binding to B82 cells lacking EGF receptors and was generally ~ 1 % of total binding. Cell number was determined by a Coulter Counter. Values for surface-bound and internalized ligand were corrected for nonspecific binding and for spillover from the interior and surface of the cell, respectively (24). Specific internalization rates were determined by plotting the integral of surface-associated ligand against the amount internalized (25). The slopes were then determined by linear regression, Correlation coefficients of internalization plots were generally >0.98.

Saturation of specific receptor internalization was established using lZ5I-EGF concentrations ranging from 1 to 120 ng/ml. Internalization plots were obtained a t each concentration using 1-min intervals for a total of 5 min. The internalization velocity was plotted against the specific internalization rate, yielding a Satin plot (26). These plots were analyzed with the program ProFit (QuantumSoft, Zurich) as a two- component process by nonlinear regression using the Levenberg-Mar- quardt algorithm. Initial parameter estimates were based on previous analyses and were calculated using Monte Carlo simulations. Parame- ters were estimated with the low affinity endocytic component either fixed or variable. No significant differences in the estimates of the high affinity component resulted. Chi-square values of the fits all ranged between 1 x and 3 x

Simulations of Satin plots were run using the two-component endocytic model previously described (26). Model parameters were 1.2 min" for the specific internalization rate of coated pits, 0.03 min-' for nonspecific internalization, 20,000 for the total number of internalization components, and a total EGF receptor complement of 1 x 106/cell. Simulations were run in Microsoft Excel by varying the number of occupied receptors and assuming that equilibrium of receptors with coated pit components occurs faster than internalization. All templates for the simulations and data analyses are available from the authors upon request.

Down-regulation of EGF Receptors-Cells expressing various EGF receptors were treated with 50 nM EGF for the indicated times a t 37 "C. Dishes were then placed on ice, the medium was removed, and surface- bound EGF was stripped by incubation with 50 mM acetic acid (pH 2.5) containing 135 mM NaCl and 2.5 mM KC1 for 5 min. Cells were rinsed, and residual surface receptors were detected by incubation with 0.5 mM lz5I-EGF (9, 15). Nonspecific binding was estimated in the presence of a 200-fold molar excess of unlabeled EGF and was <5%. Data points represent the mean * S.E. of four or more experiments, each containing triplicate dishes for each data point. Measurements were made in at least two independent clonal B82 cell lines expressing each mutant EGF receptor.

Analysis of Tf Internalization under Steady-state Conditions "Diferric human Tf was labeled with lZ5I to a specific activity of 350- 700 cpdpmol using Enzymobeads (Bio-Rad) according to the manufacturer's directions. Chicken embryo fibroblasts expressing wild-type and mutant Tf receptors were plated in triplicate a t a density of 7.5 x lo4 cells/cm2 in 24-well Costar tissue culture plates 24 h prior to the assay. For the assay, the cells were first incubated for 1 h a t 37 "C in serum- free Dulbecco's modified Eagle's medium and were then incubated with 4 pg/ml 1251-labeled Tf in 0.1% bovine serum albumin in Dulbecco's modified Eagle's medium for 1 h a t 37 "C. The medium was removed, and the cells were washed three times with 1 ml of ice-cold 0.1% bovine serum albumin in 10 mM sodium phosphate (pH 7.4), 0.15 M NaCl and incubated twice for 3 min with 0.5 ml of 0.2 M acetic acid, 0.5 M NaCl (pH 2.4) to remove surface-bound lZ5I-labeled Tf (27). Cells were then removed from the wells with 1 M NaOH, and radioactivity in the acid wash and the cell lysate was determined. Prolonged incubation with the acid wash did not affect the radioactivity released (7). Because at steady state the net rate of internalization of the Tf receptor (k,[TfR].,,) is equal to the net rate of recycling (k,[TfRI,), the specific internalization rate (k,) of surface Tf.Tf receptor complexes is directly proportional to the [TfRli,,/[TfRl,,, ratio (28).

