|
|
|
|||||||
|
|||||||||
(Received for publication, February 12, 1996; and in revised form, March 7, 1996) From the
The role of Eps15 in clathrin-mediated endocytosis is supported
by two observations. First, it interacts specifically and
constitutively with the plasma membrane adaptor AP-2. Second, its
NH
Eps15 is the prototype of a new family of signal transducers
characterized by their ability to interact with a large number of
proteins(1) . It was initially described as a substrate of the
epidermal growth factor (EGF) ( Besides its possible function in
signal transduction, Eps15 may play a role in endocytosis. First, there
is 62% homology between Eps15 EH domains and the NH
For limited proteolysis of the AP-2 complex,
MOLT16 cells were lysed in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5% Triton X-100, in the absence of protease inhibitors.
Cell lysates were then digested with trypsin (Life Technologies, Inc.,
Eragny, France) at a 1:500 protein ratio at 37 °C for various
periods of time. Digestion was stopped by addition of a mix of protease
inhibitors (see above) and 10% fetal calf serum (Life Technologies).
Digested lysates and control undigested lysates were precipitated by
GST fusion proteins. For precipitation, cell lysates were cleared
with protein A-Sepharose or GST coupled to glutathione-Sepharose 4B
beads (Pharmacia Biotech Inc.) and then incubated overnight with mAb
6G4 (10 µg/3 For Western blotting, acrylamide gels were transferred
onto nitrocellulose membranes (Schleicher &
Schüll) in 10 mM Tris, 0.2 M glycine, and 30% methanol. Nonspecific binding sites were blocked
by incubation in Tris-HCl, pH 7.6, containing 5% bovine serum albumin
and 0.2% Tween (Sigma). The blots were then sequentially incubated for
1 h, either with mouse mAbs at the indicated dilutions, followed by
peroxidase-labeled sheep anti-mouse antiserum (1:20,000) (Amersham) or
with rabbit antiserum Ab32 followed by swine anti-rabbit immunoglobulin
antiserum (1:5,000) (Dakopatts, Trappes, France). Labeled bands were
revealed using ECL (Amersham).
Figure 1:
Structural organization of Eps15 and
characterization of fusion proteins derived from Eps15 domains. A, three different constructs, GST-DI (1-315), GST-DII
(305-538), and GST-DIII (529-896), were derived from each
of the three domains of Eps15. B, lysates of bacteria
transformed with GST-DI (lane 1), -DII (lane 2), or
-DIII constructs (lane 3) were tested by Western blotting (WB) with mAb 6G4 (0.5 µg/ml) (upper panel) or
with commercial anti-Eps15 mAb (2 µg/ml) (lower panel) as
indicated under ``Experimental
Procedures.''
GST-DI, -DII, and -DIII fusion proteins and
the anti-Eps15 antibody 6G4 were used to precipitate lysates of
Figure 2:
Interaction of AP-2 with the COOH-terminal
domain of Eps15. A, lysates of biosynthetically labeled MOLT16
cells were first cleared with protein A-Sepharose (lane 1) or
with GST coupled to glutathione-Sepharose 4B beads (lane 6)
and then precipitated (PP) with mAb 6G4 coupled to protein
A-Sepharose (lane 2) or GST-DI (lane 3), GST-DII (lane 4), or GST-DIII (lane 5) coupled to
glutathione-Sepharose 4B beads, as described under ``Experimental
Procedures.'' Precipitated proteins were separated on a
7-15% gradient SDS-PAGE under reducing conditions and
autoradiographed. B and C, MOLT16 cell lysates were
precipitated by GST-DIII (B and C, lane 2)
and/or GST (C, lane 1). Precipitated proteins were
separated on a 6.5% SDS-PAGE and transferred on a nitrocellulose
membrane as indicated in experimental procedures. In B, the
102-kDa band precipitated by GST-DIII was immunoblotted with pan
anti-
Besides the components of AP-2, GST-DIII
coprecipitated polypeptides with molecular masses of 180 and
30-35 kDa (Fig. 2A, lane 5). The
molecular mass of these polypeptides were comparable to those of the
heavy and light chains of clathrin, which interact with the AP-2
complex(13) . The presence of clathrin in the precipitate of
GST-DIII was confirmed by immunoblotting with the TD.1 antibody
specific for the clathrin heavy chain, which detected a 180-kDa band in
the GST-DIII precipitate but not in a control GST precipitate (Fig. 2C, upper panel, lane 2).
