©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Roles for a Cytoplasmic Tyrosine and Tyrosine Kinase Activity in the Interactions of Neu Receptors with Coated Pits (*)

(Received for publication, November 4, 1994)

Lilach Gilboa (1) Rachel Ben-Levy (2) Yosef Yarden (2) Yoav I. Henis (1)(§)

From the  (1)Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel and the (2)Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The neu proto-oncogene product, p185 (HER2, c-ErbB-2), encodes a cell-surface tyrosine kinase receptor with high oncogenic potential, which correlates with increased tyrosine kinase activity and a rapid receptor internalization rate. To investigate the interactions and signal(s) leading to the endocytosis of Neu receptors, we employed lateral mobility and internalization studies. Fluorescence photobleaching recovery measurements revealed that activation of Neu receptors (induced by mutation or by agonistic antibodies) markedly reduced their mobile fractions. To elucidate the signals involved, other mutants, all carrying a constitutively dimerizing oncogenic mutation, were analyzed. A kinase-negative mutant and a mutant lacking all cytoplasmic tyrosine phosphorylation consensus sequences exhibited high mobile fractions, similar to nonactivated Neu. Retention of a single tyrosine autophosphorylation site (Tyr-1253) out of the five known such sites was sufficient to immobilize a large fraction of the receptor. For all mutants, internalization correlated with receptor immobilization and was blocked by treatments that interfere with coated pit structure, indicating that the immobilization is due to interactions with coated pits. This was supported by the coimmunoprecipitation of alpha-adaptin only with the constitutively activated Neu mutants. We conclude that activated Neu receptors become stably associated with coated pits via plasma membrane adaptor complexes (AP-2). Efficient Neu receptor endocytosis requires activation, a functional kinase domain, and at least one tyrosine autophosphorylation site.


INTRODUCTION

The life cycle of growth factor receptors plays an important role in the control of cell proliferation and carcinogenesis. A crucial part of this cycle is receptor-mediated endocytosis, which provides a major mechanism for receptor down-regulation and signal termination (reviewed in (1, 2, 3, 4) ). The first stage of this process involves recruitment of specific receptors into plasma membrane clathrin-coated pits, whose structure has been characterized morphologically and biochemically (5, 6, 7) . Accumulating evidence suggests that a signal necessary and sufficient for internalization through coated pits exists in the cytoplasmic domains of many receptors (reviewed in Refs. 3, 4, 7, and 8). This short peptide ``recognition signal'' contains at least one aromatic residue (usually a tyrosine) in a sequence typical of a beta-turn(3, 9, 10, 11, 12, 13) . In vitro studies demonstrated interactions between AP-2 (the adaptor protein complex associated with plasma membrane coated pits) or their adaptin subunits and the cytoplasmic internalization signals of several receptors(14, 15, 16, 17, 18) . Furthermore, AP-2 binding to plasma membrane fragments has been reconstituted in broken cell systems(19, 20, 21, 22) .

To characterize the interactions of receptors with coated pits at the surface of intact cells, we have recently developed an approach based on comparative studies of the lateral mobilities of native and specifically mutated membrane proteins and demonstrated that such studies can provide information on their interactions with coated pits in live cells(23) . In the present study, we applied this approach (combined with internalization and coimmunoprecipitation experiments) to investigate the internalization of the neu proto-oncogene product (Neu receptor; also designated p185, c-ErbB-2, or HER2). This protein is a 185-kDa transmembrane tyrosine kinase that is closely related to the epidermal growth factor (EGF) (^1)receptor, but is not activated by EGF(24, 25, 26) . A point mutation in the transmembrane domain of the rat Neu receptor (Neu*; Val-664 replaced by Glu) results in a constitutively dimerized and permanently active receptor(25, 27, 28) . Unlike constitutively endocytosed receptors, both Neu and EGF receptors undergo endocytosis only after activation by the binding of ligand or specific agonistic monoclonal antibodies (reviewed in (2) ). Activation of the Neu receptor is thought to involve its dimerization (28, 29, 30) and is manifested by enhanced tyrosine kinase activity, leading to autophosphorylation as well as to phosphorylation of other cellular proteins(31) . Autophosphorylation occurs on five tyrosine residues located in the Neu protein C-terminal region(32, 33, 34) . Some receptor dimerization may occur when it is expressed at very high levels; it was speculated (35) that this can lead to elevated basal tyrosine kinase activity, which may be relevant to the overexpression of Neu receptors in many adenocarcinomas (36, 37) and to the correlation between elevated Neu expression and poor prognosis in cases of breast cancer(38, 39) .