Immunofluorescence Microscopy-Cells expressing various mutant

19314 Endocytic Codes in EGF Receptor

EGF receptors were incubated with anti-human EGF receptor monoclonal antibody 13A9 for 60 min at 4 "C with or without 50 nM EGF. Monoclonal antibody 13A9, which recognizes a protein epitope in the ectodomain, does not compete with EGF for binding to the EGF receptor (29) and does not affect the rate of internalization of 'T-EGF (data not shown). Cells were then washed three times with cold phosphate-buffered saline, warmed by adding 38-39 "C medium with or without 50 nM EGF, and incubated at 37 "C for 60 min. Cells were fixed by periodate- lysine-paraformaldehyde (30); scraped from the culture dish; pelleted; mixed with 2.3 M sucrose in 0.1 M potassium phosphate (pH 7.4), 20% polyvinylpyrrolidone; and frozen in liquid nitrogen (31). Semithin cryosections (1 p m thick) were placed on gelatin-coated glass slides and incubated with rabbit anti-lgpl20, a lysosomal marker (32) (provided by Dr. W. A. Dunn, Jr.), for 1 h at room temperature, followed by a combi- nation of fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Zymed, San Mateo, CA) and tetramethylrhodamine B isothiocyanate- conjugated F(ab')z fragments of goat anti-rabbit IgG (TACO, Inc., Bur- lingame, CA).

RESULTS Endocytic Codes Are Distributed in Three Regions of Car-

boxyl Terminus of EGF Receptor-Progressive carboxyl-terminal truncation of the EGF receptor decreases the rate of ligand- induced endocytosis (9, 33, 34). Analysis of a 48-amino acid region located between residues 973 and 1022 showed that both the proximal and distal halves of this sequence contained motifs that restored high affinity saturable endocytosis to internalization-defective EGF receptors (15). Tyrosine self-phosphorylation was not an absolute requirement for ligand-induced endocytosis because internalization-competent mutants did not necessarily contain any self-phosphorylation sites (15). A schematic representation of the domains implicated in endocytosis of the EGF receptor is shown in Fig. 1. The region distal to residue 1022 contains three NPXY motifs analogous to the endocytic code found in the LDL receptor, which is FXNPXY (31, that are thus candidate internalization sequences in the EGF receptor. The endocytic function of the distal carboxyl-terminal 162 amino acid residues from positions 1024 to 1186 was in- vestigated by fusing them to residue 958 at the end of the kinase domain. The fusion receptor c'958 fl024-1186 contains four self-phosphorylation sites, allowing analysis of this region of the EGF receptor without the decrease in tyrosine kinase activity that occurs with mutations and partial truncations that remove the self-phosphorylation sites (33, 40). Reported decreased rates of internalization of EGF receptors lacking these self-phosphorylation sites (33, 40) may have resulted from either decreased tyrosine kinase activity or loss of endocytic codes.

Internalization rates were measured at varying EGF concentrations, and the data are presented in Fig. 2 as saturation- internalization plots (26). At low EGF concentrations, the specific internalization rate ( K , ) of EGF holoreceptors was more

EGF receptor and carboxyl-terminal FIG. 1. Schematic representation of

regions that determine endocytosis. The core tyrosine kinase domain is bounded by exon-intron junctions at residue 957 (35) and at residue 663 as analyzed by polymerase chain reaction (data not shown). Mutation of LYS'~' to methionine abolishes kinase activity. The regulatory carboxyl terminus contains five identified sites of tyrosine phosphorylation (residues 992, 1068, 1086, 1148, and 1173 (indicated by asterisks)) (36-39). Three NPXY motifs similar to the NPXY internalization code of the LDL receptor are indicated (NPVY'086, NPEY"14, and NPDY114*). Fragments that provide gain of endocytic function are designated regions 1 , 2, and 3. TM, transmembrane segment.