Figure 3:
Localization of the AP-2 binding site to
Eps15 amino acids 667-739. A, different constructs
encoding parts of the COOH-terminal domain of Eps15 were used to
precipitate MOLT16 cell lysates. The presence of the AP-2 complex in
the precipitates was revealed by Western blotting (WB) using
the anti-
Figure 4:
Binding of Eps15 with the ear domain of
Figure 5:
Binding of the COOH-terminal domain of
Eps15 to fusion proteins derived from
We have recently demonstrated a specific and constitutive
interaction between Eps15 and the plasma membrane adaptor,
AP-2(6) . In the present study, the binding site of Eps15 for
AP-2 was localized to a domain of 72 amino acids in the COOH terminus
of Eps15. In addition, we have identified The predicted primary sequence of Eps15 identifies a
modular protein with three domains, and each may be involved in
specific protein interactions (1, 2, 3) (Fig. 6A). An EH
domain comparable to those observed in the NH
Figure 6:
Schematic representation of the
interaction between Eps15 and AP-2. A, the localization of the
AP-2 binding site on Eps15 is indicated and its amino acid sequence is
shown. The four DPF repeats are underlined. B, interactions
between AP-2 and Eps15 are shown
schematically.
Clathrin-coated pits and vesicles bud from two membrane
compartments, the plasma membrane and the trans-Golgi network. Two
distinct adaptor complexes link the clathrin lattice to the appropriate
membrane: AP-2, associated with the plasma membrane coated vesicles,
and AP-1, associated with trans-Golgi coated
vesicles(7, 8) . Both adaptors are heterotetramers
consisting of two 90-110-kDa adaptins ( The
association of Eps15 with AP-2 and its homology with End3p strongly
suggest that this protein has a function in clathrin-mediated
endocytosis. Furthermore, the presence of the clathrin heavy chain in
the GST-DIII precipitate indicates that Eps15 may be a component of
clathrin-coated pits and vesicles where AP-2 and clathrin may interact,
a hypothesis supported by preliminary electron microscopic data. (
Volume 271,
Number 20,
Issue of May 17, 1996 pp. 12111-12116
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-Adaptin Interacts with the COOH-terminal Domain of the Eps15
Protein (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
terminus shows significant homology to the NH terminus of yeast End3p, necessary for endocytosis of
-factor. To gain further insight into the role of Eps15-AP-2
association, we have now delineated their sites of interactions. AP-2
binds to a domain of 72 amino acids (767-739) present in the COOH
terminus of Eps15. This domain contains 4 of the 15 DPF repeats
characteristic of the COOH-terminal domain of Eps15 and shares no
homology with known proteins, including the related Eps15r protein.
Precipitation of proteolytic fragments of AP-2 with Eps15-derived
fusion proteins containing the binding site for AP-2 showed that Eps15
binds specifically to a 40-kDa fragment corresponding to the ear of
-adaptin, a result confirmed by precipitation of Eps15 by
-adaptin-derived fusion proteins. Our data indicate that this
specific part of AP-2 binds to a cellular component and provide the
tools for investigating the function(s) of the association between AP-2
and Eps15 .
)and platelet-derived growth
factor tyrosine kinase receptors endowed with transforming
properties(2, 3) . A novel protein, Eps15r, with 47%
identity to Eps15 has recently been cloned using a probe derived from
the region encoding the NH-terminal domain of
Eps15(1) . Both Eps15 and Eps15r are organized into three
distinct structural domains. The amino terminus domain of Eps15 (amino
acids 1-300) displays 70% identity to the amino terminus of
Eps15r and is composed of three imperfect repeats of approximately 100
amino acids with candidate tyrosine phosphorylation sites and two
EF-hand type calcium binding sites. Each repeat contains a domain of 70
amino acids, which is conserved not only in Eps15 and Eps15r, but also
in several proteins in yeast and nematodes and is therefore designated
EH for Eps15-Homology domain. The first domain of
Eps15 interacts with several unidentified cytosolic
proteins(1) . The homology between the two proteins drops to
45% in the second domain, but the heptads required for coiled-coil
structure are conserved. A possible function of these heptads in homo-
or heterodimerization has been hypothesized(2, 3) .
