(Received for publication, November 4, 1994)
From the
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
-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.
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
-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) (
)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 -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.
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-P
. 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
) 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.
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 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
values, the Neu and Neu* receptors displayed
similar lateral diffusion rates (measured by D). Such a
reduction in R
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
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
-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 -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
-adaptin. Blots
were labeled by AC1-M11 monoclonal antibodies directed against
-adaptin followed by protein A-peroxidase. Detection was by
enhanced chemiluminescence. A densitometric analysis was performed
employing the ratio between the
-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
-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
-adaptin coprecipitated with P1F is 16.5% relative
to the level of coprecipitation with P1. No signals were detected for
either Neu proteins or
-adaptin when immunoprecipitation and
immunodetection were performed on untransfected NIH 3T3 cells; similar
levels of
-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
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
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
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 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
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
10
/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
-adaptin (specific to the
plasma membrane coated pit adaptor complex, AP-2) were found in all
cell lines (see legend to Fig. 6).
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).
The association of membrane proteins with structures that are
immobile in lateral mobility measurements can affect either D or R, 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
(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
(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
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
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
-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 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 value, and physical
association with
-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
-P
tyrosines(34) , and the QQGFF stretch proposed to be one
of several internalization signals in the EGF receptor(44) .
However, the rather similar R
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 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.