Domains: 1

than &fold greater than that of c'958 EGF receptors (Fig. 2, upper lefc panels). Internalization of EGF holoreceptors was a saturable, second-order process, indicating that it is induced by receptor occupancy. In contrast, internalization of c'958 EGF receptors was unaffected by receptor occupancy and resembles the constitutive internalization of kinase-inactive EGF receptors (41). Fusion of residues 1024-1186 (region 3) to c'958 restored high affinity saturable endocytosis (Fig. 2, lower left panels). At low concentrations of EGF, endocytic rates of the c'958 fl024-1186 EGF receptor were similar to those observed for EGF holoreceptors. They were also similar to those observed for c'958 f993-1022 EGF receptors, in which region 2 is fused to c'958. EGF receptors containing region 1 (~'991) exhibited somewhat lower K , values, but displayed a clear decrease in FE, with increasing internalization velocity, characteristic of high affhity saturable endocytosis (26). Mutation of the three NPXY sequences (NPW'0s6, NPEY1114, and NPDY114s) to VMXA had no effect on the ability of residues 1024-1186 to restore high affinity saturable endocytic function to c'958 EGF receptors. These results indicate that at least one endocytic code distinct from NPXY is located within residues 1024-1186.

The activity of NPVY was directly tested by adding a 14- amino acid segment to the carboxyl terminus of c'973 EGF receptors, which are defective in ligand-induced endocytosis (9, 15). This sequence in the context of 971SNPNPWR- RPQSKLGSL failed to restore ligand-induced saturable endocytosis to c'973 EGF receptors (see Table I and below). The lack of activity of this 29-amino acid addition to the carboxyl terminus of c'958 EGF receptors indicates the importance of specific sequences and their surrounding context.

Ligand-induced down-regulation of surface EGF receptors correlated with endocytic function (Fig. 2, right panels). The extent of down-regulation of fusion mutant EGF receptors containing region 2 or 3 resembled that of EGF holoreceptors. Down-regulation of c'991 EGF receptors containing region 1 was less than that exhibited by EGF receptors containing region 2 or 3.

Identification of n o Endocytic Sequence Codes in EGF Re- ceptor Carboxyl Terminus-To identify specific amino acid residues necessary for ligand-induced endocytosis of EGF receptors, additional mutations were placed in the 29-amino acid fragment containing residues 993-1022. Chou-Fasman analysis (42) of the EGF receptor predicts that this region contains turns at both termini and serine and threonine residues in this fragment are phosphorylated in vivo (43). Substitution of ala- nine and asparagine for serine and threonine in the distal part, however, did not affect function (Fig. 3). When the carboxyl terminus of the fragment from residues 1001-1022 was directly fused to the kinase domain at residue 958, endocytosis and

EGF Receptor

Ecto TM Kinase Regulatory 622 644 663 K7" 957 1186

. . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

NPDY' Y' NPVY' NPEY Y'

/ I ) j 958 973 992 1022 1068 1086 1114 1148 1173 1186

UUrl I 1 2 3


Satin Plots Down-Regulation

O a 4 0 7 c‘99 1

1

0.40

n ~ ‘ 9 5 8 f 1024-1 186

0.20/k 1

0.00 I 0 ‘ 1Xio4 ’ 2 x i ~ 4 0

c‘958 1 t Wild Type

P 1 . I - I

r c‘958

C’958 f 993-1022 i

1 1 ~ ‘ 9 5 8 11024-1 186

t Internalization velocity (molecules x min -1)

L ~ ‘ 9 5 5 11024-1186

L C’958 1993-1022

\ C’958 f1024-1186

3-VMXA

0 20 40 60 0 20 40 60 Treatment time with EGF (min)

. l o o

.50

100

50

0

ration-internalization plots for the indicated mutant EGF receptors; right, down-regulation of these receptors (*S.D.). Internalization rates were FIG. 2. Internalization and down-regulation of mutant EGF receptors that contain various carboxyl-terminal regions. L e f t , satu-

measured using 0.20-20 nM 1251-EGF, and calculated k, values were plotted against the velocity of internalization. In the mutant receptor c’958 fl024-1186, the three NPXY motifs were changed t o VMXA.

down-regulation were not significantly restored. The amino- terminal 8 amino acids (residues 993-1000) were, however, able to provide a significant gain of endocytic function when fused to residue 958, as indicated by the steep downward slope of the curve (Fig. 3). This fragment also partially restored receptor down-regulation (see Fig. 6). The ability of residues 993- 1000 to restore endocytic function, in contrast to the inactive residues 1001-1022, indicates that the endocytic code is found in LIPQQGFF. Significantly, the sequence QQGFF is predicted to display a tight turn, a structural feature of the Tf, LDL, and insulin receptor endocytic codes (4, 5, 14).