Finally, there is little conservation between the COOH-terminal domains
(amino acids 520-896) of Eps15 and Eps15r, with two notable
exceptions. First, multiple DPF motifs are present in both proteins.
Second, the two proteins contain a proline-rich domain, PALPPK, which
binds the Src homology 3-domain of the crk protooncogene(4) . terminus of End3p, a protein required for clathrin-mediated
internalization of -factor in Saccharomyces cerevisiae.
In addition, the temperature-sensitive internalization defect of end3 mutants can be complemented with wild type End3p but not
with mutated End3p bearing small deletions in the EH
domain(5) . Furthermore, we have recently observed that, in all
cell types tested and in several species, Eps15 is constitutively
associated with the plasma membrane adaptor complex AP-2(6) ,
which serves several functions in endocytosis. On the one hand, AP-2
favors assembly of the clathrin triskelion and association of the
clathrin lattice to the plasma membrane. On the other hand, AP-2
participates in the recruitment of endocytosed receptors in
clathrin-coated pits(7, 8) . To gain insight into the
mechanisms of interaction between AP-2 and Eps15, and thereby into the
function of Eps15 and related proteins in endocytosis, a precise
knowledge of the sites of interaction of the two proteins is required.
With this goal in mind, we constructed a large series of GST fusion
proteins derived from Eps15 and tested them for their ability to
precipitate the AP-2 complex from cell lysates. Eps15-derived fusion
proteins containing the binding site for AP-2 were then used to
precipitate fragments derived from the limited proteolysis of AP-2 to
define the component of AP-2 interacting with Eps15. The binding site
of Eps15 was localized to the COOH-terminal appendage (ear) of
-adaptin, a result confirmed using fusion proteins derived from
-adaptin.
Cells and Antibodies
All studies were performed
using the human MOLT16 leukemic T cell line (gift of Dr. Minowada,
Fujisaki Cell Center). Production and characterization of the 6G4
anti-Eps15 monoclonal antibody (mAb) has been previously
reported(6) . Another anti-Eps15 mAb produced against a
COOH-terminal fragment (residues 717-896) of murine Eps15 was
obtained from Affiniti Research Products Ltd (Nottingham, UK). Mouse
mAbs, AC2-M15, against -adaptin A, and AC1-M11 against the
NH-terminal domains of -adaptins A and C, were kind
gifts of Dr. M. S. Robinson(9) . mAb 100/2, against the COOH
termini (ears) of -adaptins A and C; mAb 100/1, against the
NH terminus of 
-adaptin; and mAb 100/3, against
-adaptin, were purchased from Sigma (Saint Quentin Fallavier,
France). Antiserum Ab32, against the COOH terminus of -adaptin,
was given by Dr. A. Sorkin(10) . Mouse mAb TD.1, against the
clathrin heavy chain, was provided by Dr. F. Brodsky(11) .Construction of Glutathione S-transferase (GST) Fusion
Proteins
Different fusion proteins were derived from eps15 using the GST gene fusion system and the PGEX5.1 vector
(Pharmacia-LKB, Les Ulis, France). The cDNA of human eps15 subcloned in pBluescript II KS (Stratagene) was obtained in the
laboratory ()and used as a template to generate different
cDNA fragments encoding domains DI, DII, and DIII of eps15 and
truncated forms of DIII. A BamHI and a XhoI site were
introduced in the upper and lower primers, respectively, to allow
subcloning of the PCR products in the PGEX5.1 vector in frame with the
GST moiety. The constructs were checked by nucleotide sequencing
(Thermosequenase, Amersham Corp., Les Ulis, France). GST fusion
proteins encoding the COOH domain of -adaptin or parts of this
domain were similarly generated by PCR using the cDNA of mouse
-adaptin C, the nonalternatively spliced ubiquitous form of
-adaptin(12) , subcloned in pBluescript II SK as a
template (a kind gift of Dr. M. Robinson). Sequences of the used
primers are available on request. Production of fusion proteins in
DH5 bacteria and purification were performed as described
elsewhere(6) .Biochemical Procedures
For biosynthetic labeling,
MOLT16 cells were incubated with 
S-labeled amino acids
(TransS-label, Amersham) for 90 min. After a 3-h chase,
cells were lysed in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, containing a mix of
protease inhibitors (4 mM phenylmethylsulfonyl fluoride, 10
µg/ml aprotinin, leupeptin, pepstatin, 50 µg/ml trypsin
inhibitor (Sigma)).