While numerous studies were performed on the internalization and recycling of EGF receptors(40, 41, 42, 43, 44) , only a few have addressed these issues in the Neu receptor(31, 45, 46) . The signals and interactions leading to Neu receptor endocytosis are not yet clear, and the possible role of tyrosine autophosphorylation in this process has not been explored. In this study, we investigated the interactions leading to Neu receptor endocytosis. By combining fluorescence photobleaching recovery (FPR) studies on the lateral mobilities of native and mutated Neu proteins with studies on their internalization and interactions with alpha-adaptin, we explored the mode of their interactions with coated pits. The results indicate that activated Neu receptors become stably associated with coated pits and adaptor complexes. These interactions depend on Neu kinase activity and can be efficiently mediated by a single tyrosine autophosphorylation site.


EXPERIMENTAL PROCEDURES

Materials

Tetramethylrhodamine (TMR) 5-isothiocyanate was from Molecular Probes, Inc. (Eugene, OR). Hanks' balanced salt solution (HBSS), protein G-Sepharose 4B, protein A-peroxidase, amiloride hydrochloride, and bovine serum albumin (BSA) were from Sigma. Chemiluminescence reagent (Renaissance) was from DuPont NEN.

Antibodies

B-10 and C-11 mouse monoclonal antibodies directed against extracellular epitopes of the rat Neu receptor were described earlier(31) . Rabbit anti-peptide antibodies against a peptide corresponding to the C-terminal amino acids of human Neu (designated NCT)(47) , which cross-react with rat Neu proteins, were also employed. AC1-M11 mouse monoclonal antibodies against alpha-adaptin (48) were donated by Dr. Margaret S. Robinson (University of Cambridge, Cambridge, United Kingdom). Affinity-purified goat IgG directed against mouse F(ab`)(2) (GAM IgG) was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). I-F(ab`)(2) sheep anti-mouse IgG (I-SAM F(ab`)(2); 10 µCi/µg) and peroxidase-conjugated donkey anti-rabbit IgG were from Amersham (Buckinghamshire, United Kingdom). IgG fractions of B-10 and C-11 were prepared from mouse ascites by ammonium sulfate precipitation and DEAE-cellulose chromatography(49) . F(ab`)(2) fragments were generated by pepsin digestion as described by Kurkela et al.(50) for B-10 mouse IgG or by Henis et al.(51) for GAM IgG. All the F(ab`)(2) preparations were reduced with mercaptoethanol and alkylated with iodoacetamide to generate monovalent Fab` fragments(51) . To eliminate possible IgG traces, they were treated with protein G-Sepharose. The resulting Fab` fragments were free of contamination by IgG or F(ab`)(2) as judged by SDS-PAGE under nonreducing conditions. The GAM Fab` fragments were tagged with TMR isothiocyanate (to generate TMR-GAM Fab`) following standard procedures(52) .

Cell Culture

Several previously described cell lines, stably expressing rat Neu receptors or their mutants, were employed. The various mutants are depicted in Fig. 1. DHFR-G8 (designated G8) (53) and NE19 (25) cells express normal Neu receptors. The B104-1-1 (25) and RB22 (47) cell lines express Neu*. K758A cells (47) express a kinase-negative Neu*. The P1 cell line expresses a truncated Neu* containing only the first tyrosine autophosphorylation site (Tyr-1253), and P1F cells express P1 with Tyr-1253 replaced by Phe(54) . All cell lines were grown in Dulbecco's modified Eagle's medium (Biological Industries, Beth Haemek, Israel) containing 10% calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml streptomycin (Biological Industries). Prior to all experiments, cells were incubated overnight in the same medium containing 1% calf serum.


Figure 1: Schematic representation of mutant Neu proteins. The sequence of the rat Neu receptor (1260 amino acids long) is from (77) . The domains, starting from the extracellular N terminus (position 1), are represented by boxes corresponding to the signal peptide, the cysteine-rich domains (CRD1 and CRD2), the transmembrane domain (TM), and the tyrosine kinase (TK) sequence. The cytoplasmic C-terminal tail (CT) is shown with the five known autophosphorylation sites(34) , designated P(1)-P(5). The constitutively active mutant Neu-E664 (Neu*) has one point mutation (in the transmembrane region), where Val-664 is replaced by Glu. All the other mutants are derived from Neu*. The construct P1 lacks 243 amino acids between the tyrosine kinase domain and the C terminus of Neu* and includes only a short cytoplasmic tail (the 12 C-terminal amino acids) with a single tyrosine phosphorylation consensus sequence. P1F is identical to P1, except that the tyrosine originally located at position 1253 (P(1)) was replaced by Phe. K758A is a full-length Neu* whose tyrosine kinase was inactivated by replacing Lys-758 at the ATP-binding site by Ala.