Visual inspection of Satin plots indicated a quantitative difference between receptors containing residues between positions 1024 and 1186 and those with residues between positions 958 and 1022. Receptors lacking residues 1024-1186 had lower internalization rates, but displayed very steep Satin plots, in- dicative of high affinity for coated pits. Quantitative analysis of Satin plots by nonlinear regression confirmed this impression. As shown in Table I, the mutant EGF receptors could be grouped into three broad categories based on both affinity for the endocytic apparatus (Kcp) and the V,, of endocytosis through the high affinity pathway. Group A included wild-type receptors and those containing residues between positions 1024 and 1186. These receptors displayed affinities of between 2 and 3 x and a V,, of -10,000 receptors cell” min”. This is very close to previously reported values in B82 cells (26, 41). Group B had a high V,,,, but low affinities ( 4 x Trun-

cated receptors such as c’973 and e958 could probably be included in this group, but our parameter estimation routines would not converge using data from these receptor types because of their affinities. Group C included receptors with sequences between positions 973 and 1022. This group displayed receptor affinities equivalent to or higher than those of wild- type receptors, but with substantially lower V,, values.

There are several possible explanations for the difference in behavior between the mutant EGF receptors. There could be multiple binding sites for EGF receptors in coated pits that depend on different receptor sequences. Alternatively, the affinity of the EGF receptor for coated pit structures could depend on two processes: the binding of the receptor to a specific internalization component (IC) and the binding of the receptor-IC complex to coated pits. This is the model we have previously proposed based on both kinetic considerations and the ability of the endocytic apparatus to discriminate between receptor types (26, 41). In this case, the overall “affinity” of a receptor for coated pits depends on two sequential binding steps. To determine how changes in these two interactions could affect Satin plots, we performed a simulation in which the affinity of EGF receptors for ICs was varied. We also simulated changes in the affinity of the receptor-IC complex for coated pits. As shown in Fig. 4A, changing the affinity of the complex for coated pits changes the apparent V,,, of the process. In contrast, simply changing the affinity of the receptor for the IC results in changes in the slopes of the Satin plots without a


I 958 993 LIPPQGEESSPSTSRTPLLSSLSATSNNST 1022

1 993 AN A AA 1022

I1

1001 I11

1022

993 1000 IV

0.00 I I I I I

0.0 0.5 1 .o 1.5 2.0 2.5

Velocity (molecules x min"x 1 0 . ~ )

boxyl terminus of EGF receptor. The sequences fused to residue 958 FIG. 3. Localization of endocytic sequence in region 2 of car-

of the EGF receptor are indicated (upper panel). The saturation-internalization plots were done using 0.20-20 nM lZ5I-EGF as described under "Materials and Methods."

TABLE I Relative afinity and capacity of the endocytic system

for mutant EGF receptors

fected into B82 cells as described under "Materials and Methods." The Various mutants of the EGF receptor were constructed and trans-

relative affinity and capacity of the high affinity component of the endocytic system for these mutants was determined by Satin plot analysis. Each measurement is the average of two independent experiments using 10 internalization plots each (total of 100 separate measurements for each mutant). The groupings were set using criteria discussed in the text.

Mutant receptor Kep V,, Grouping

Wild tvDe molecule" cell receptors cell" min"

2.6 x 10-5 11,600 A c'958 gO24-1186 2.8 x 11,200 A

3-VMXA c'958 fl024-1186 3.3 x 8500 A

c'973 rnPvY" 7.8 x 19,700 B ~ '958 flOOl-1022 7.6 X lo-' 13,700 B

c'991 1.9 x 10-5 3600 C c'1000 2.8 x 10-5 3700 C c'1010 3.6 x 3800 C 958 f993-1000 4.0 x 3200 C c'958 f993-1022 5.2 x 3200 C

871SNPNPVYRRPQSKLGSL.

corresponding change in the V,,, (Fig. 4B). I t seems likely that alterations in EGF receptor structure would affect both receptor-IC interactions and the ability of receptors to form ternary complexes with coated pits. EGF receptors that retain endocytic sequences located between residues 958 and 1022 retain high affinity for the endocytic apparatus even though there