10
cells) coupled to protein
A-Sepharose (Pharmacia Biotech Inc.) or with GST fusion proteins
(5-10 µg/10 cells) coupled to
glutathione-Sepharose 4B beads (20-30 µl/10 cells). Precipitated proteins were separated by
SDS-polyacrylamide gel electrophoresis (PAGE) under reducing
conditions.
Eps15 Binds AP-2 via Its Third COOH-terminal
Domain
Previous studies have shown that the anti-Eps15 mAb 6G4
as well as a fusion protein encompassing the full-length of Eps15
precipitate the various polypeptides of the AP-2 complex(6) .
To determine which domain of Eps15 interacts with AP-2, three GST
fusion proteins, each one comprising one of the three structural
domains of Eps15, GST-DI (amino acids 1-315), GST-DII (amino
acids 305-538), and GST-DIII (amino acids 529-896), were
prepared (Fig. 1A). Correct translation of the fusion
proteins was checked by Coomassie Blue staining and/or immunoblotting.
The GST-DI protein had the predicted size of 61 kDa and reacted with
mAb 6G4 (Fig. 1B, lane 1), indicating that
this antibody recognizes the NH terminus of Eps15. The
epitope was further mapped to the first 97 NH-terminal
amino acids (not shown). The GST-DII fusion protein, visualized by
Coomassie Blue staining, had the expected size of 54 kDa (not shown).
The GST-DIII protein reacted with a mAb produced against the C terminus
of Eps15 (Fig. 1B, lane 3). Its apparent
molecular mass of 97 kDa contrasted with its expected size of 67 kDa.
However, the delayed migration of GST-DIII is reminiscent of the
behavior of the full-length fusion protein which has an expected size
of 130 kDa and migrates as a 160-170-kDa polypeptide in SDS-PAGE
even in the absence of post-translational modification(2) . The
delayed migration of Eps15 and its third domain is thus likely due to
the amino acid composition of the latter domain and particularly to its
high number of prolines, 15 of which are adjacent to an acidic residue
in the 15 DPF repeats.
S-labeled MOLT16 cells (Fig. 2A). As
described previously(6) , the anti-Eps15 antibody precipitated
two major bands of 140 and 102 kDa (lane 2). The 140-kDa band
corresponds to Eps15 since it reacts with 6G4 (6) and can be
cleaved by endoprotease-Lys C into the same peptides as the in
vitro translation product of eps15 cDNA. The
102-kDa band contains the - and -adaptins, as
previously demonstrated by microsequencing and
immunoblotting(6) . A large band with the same molecular mass
of 102 kDa was also precipitated by GST-DIII (lane 5) but not
by GST-DI, GST-DII, or the control GST (lanes 3, 4,
and 6). The identity of the 102-kDa band precipitated by
GST-DIII with the adaptins of the AP-2 complex was demonstrated by
immunoblotting experiments, which showed that it reacts with anti- (Fig. 2B, lanes 1 and 2) and
anti- (Fig. 2B, lane 4) but not with an
anti- adaptin antibody (Fig. 2B, lane 3).
In addition, GST-DIII precipitated bands of 50 and 17 kDa (lane
5). Bands of comparable molecular mass were previously observed in
6G4 immunoprecipitates(6) , although only the 50-kDa band is
visible in the 6G4 immunoprecipitate shown in this experiment (lane
2). These two bands have a molecular mass compatible with that of
the two small components of AP-2, µ and
.