Immunofluorescent Labeling

Subconfluent cells were split 1:10 and grown on glass coverslips for 1 day. They were washed twice with cold HBSS supplemented with 20 mM Hepes and 2% BSA (HBSS/Hepes/BSA, pH 7.2) and labeled successively in this buffer at 4 °C (washing three times after each incubation) with the following antibodies: 1) B-10 Fab` or IgG (60 µg/ml, 60 min) and 2) TMR-GAM Fab` (100 µg/ml, 60 min). All labeling steps were carried out in the cold to allow only surface labeling and to eliminate endocytosis. The coverslips carrying the live cells were taken for lateral mobility measurements (see below).

Fluorescence Photobleaching Recovery

Lateral diffusion coefficients (D) and mobile fractions (R(F)) of Neu and Neu mutants were measured by FPR (55, 56) using an apparatus described previously(57) . After labeling the cells as described above, the coverslip was placed over a serological slide with a depression filled with HBSS/Hepes/BSA equilibrated at 22 °C. Measurements were performed within 20 min of labeling to minimize internalization of activated Neu, which could lead to receptor immobilization. The similar values of D and R(F) at the beginning and end of this period demonstrated that the contribution of such effects to the measurements was negligible. Temperatures below 22 °C were avoided since earlier work on another protein (23) has shown that the interactions with coated pits were greatly reduced at lower temperatures. The monitoring laser beam (Coherent Innova 70 argon ion laser; 529.5 nm, 1 microwatt) was focused through the microscope (Zeiss Universal) to a gaussian radius of 0.61 ± 0.02 µm using a 100 times oil immersion objective. A brief pulse (5 milliwatts, 30 ms) bleached 50-70% of the fluorescence in the illuminated region. The time course of fluorescence recovery was followed by the attenuated monitoring beam. D and R(F) were extracted from the fluorescence recovery curves by nonlinear regression analysis(58) . Incomplete fluorescence recovery was interpreted to represent fluorophores that are immobile on the FPR experimental time scale (D leq 5 times 10 cm^2/s).

Treatments That Alter Coated Pit Structure

The treatment employed was incubation in a hypertonic medium (59, 60, 61) or acidification of cytosol(61, 62, 63) . Hypertonic treatment, which blocks endocytosis via coated pits by dispersing the underlying clathrin lattices(60, 61) , was performed by 15-min incubation (37 °C) in HBSS/Hepes/BSA supplemented with 0.225 M NaCl or 0.45 M sucrose. The cells were kept in hypertonic medium during labeling and internalization assays. Cytosol acidification, which alters the coated pit structure and eliminates endocytosis by blocking the pinching off of clathrin-coated vesicles, was performed as described earlier(23, 62) . Cells were incubated in Hepes-buffered Dulbecco's modified Eagle's medium, pH 7.2, containing 30 mM NH(4)Cl for 30 min at 37 °C, followed by 5 min at 37 °C in potassium/amiloride buffer (0.14 M KCl, 2 mM CaCl(2), 1 mM MgCl(2), 1 mM amiloride HCl, 20 mM Hepes, pH 7.2). This buffer was replaced by cold potassium/amiloride buffer containing 2% BSA, which was also used for I-Fab` labeling and throughout the internalization experiments.

Internalization Assays

Cells were plated 2 days before the experiment in collagen-coated 24-well trays and grown to 80-90% confluence. They were treated to alter coated pit structure as described above, while untreated control dishes were incubated instead in HBSS/Hepes/BSA. Cells were labeled at 4 °C in the buffers specific to each treatment with B-10 Fab` or IgG (60 µg/ml, 60 min), washed, and labeled with I-SAM Fab` (1 µg/ml, 60 min, 4 °C). The plates were washed, shifted to 37 °C in the appropriate buffers for different periods, and returned to 4 °C. The amount of Fab` at the cell surface was determined by incubating the cells in an acidic stripping medium (HBSS buffered with 20 mM Mes, pH 2.5) for 10 min at 4 °C. Iodinated antibody retained within the cells (internalized Fab`) was determined by solubilizing the cells with 1 N NaOH and counting aliquots in a -counter (Pharmacia Biotech 1272). The contribution of nonspecific binding (<10% in all cases) was measured in untransfected NIH 3T3 cells, which do not express rat Neu receptors, or in control wells labeled only with secondary I-SAM Fab`.