0.30

A

B

/ Receptor:IC affinity

- 0.00

0 1.1 04 2.1 04

Velocity (molecules x rnin-1 x

FIG. 4. Effect of receptor-internalization component affinity and receptor-internalization component-coated pit affinity on shape of Satin plots. Shown are simulations of a model in which the EGF receptor forms a complex with an IC, followed by the formation of a ternary complex with coated pits. A, effect of varying the steady-state fractional association of the receptor-IC complex with coated pits from 25% (bottom curve) to 75% (top curve) using a receptor-IC affinity of 2 x B , effect of increasing receptor-IC affinity from 2 x (bottom curue) to 7 x (top curve) with a fractional coated pit association of 25%. Other parameters were as described under "Materials and Meth- ods."

is a decrease in the Vm=. Analysis of sequences in this region thus appeared more likely to identify motifs involved in the binding of the EGF receptor to specific IC proteins.

The endocytic code located in region 1 of the EGF receptor is most likely present between residues 973 and 991 because c'973 EGF receptors lack endocytic function and do not down- regulate (9, 15). Comparisons between this region and the Tf receptor endocytic code suggested that the sequence 972NFYRAL as a likely candidate. This sequence is predicted to be part of a turn that precedes an acidic helix in the EGF receptor structure. To test the function of F Y R A L , it was substituted for QQGFF in fragment 993-1022, and fusion mutants of the EGF receptors containing this sequence were analyzed for endocytic function. Fig. 5 shows that whereas FYRAL was less effective than QQGFF, it was able to function weakly in the context of fragment 993-1022. This is in agreement with the rank order of regions 1 and 2 in down-regulation. The endocytic code of the Tf receptor YTRFS could also replace QQGFF and function weakly in the context of the EGF receptor 993-1022 fusion protein. A synthetic sequence (NNAYF) that contains features similar to the tight turn endocytic code of the LDL receptor (5) was also substituted for QQGFF. This sequence resulted in somewhat stronger endocytic function compared with the native sequence.

Tyrosine kinase activity was required for function of these sequences (Fig. 5). When analyzed in the context of kinase-


0.20

0.15

- - .- E 0.1c -

Y X

O.O!

O.O(

5 1

I I 0.0

QQGFF->YTRFS 0 QQGFF->NNAYF + QQGFF->FYRAL a 958/993-1022 (QQGFF) A M721 QQGFF->MRFS

FIG. 5. Comparison of endocytic function of various sequences placed within region 2 of carboxyl terminus of EGF receptor. The indicated substitutions were made within the fragment containing residues 993-1022 in the mutant c'958 f993-1022 EGF receptor. A construction with a QQGFF-to-YTRFS substitution made in kinase- inactive EGF receptors that contain a Lys721 - Met substitution is shown. Saturation-internalization plots were generated as described under "Materials and Methods."

M ~ ~ ~ Y T R F S

f1001-1022 C'958

f993-1000 c'958

YTRFS

NNAYF

F t R A L

O l I I I -I 0 20 40 60 80

Time (min)

FIG. 6. Ability of endocytic sequences placed within region 2 of carboxyl terminus of EGF receptor to mediate receptor down- regulation. The indicated substitutions were made within the fragment containing residues 993-1022 in the mutant c'958 f993-1022 EGF receptor. The c'958 flOOl-1022 and c'958 f993-1000 receptors are also shown. A construction with a QQGFF-to-YTRFS substitution made in kinase-inactive EGF receptors that contain a LYS'~~ + Met substitution is shown for comparison. The relative numbers of EGF receptors remaining (&.D.) aRer treatment with 50 nM EGF for the indicted times were determined as described under "Materials and Methods."

inactive EGF receptors created by substituting methionine for lysine at residue 721, all sequences exhibited low constitutive internalization. As was the case for EGF holoreceptors, intrinsic tyrosine kinase activity is strictly required for high affinity saturable endocytosis of EGF receptors that contain native sequence codes.