Immunoprecipitation and Western Blotting

Cells grown to 90% confluence in 140-mm dishes (two to five dishes, depending on the level of Neu receptor expression in the specific cell line) were washed with HBSS/Hepes and solubilized in 1 ml of ice-cold lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% (w/v) Triton X-100, 10% glycerol, 1.5 mM MgCl(2), 1 mM EGTA, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Extracts were precleared by incubation (2 h, 4 °C) with 80 µg/ml protein G-Sepharose. After centrifugation, the supernatant was incubated (4 h, 4 °C) with protein G-Sepharose coupled to B-10 IgG (80 µg/ml of beads; this amount is sufficient for complete precipitation of the Neu receptors). Immunocomplexes were washed three times with 20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% (w/v) Triton X-100, and 10% glycerol. The pellets were dissolved in Laemmli gel electrophoresis loading buffer (containing SDS and mercaptoethanol), boiled for 5 min, and subjected to SDS-PAGE on 10% acrylamide gels. The proteins were electrophoretically transferred onto nitrocellulose filters presaturated (1 h, 22 °C) with TBST buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 0.1% Tween 20) supplemented with 5% dry milk (low fat) and 2% BSA. NCT anti-Neu (1:5000 dilution) or AC1-M11 anti-alpha-adaptin (1:100 dilution) ascites were then added. The filters were incubated with the antisera overnight at 4 °C and washed extensively. For detection, NCT rabbit antibodies were labeled with peroxidase-conjugated donkey anti-rabbit IgG (1:25,000, 1 h, 22 °C), while AC1-M11 antibodies were labeled with protein A-peroxidase (1 µg/ml, 1 h, 22 °C). After washing with TBST buffer three times, the filters were reacted for 1 min with enhanced chemiluminescence reagent (ECL) and exposed to autoradiography film for 2 min. Densitometry measurements were performed on a Pharmacia Biotech Ultrascan XL.


RESULTS

Selective Lateral Immobilization of Activated Neu Receptors and Neu Mutants

We have recently developed an approach that, based on comparative studies on the lateral mobilities of membrane proteins mutated to alter their internalization signals, is capable of characterizing their interactions with coated pits in intact cells (23) . Interactions of membrane proteins with immobile structures retard their lateral motion(64, 65) . Plasma membrane coated pits, which are immobile in lateral diffusion measurements by FPR(66) , can induce such effects and were shown to retard the lateral motion of a mutant influenza hemagglutinin protein containing a tyrosine recognition signal(23) . Therefore, we designed experiments comparing the lateral mobilities of Neu receptors under conditions in which they are or are not internalized and investigated the effects of mutations interfering with potential internalization signals. The experiments employed several NIH 3T3 cell lines stably expressing normal rat Neu receptors (whose activation requires binding of agonistic antibodies), constitutively dimerized and activated Neu*, or several double mutants of Neu*. The constructs employed are depicted in Fig. 1.

To determine the lateral mobilities of the various Neu mutants, they were labeled by monovalent Fab` fragments (see ``Experimental Procedures''). The use of monovalent Fab` is essential to avoid possible cross-linking by bivalent IgG (which may affect mobility) and is of special importance in this study to eliminate the agonistic effect of dimerization by B-10 IgG. The results of the FPR experiments performed on cell lines expressing various Neu receptor mutants are depicted in Fig. 2. Several observations can be made on the basis of the data presented. First, it is apparent that native Neu receptors exhibit significantly higher R(F) values than constitutively activated Neu* (for G8 (Neu) versus B104-1-1 (Neu*) and for NE19 (Neu) versus RB22 (Neu*), Student's t test yielded p < 0.0005). In spite of the different R(F) values, the Neu and Neu* receptors displayed similar lateral diffusion rates (measured by D). Such a reduction in R(F) with no effect on D is expected if a subpopulation of Neu* (but not Neu) molecules is entrapped by stable interactions with immobile structures (presumably coated pits) for the entire duration of the FPR measurement (about 1 min). The remainder of the Neu* molecules present at the cell surface are free to diffuse without being slowed down by dynamic interactions with these structures (e.g. transient exchange into coated pits), which would slow down the average lateral diffusion rate(23, 64, 65) . These results hold for two pairs of cell lines, each derived from a different NIH 3T3 subline (compare G8 with B104-1-1 and NE19 with RB22), demonstrating that they are not due to cell line variations. The notion that the reduction in R(F) of Neu* is due to interaction with coated pits is supported by the correlation between this parameter and Neu receptor internalization as well as by the coprecipitation of alpha-adaptin together with Neu* (see Fig. 4and Fig. 6).


Figure 2: Lateral diffusion of Neu, Neu*, and Neu* mutants. Cells grown on glass coverslips were labeled with B-10 Fab` (blackbars) or B-10 IgG (stripedbars) followed by TMR-GAM Fab` as described under ``Experimental Procedures.'' The FPR measurements were performed in HBSS/Hepes/BSA at 22 °C. Each bar is the mean ± S.E. of 25-35 measurements. A, R values; B, D values.




Figure 4: Internalization of Neu receptor mutants labeled by B-10 Fab` or B-10 IgG. Cells were incubated successively (1 h, 4 °C) with B-10 Fab` (blackbars) or B-10 IgG (stripedbars) followed by I-SAM Fab`. The cells were washed, warmed for 10 min to 37 °C, and returned to 4 °C. Surface-bound and internalized Fab` fragments were determined as described in the legend to Fig. 3. Results are the means ± S.E. of three experiments, each performed in triplicate.