Function of Endocytic Codes in EGF Receptor Down-regulation-To establish the functional role of the different endocytic codes in EGF receptor regulation, we compared their relative efficiency in mediating receptor down-regulation. As shown in

Fig. 6, receptor regions that specified ligand-induced internalization when transferred to c'958 receptors also specified receptor down-regulation. The rank order of their efficiency (residues 993-1022 > 993-1000 > 1001-1022) was also similar. All endocytic codes that could substitute for the QQGFF sequence were similar in their ability to mediate receptor down-regulation. However, the mutants appeared to be more similar to the EGF holoreceptors with respect to down-regulation compared with internalization. The stronger effects on down-regulation reflect the longer times necessary for this measurement, which encompasses multiple rounds of internalization and recycling. As was the case for ligand-induced endocytosis, receptor kinase activity was strictly required for the function of the foreign sequences in the context of the EGF receptor.

Endocytic Codes from EGF Receptor Function in Tf Receptor -To investigate the ability of the identified endocytic codes to function in another context, codes from the EGF receptor were substituted for the endogenous sequence code of the Tfreceptor. Table 11 shows a comparison of the function of the two identified EGF receptor codes with that of the wild-type Tf receptor. Both EGF receptor endocytic codes function in Tf receptors, with the FYRAL sequence being somewhat stronger (125%) than the native Tf receptor code YTRF. Whereas QQGFF was less effective (63%) than YTRF, it was clearly active when compared to Tf receptors either lacking the endogenous endocytic code or containing the sequence AADAA.

These results indicate that endocytic codes are exchangeable between a Type I receptor that is constitutively internalized and a Type I1 receptor that exhibits ligand-induced internalization. In addition, tyrosine kinase activity that is required for internalization sequences to function in the context of EGF receptors is not required for sequences to function in the context of Tf receptors.

Both Tyrosine Kinase Activity and Endocytic Codes Are Re- quired for Down-regulation of EGF Receptors-The requirements for internalization and down-regulation of EGF receptors were examined further using immunocytochemistry with dual staining of EGF receptors and the lysosomal marker lgp120 (32). For these experiments, a monoclonal antibody directed against the ectodomain of the EGF receptor was used (29). This antibody (13A9) does not compete with lZ5I-EGF for binding to EGF receptors and does not affect the rate of internalization of ligand-receptor complexes. The monoclonal antibody thus recognized both occupied and unoccupied EGF receptors. As shown in Fig. 7 , wild-type EGF receptors, which

TABLE I1 Comparisons of steady-state distributions

of human T f receptor mutants

Human TW construct Human TfR" Relative internalized internalization

a t steady state efficiency

% Wild-type TfR (16EPLSYTRFS24)b 65.1 * 1.7 c(6)d 100 EGFR 1 (16LIPQQGFFS24) EGFR 2 (18NFYFf.AL23)

54.2 f 1.6 (4) 63 70.0 f 1.6 (6) 125

Nonspecific (l9AADAAZ3) 24.7 f 1.0 (3) 18 Tailless TfR (A3-59) 19.8 f 1.5 (3) 13

a TfR, Tf receptor; EGFR, EGF receptor.

9c

The letters in parentheses represent the single-letter code for the Tf receptor sequence from residues 16 to 24 of the wild-type receptor. This region of the cytoplasmic domain contains the Tf receptor tetrapeptide internalization signal YTRF (5). For the mutant Tf receptors containing the putative EGF receptor signals, the Tf receptor signal was replaced with the sequences shown using oligonucleotide-directed mutagenesis (in EGF receptor 1, the sequence l6EPLSYTRFSZ4 was replaced with 16LIPQQGFFS24; in EFG receptor 2, 1 s L S 3 F 2 3 was replaced with lRNFYRALZ3; and in nonspecific, l9S= was replaced with '9AADAA23)) .

Mean f S.E. Number of independent experiments.