Figure 6: Coimmunoprecipitation of alpha-adaptin with constitutively activated Neu receptors. Neu receptors were immunoprecipitated from detergent extracts of G8, B104-1-1, P1, or P1F cells as described under ``Experimental Procedures.'' Immunoprecipitates were analyzed by 10% SDS-PAGE followed by electroblotting. A, immunodetection of Neu. Blots were labeled by NCT rabbit antiserum followed by horseradish peroxidase-conjugated donkey anti-rabbit IgG. The bands were visualized by enhanced chemiluminescence. B, immunodetection of alpha-adaptin. Blots were labeled by AC1-M11 monoclonal antibodies directed against alpha-adaptin followed by protein A-peroxidase. Detection was by enhanced chemiluminescence. A densitometric analysis was performed employing the ratio between the alpha-adaptin and Neu protein bands in each lane for internal calibration (to compensate for different levels of Neu receptors). This analysis showed that the amount of alpha-adaptin coprecipitated with Neu is only 4.3% of that precipitated with Neu*; a similar comparison between the truncated versions of Neu* shows that the alpha-adaptin coprecipitated with P1F is 16.5% relative to the level of coprecipitation with P1. No signals were detected for either Neu proteins or alpha-adaptin when immunoprecipitation and immunodetection were performed on untransfected NIH 3T3 cells; similar levels of alpha-adaptin were observed in all the cell lines upon detergent solubilization (without immunoprecipitation) and immunodetection by AC1-M11 (data not shown).




Figure 3: Time course of the internalization of Neu, Neu*, and K758A. NE19, RB22 and K758A cells were incubated successively (1 h, 4 °C) with B-10 Fab` followed by I-SAM Fab`. The cells were washed with cold buffer, warmed to 37 °C for the indicated times, and returned to 4 °C. Surface-bound and internalized Fab` fragments were determined by acid stripping of Fab` from the cell surface (see ``Experimental Procedures''). Nonspecific binding (<10% in all cases) was subtracted. Results are the means ± S.E. of three experiments, each performed in triplicate.



To further characterize elements in the Neu receptor C-terminal region that might function in the mobility-restricting interactions, the lateral mobility of several Neu mutants was investigated. The constructs (Fig. 1) were made on the basis of Neu*; namely, they all contain the Val-664 Glu mutation, which results in Neu receptor dimerization(29, 31, 67) . The P1 mutant, in which Tyr-1253 is the only tyrosine in a phosphorylation consensus sequence, yielded a low R(F) not significantly different from that of Neu* (p > 0.1 according to Student's t test) (Fig. 2A). This tyrosine is found in an NPXY sequence, which has been shown to serve as an internalization signal for the low density lipoprotein receptor(3, 68) . Tyr-1253 is clearly involved in the mobility-restricting interactions since P1F, in which Tyr-1253 was replaced by Phe, exhibited a high R(F) value similar to that of wild-type Neu expressed in NE19 cells (p > 0.1) (Fig. 2). The inability of Phe to replace Tyr-1253 in these experiments indicates that tyrosine phosphorylation may be involved. This possibility is supported by the finding that K758A, which is a kinase-negative Neu*(47) , yields a high R(F) value similar to that of normal Neu (p > 0.1) (Fig. 2A). All these mutants exhibited similar D values (Fig. 2B), in accord with the notion that the interactions involved (most likely with coated pits) are stable rather than transient.

To exclude the possibility that the differences between the R(F) values of the various mutants are due to different levels of surface expression, we quantified the relative surface densities of the Neu mutants in the different cell lines. This was done using the prebleach fluorescence level in the FPR experiments (measuring over 30 cells in each sample), which is directly proportional to the surface density of the Fab`-labeled protein(65) . Neu surface density in most cell lines (B104-1-1, NE19, RB22, P1F, and P1) was rather similar (630, 640, 440, 900, and 780 counts/150 ms, respectively; the standard error was <10% in all cases). G8 cells exhibited a somewhat higher density (1350 counts/150 ms), and the lowest level was observed on K758A cells (300 counts/150 ms). All these values are of the same order of magnitude. The fact that G8, NE19, P1F, and K758A all yielded similar R(F) values suggests that the differences in surface density (4.5-fold between the extremes) do not determine the observed behavior of the receptors at the cell surface. This conclusion is supported by the similar FPR results obtained in each cell line on individual cells expressing lower and higher levels of Neu receptors. The total receptor content in G8 cells (the highest expressors) is in the range of 2-4 times 10^5/cell, as estimated by immunoprecipitation and comparison with cells expressing chimeric receptors that bind EGF (data not shown). Considering that only a fraction of the receptors are at the cell surface, this level most likely does not saturate the coated pits. Differences between the various cell lines in the amount of plasma membrane coated pits could be ruled out since similar levels of alpha-adaptin (specific to the plasma membrane coated pit adaptor complex, AP-2) were found in all cell lines (see legend to Fig. 6).

Effect of Bivalent B-10 IgG on the Lateral Mobility of Neu Receptors

The above results indicate that immobilization is observed only in Neu mutants that are constitutively dimerized and activated (i.e. have undergone tyrosine phosphorylation). To further examine this point, we performed FPR experiments employing intact bivalent agonistic B-10 IgG in place of B-10 Fab` as primary antibody. Fig. 2demonstrates that B-10 IgG reduces R(F) of wild-type Neu to a level not significantly different from that of Neu* (p > 0.1 for G8 versus B104-1-1 and for NE19 versus RB22). Only a minor fraction of this reduction can be attributed to the formation of immobile aggregates due to cross-linking of Neu by IgG since another monoclonal antibody that lacks agonistic activity (C-11 IgG) (31) reduced R(F) of Neu receptors on G8 cells by <20% (data not shown). The lack of formation of large aggregates is also indicated by the finding that the D values are similar for B-10 IgG and B-10 Fab` labeling for all the mutants (Fig. 2B). Interestingly, R(F) of the P1F mutant, which is devoid of all the potential tyrosine phosphorylation sites, was not significantly reduced by B-10 IgG (p > 0.05), in keeping with the suggestion that a tyrosine signal is involved. As for Neu* and P1, these mutants displayed the same low R(F) value with B-10 IgG as with Fab` (Fig. 2), in accord with their constitutive dimerization and activation even prior to B-10 IgG binding. In the case of K758A, which lacks kinase activity, the B-10 IgG-mediated reduction in R(F) may reflect the combined action of two processes. 1) The dimeric structure of K758A makes it more prone to cross-linking into higher aggregates by bivalent B-10 IgG (for a monomer, such as normal Neu, a monoclonal IgG would generate only dimers). 2) K758A has all five potential tyrosine phosphorylation sites, which could be slowly phosphorylated by other tyrosine kinases, a possibility supported by the slow but detectable internalization of K758A (Fig. 3).

Internalization of Neu Receptor Mutants Correlates with Their Activation and Lateral Immobilization

We have shown formerly that interactions with coated pits can retard or immobilize specific membrane proteins(23) . If coated pits are involved in the selective immobilization of Neu receptor mutants, the reduction in R(F) should correlate with endocytosis of the activated mutants. Fig. 3depicts the internalization time course of representative Neu mutants. Since the highest differences in internalization level were observed after 10 min at 37 °C, this time point was employed to compare all the Neu mutants (Fig. 4). It should be noted that this is therefore not a rate determination, but a qualitative measure for the ability of the mutants to be internalized. When B-10 Fab` and I-SAM Fab` were employed to label the receptors, a good correlation of the internalization with the reduction in R(F) was observed: receptors yielding high R(F) values exhibited internalization levels significantly lower than those characterized by low R(F) values (p < 0.0005 for G8 versus B104-1-1, NE19 versus RB22, and P1F versus P1). The kinase-negative mutant K758A displayed a low internalization level after 10 min, in correlation with its high R(F) when labeled by monovalent Fab`. However, it could still undergo endocytosis, as is evident at longer times (Fig. 3). This could be due to slow phosphorylation by other tyrosine kinases, e.g. via mixed dimers with endogenous mouse Neu receptors present in the 2.2 NIH 3T3 subline used to generate K758A cells(47) . Cross-phosphorylation among EGF-Neu receptor mixed dimers (69, 70) can be excluded since the 2.2 subline is devoid of EGF receptors. Alternatively, the residual internalization of K758A could indicate the existence of additional internalization signal(s) that do not depend on phosphorylation and that require dimer formation. The slow endocytosis of K758A relative to Neu* is not due to increased recycling of K758A since the addition of monensin to the internalization assay had no effect on its internalization (data not shown). When activating B-10 IgG replaced B-10 Fab` in the labeling, the internalization of wild-type Neu increased to levels not significantly different from those of Neu* (p > 0.1 for G8 versus B104-1-1 and for NE19 versus RB22) (Fig. 4). The endocytosis of Neu* was not affected significantly (p > 0.1 for Fab`- versus IgG-labeled Neu* in B104-1-1 and RB22), while that of P1 was only slightly reduced (p leq 0.05; the internalization of IgG-labeled P1 was similar to that of Neu* in RB22 cells, with p > 0.1), in accord with the lack of B-10 effect on their R(F) values (Fig. 2). Similarly, P1F, whose R(F) was not reduced by B-10 IgG, was also internalized inefficiently even when labeled with B-10 IgG (p > 0.1) (Fig. 4). The internalization of K758A was not changed by B-10 IgG (p > 0.1), supporting the suggestion that its partial immobilization by B-10 IgG is not due to interactions with coated pits (see above). To establish that the Neu receptor mutants are internalized via coated pits, we examined the effects of treatments that block coated pit-mediated endocytosis. The procedures used were hypertonic treatment, which blocks internalization through coated pits by dispersing the clathrin lattices(60, 61) , and cytosol acidification, which blocks the pinching off of clathrin-coated vesicles(61, 63) . The internalization of all the Neu mutants was effectively blocked by either one of these treatments (Fig. 5).