FIG. 7. Immunofluorescence analysis of EGF-induced internalization of wild-type and mutant EGF receptors. Cells expressing the indicated EGF receptors (EGFR) were incubated with EGF receptor monoclonal antibody 13A9 (1.6 pg ml") for 60 min at 4 "C. Cells were washed to remove unbound antibody and placed a t 37 "C with or without 50 nM EGF for 60 min. Cells were then fixed, and semithin sections (1 pm thick) were prepared and incubated with fluorescein isothiocyanate-conjugated goat anti- mouse IgG and with tetramethylrhodamine B isothiocyanate-conjugated F(ab'), fragments of goat anti-rabbit IgG to detect EGF receptors and the lysosomal marker lgp120, respectively. Cells were photographed using appropriate filters for the two color fluorescent dyes. In each case, the left panel shows the distribution of EGF receptors in the absence of EGF, and the middle and right panels show the same cells dually stained for the EGF receptor and lgp120. In all cases, EGF receptors are distributed primarily a t the cell surface in the absence of EGF. After incubation with EGF, receptor distribution varies: in the case of wild type (WT) and c'958 f993-1022, the receptors are distributed both a t the cell surface and in lysosomes, whereas remaining mutant receptors are found largely at the cell surface.

EG FR -EG F

WT Kin+

EGFR Lgp 120 +EGF

1 +EGF

c l 9 5 8 r Kin+

were diffusely distributed on the cell surface in the absence of EGF, were redistributed to endocytic vesicles and subsequently segregated to lysosomes in the presence of EGF. In contrast, kinase-active c'958 EGF receptors remained diffusely distributed on the cell surface after addition of EGF (Fig. 71, similar to kinase-inactive Met721 EGF receptors (44). Fusion of region 2 containing QQGFF to residue 958 (c'958 f993-1022) restored ligand-induced internalization and ultimate targeting of internalized receptors to lysosomes. The extent of clearance of mutant EGF receptors from the cell surface was less than that seen with wild-type EGF receptors, compatible with weaker activity of a single endocytic code. As shown in Fig. 7, EGF had no effect on kinase-inactive c'958 f993-1022 EGF receptors, which remained diffusely distributed on the cell surface. These results confirm the requirement for both an endocytic sequence and intrinsic tyrosine kinase activity for ligand-induced down- regulation of EGF receptors. As reported, c'958 f993-1022 EGF receptors lack sites of tyrosine self-phosphorylation (151, indicating that the tyrosine kinase requirement is not involved in self-phosphorylation, which could expose an endocytic code.

DISCUSSION Previous studies have shown that ligand-induced endocyto-

sis of EGF receptors requires intrinsic protein-tyrosine kinase activity and sequence information located in the carboxyl terminus distal to the kinase domain (9, 15). This study indicates

that multiple endocytic codes exist in the carboxyl terminus. We located these sequences based on their ability to restore endocytic function to an internalization-defective EGF receptor truncated a t residue 958. Using this approach, we found that both regions 993-1022 and 1024-1186 were effective in restor- ing the wild-type endocytiddown-regulation phenotype. More detailed mapping studies of the proximal region 958-1022 re- vealed two distinct endocytic sequences. "wo internalization signals have also been identified in the exon 16-encoded jux- tamembrane domain of the insulin receptor (131, in the cation- dependent mannose 6-phosphate receptor (45), and in the pol- ymeric IgA receptor (46). Additionally, endocytosis is elevated in mutant insulin receptors containing an additional endocytic sequence (47). We found that endocytosis of EGF receptors containing a single code is less efficient than that of EGF holoreceptors. "his suggests that multiple codes increase receptor affinity for components of the endocytic machinery. "his could be due to multiple interactions or could reflect different primary sequence contributions to a binding surface analogous to the aromatic residues that make up the SH3 domain binding surface (48).

Large alterations in EGF receptor structure resulted in a change in both receptor affinity for the endocytic apparatus and the maximum rate of receptor endocytosis. Simulation studies of receptor-mediated endocytosis indicate that this


could be due to a two-step mechanism for ligand-induced endocytosis. Previous studies indicate that specific internalization components are required for EGF receptor internalization because they do not compete with transferrin receptors for internalization (49). Similarly, insulin receptors do not compete with insulin-like growth factor IYmannose 6-phosphate receptors for internalization (10). Alterations in receptor affinity for specific internalization components would appear as a change in slopes of Satin plots. However, changes in the binding of receptor complexes to coated pits would appear as changes in maximum internalization rates. I t seems that receptor sequences would be involved in both steps, but the endocytic codes that we have identified seem to be involved in initial receptor binding to internalization components because of their strong effect on endocytic affinity.