Figure 5: Effect of treatments that alter coated pit structure on internalization of Neu receptor mutants. Cells expressing the various Neu proteins were subjected to hypertonic medium or cytosol acidification treatment as described under ``Experimental Procedures.'' They were labeled with B-10 Fab` and I-SAM Fab` and assayed for internalization as described in the legend to Fig. 4. The results shown (means ± S.E. of three experiments, each performed in triplicate) are for hypertonic treatment with 0.225 M NaCl. Cytosol acidification yielded similar results (data not shown).



Coimmunoprecipitation of alpha-Adaptin with Neu Receptor Mutants

To substantiate the interactions between activated Neu receptor mutants and plasma membrane coated pits, we performed immunoprecipitation studies to examine the coprecipitation of alpha-adaptin with Neu receptors (Neu, Neu*, P1, and P1F). After detergent solubilization and immunoprecipitation of the Neu receptors, the extracts were analyzed for Neu receptors and for alpha-adaptin by SDS-PAGE and immunoblotting (Fig. 6). While a large amount of alpha-adaptin coprecipitated with constitutively dimerized and activated Neu*, only trace amounts coprecipitated with wild-type Neu. Comparison between the two truncated versions of Neu, P1 and P1F, yielded similar results: a substantially higher amount of alpha-adaptin coprecipitated with constitutively activated P1 as compared with P1F. These findings are in accord with the results of the lateral mobility and internalization studies and suggest that the interactions of activated Neu receptors with coated pits occur via AP-2 adaptors.


DISCUSSION

The association of membrane proteins with structures that are immobile in lateral mobility measurements can affect either D or R(F), depending on the dissociation rate of the membrane protein from the immobile structure. Thus, transient interactions (labile complexes) are reflected by a reduction in D, while stable association (permanent entrapment on the lateral mobility time scale) reduces R(F) (see ``Results'')(23, 64) . The results of the lateral mobility measurements on Neu, Neu*, and Neu* mutants (Fig. 2) labeled with monovalent Fab` clearly demonstrate a reduction in R(F) (with no effect on D) for all the constitutively dimerized mutants that can become activated (i.e. can undergo tyrosine autophosphorylation). A similar reduction in R(F) was obtained for wild-type Neu receptors (which are normally not dimerized) upon activation by intact agonistic B-10 IgG (Fig. 2). These results are compatible with the notion that dimerized and activated Neu receptors become stably associated for the duration of the measurement with immobile structures, most likely coated pits. The identification of these structures as coated pits is supported by (i) the correlation between the reduction in R(F) and the internalization of activated Neu mutants ( Fig. 3and Fig. 4), (ii) the blockade of the internalization by treatments (hypertonic treatment and cytosol acidification) that disrupt or alter the structure of coated pits (Fig. 5)(60, 61, 62) , and (iii) the selective coprecipitation of alpha-adaptin with the constitutively dimerized and activated Neu* and P1 mutants (Fig. 6). The endocytosis data presented here are compatible with previous reports: electron microscopy studies demonstrated internalization of Neu receptors via coated pits(45) , and the rate and extent of Neu receptor internalization encountered here ( Fig. 3and Fig. 4) are in agreement with those reported recently for endocytosis of EGF by Neu/EGF receptor chimeras, which are slower than those of the EGF receptor(46) .

This study demonstrates that a membrane receptor can become stably entrapped in native coated pits at the surface of untreated cells. We have formerly shown that a mutant influenza hemagglutinin protein containing a tyrosine recognition signal interacts transiently with coated pits, shifting to stable entrapment only after ``freezing'' the coated pits by cytosol acidification(23) . Interestingly, a previous study on the related EGF receptor reported that an internalization-defective truncated receptor labeled with fluorescent EGF exhibited a significantly higher R(F) value than the normal receptor, while their D values were similar(71) . This is in accord with the present findings on Neu receptors and suggests that activated EGF receptors may also become stably associated with coated pits.