The two sequence codes identified in the EGF receptor carboxyl terminus (FYRAL and QQGFF) occur in regions of turns according to structural predictions of Chou and Fasman (42). Both sequence motifs also contain tyrosine or phenylalanine residues found in endocytic codes from a variety of receptors. The prediction of Collawn et al. (4) that the endocytic code has the structure of a tight turn is supported by NMR analysis of peptides corresponding to the functionally identified internalization signals of the LDL receptor (5), lysosomal acid phospha- tase (511, and the insulin receptor (14). The endogenous EGF receptor sequence QQGFF and the constructed sequence NNAYF have the potential for coordination of the amino group at one end of the turn to the center of the aromatic ring of tyrosine or phenylalanine on the other end, similar to the LDL receptor motif NPXY (5) .

The similarity of the two identified EGF receptor internalization sequences to those found in other proteins was confirmed by the finding that both sequences could effectively function as replacements for the native sequence of Tf receptors. Conversely, the Tf receptor internalization sequence YTRF could replace QQGFF in reconstituting ligand-induced internalization and down-regulation of EGF receptors, albeit not as well as the native sequence. The ability to functionally exchange internalization sequences between Type I and I1 receptors suggests interaction with similar components of the endocytic machinery. The observation that EGF and Tf do not compete for internalization (49) despite being internalized into the same endocytic vesicles (41) suggests that specific as well as common recognition elements exist.

All sequences that restored ligand-induced internalization in EGF receptor mutants also restored their ability to undergo down-regulation and eventual lysosomal targeting. However, we found that sequences that only weakly restored endocytosis were able to mediate extensive down-regulation of the EGF receptor. This could be due to differences in the two assays. For example, internalization plots only measure a single round of internalization on a time scale of <5 min. In contrast, down- regulation of the EGF receptor during the time of that assay involves three to five internalizationhecycling events. Because receptor sequences that mediate normal endosomal retention of occupied EGF receptors are present within all of our mutants, multiple rounds of internalization would be expected to result in substantial lysosomal transfer.2 Even weak endocytic sequences could thus produce significant levels of down-regulation out of proportion to their endocytic efflciency. Neverthe- less, because receptor mutants defective in their ability to undergo ligand-induced internalization were unable to down- regulate, endocytosis is likely rate-limiting for the process.

The exact role of receptor tyrosine kinase activity in ligand-

* J. J. Herbst, B. J. Walsh, A. R. French, G. P. Sudlow, D. A. Lauffen- burger, and H. S. Wiley, submitted for publication.

induced internalization remains unclear. Because of their ability to function in the context of both Type I and I1 receptors, endocytic codes such as NPXY, YTRFS, QQGFF, and ITFL4.L are probably analogous structures. Although their action in the context of EGF receptors is dependent on intrinsic receptor tyrosine kinase activity, these sequences work in Tf receptors that lack such an enzymatic function. The fusion mutant c'958 B93-1022 EGF receptor lacks self-phosphorylation sites and does not undergo in vivo or in vitro self-phosphorylation (15). Therefore, tyrosine kinase activity cannot be required only to remove inhibitory constraints (50) or to expose functional sequences by self-phosphorylation. Kinase activity may be required for phosphorylation of some accessory protein that fa- cilitates receptor binding to the endocytic apparatus. Clustering of receptors in coated pits could require the partic- ipation of a number of different proteins, some of which are specific to different classes of receptors and may require tyrosine phosphorylation. Because kinase-deficient receptors result from a mutation in the ATP-binding site, the requirement for kinase activity could be due to ATP binding rather than tyrosine kinase activity per se. The cell-surface distribution and internalization kinetics of EGF and Tf receptors clearly differ, but correlate with the differing enzymatic requirements for function of the same sequences in different contexts.

Acknowledgments-We thank Dr. Alan Wells (University of Alabama

Marjorie Winkler (Genentech) for the 13A9 antibody. a t Birmingham) for construction of the c'973 fusion mutant and Dr.

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