To elucidate the possible roles of cytoplasmic tyrosines in the endocytosis of Neu receptors, we employed three double mutants of Neu*. The experiments with P1 and P1F, both of which lack most of the C-terminal tail distal to the Neu* tyrosine kinase domain (Fig. 1) (54) , reveal the ability of Tyr-1253 to function as an internalization signal. P1 showed a high level of internalization, a low R(F) value, and physical association with alpha-adaptin as compared with P1F (Fig. 2, Fig. 4, and Fig. 6). The failure of P1F to interact with coated pits is not due to inactivation of its tyrosine kinase activity since it is as active as P1 on exogenous substrate (54) . Since P1F is derived from constitutively dimerized Neu*, these results indicate that dimerization alone is not sufficient to trigger entry into coated pits and internalization. Our findings demonstrate that Tyr-1253, which resides within an NPXY sequence (known to function as an internalization signal)(3, 68) , is sufficient for triggering efficient internalization of dimerized Neu receptors via coated pits. Tyr-1253 (and the immediate amino acids around it) is not necessarily the sole internalization signal in the intact Neu receptor since P1 and P1F lack a large part of the C-terminal domain, including the P(2)-P(5) tyrosines(34) , and the QQGFF stretch proposed to be one of several internalization signals in the EGF receptor(44) . However, the rather similar R(F) values (Fig. 2) and internalization levels (Fig. 4) of P1 and untruncated Neu* suggest that Tyr-1253 is a major contributor to the interactions with coated pits.

The failure of Phe to replace Tyr-1253 in the internalization signal of P1 (compare P1F with P1) provides an indication that phosphorylation of this tyrosine might be involved. To explore this possibility, the kinase-negative Neu* mutant K758A (47) was examined. This mutant has a high R(F) value (similar to that of Neu) when labeled with Fab` (Fig. 2), and its internalization is impaired relative to Neu* or P1 ( Fig. 3and Fig. 4). These results imply that the Neu receptor tyrosine kinase activity is required for its efficient internalization. The effect could be direct (via autophosphorylation) or indirect (by phosphorylation of other proteins related to the clathrin-coated pit pathway). The combined results of this study appear to favor the notion that a phosphorylated tyrosine is involved in the internalization of Neu receptors since their endocytosis requires activation, a functional Neu kinase domain, and at least one autophosphorylation site (Tyr-1253). This does not necessarily mean that a phosphotyrosine serves directly as the endocytosis signal; it is also possible that phosphorylation triggers a conformational change that exposes the internalization signal(3, 46) . It should be noted that the role of kinase activity in the endocytosis of the related EGF receptor is still controversial. While one group reported inhibition of the endocytosis of a kinase-negative EGF receptor(40, 72) , another attributed the inefficient down-regulation of such a mutant to an elevated recycling rate(41, 73, 74) . A lower internalization rate was found for EGF receptors mutated at three major autophosphorylation sites; however, these mutants were also deficient in kinase activity, rendering the separation between the two effects inadequate(43, 75) . However, the lower internalization rate of Neu receptors (or chimeras containing the Neu receptor C-terminal region) (46) suggests that the internalization signals of the two receptors may be different at least to some degree.

Interestingly, the internalization competence (down-regulation) of the Neu receptor mutants investigated here is correlated with their ability to induce transformation. P1 cells developed tumors in athymic mice at a rate similar to that of Neu*-expressing cells, while P1F cells developed tumors at a much lower rate and were unable to form colonies in soft agar(54) . Similarly, K758A cells were inactive in these transformation assays. Tyr-1253, which allows full activation of Neu in P1 cells through effector molecules such as phospholipase C- or phosphatidylinositol 3`-kinase(47, 76) , is sufficient for efficient entrapment in coated pits and down-regulation. It is possible that effector molecules that bind to activated Neu via Tyr-1253 and that mediate signal transduction also connect the receptor indirectly to the endocytic pathway. Alternatively, the effector molecules and AP-2 complexes may compete for the same binding site(s) or bind to sites that are exposed simultaneously upon receptor activation. In either case, signal transduction and down-regulation would be coupled, and activated receptors would be selected for down-regulation.


FOOTNOTES

*
This work was supported in part by a project grant from the Israel Cancer Research Fund (to Y. I. H.) and by National Institutes of Health Grant CA51712 (to Y. Y.). 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. Tel.: 972-3-6409053; Fax: 972-3-6415053.

(^1)
The abbreviations used are: EGF, epidermal growth factor; FPR, fluorescence photobleaching recovery; TMR, tetramethylrhodamine; HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; GAM, goat anti-mouse; SAM, sheep anti-mouse; PAGE, polyacrylamide gel electrophoresis; Mes, 2-(N-morpholino)ethanesulfonic acid.


ACKNOWLEDGEMENTS

We are grateful to Dr. Margaret S. Robinson for the AC1-M11 antibodies.


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