-adaptin mAb 100/2 (ascitis 1:2,000) (lane 1),
anti--adaptin A mAb AC2M15 (ascitis 1:2,000) (lane 2),
anti--adaptin mAb 100/3 (ascitis 1:1,000) (lane 3), and
anti--adaptin 100/1 (ascitis 1/5000) (lane 4). In C, precipitates of GST (lane 1) and GST-DIII (lane 2) were immunoblotted with anti-clathrin heavy-chain mAb
TD.1 (10 µg/ml) (upper panel) or pan anti--adaptin
mAb 100/2 (lower panel).
Characterization of the Binding Site for AP-2 in the
COOH-terminal Domain of Eps15
The results described above
indicate that the COOH-terminal domain of Eps15 is sufficient for
mediating the association between Eps15 and AP-2. To further
characterize the region of Eps15 responsible for the association, a
series of truncated forms of the GST-DIII proteins was used to
precipitate cold cell lysates, and the precipitation of AP-2 was
detected by immunoblotting using the 100/2 mAb specific for
-adaptin. As summarized in Fig. 3B, a segment
comprising amino acids 667-739 and including four of the 15 DPF
repeats was required for AP-2 binding. Further trimming of this segment
by removing either amino acids 667-675 (Fig. 3A, upper panel, lane 7) or amino acids 712-739 (upper panel, lanes 9 and 16) prevented AP-2
binding. However, the amount of -adaptin precipitated by the
fusion protein comprising only amino acids 667-763 (lanes 6 and 15) was significantly less than the amount of
-adaptins precipitated by fusion proteins containing a larger
NH-terminal fragment of 43 amino acids (lanes 5 and 14). The NH-terminal fragment does not
appear to provide a second binding site for AP-2, since GST-529/682
failed to precipitate -adaptin (lane 3). More likely, the
presence of this fragment influences the conformation and/or the
accessibility of the binding site present on segment 667-739.
However, it was not possible to define more precisely the minimal size
of the NH-terminal fragment allowing optimal binding of
AP-2, since the presence of 6 DPF repeats, very close to each other
between amino acids 624-667, precluded the design of specific
primers for fusion proteins of intermediate size. Finally, the amount
of -adaptins precipitated by GST-667/739 (lane 17) was
consistently less than that precipitated by GST-667/763 (lane
15). Since GST-624/739 precipitated very efficiently -adaptin (lane 8), the fragment 740-763 is not normally required
for binding AP-2. Nonetheless, when the fusion protein does not contain
segment 624-667, the presence of segment 740-763 may
contribute to the conformation of the binding site present in segment
667-739.
-adaptin antibody 100/2 (upper panels).
Coomassie Blue staining of the membranes revealed that similar amounts
of fusion proteins were used in all precipitations (lower
panels). In addition, in lanes 1, 2, 4, 5, and 8, it revealed a doublet of approximately 100
kDa which likely corresponds to the adaptins revealed by Western
blotting in the upper panel. B, the different constructs used
and the results obtained with the fusion proteins are
summarized.
The Eps15-derived Fusion Protein Binds to the Proteolyzed
Ear Domain of
Precipitation studies did not allow
us to identify the component of AP-2 that binds directly to Eps15,
since the four components of AP-2 are dissociated only under strong
denaturing conditions and therefore coimmunoprecipitate with
Eps15(6, 14, 15) . Previous studies have
shown that AP-2 consists of a brick corelike structure or head with two
small appendages or ears linked to the head by hinges containing sites
for proteolytic cleavage. The head and ears can thus be separated by
limited proteolysis of AP-2. The head consists of the
NH-Adaptin-terminal domains of - and -adaptins associated
with the medium and small chains, whereas the ears correspond to the
COOH-terminal domains of the two adaptins. Proteolysis does not affect
the interactions between the truncated and subunits and the
protease resistant 50- and 17-kDa subunits (14, 15) .
Therefore, to define the AP-2 domain that binds to Eps15, the head and
the ears were prepared by limited proteolysis of AP-2 with trypsin. As
shown in Fig. 4, lysates treated with trypsin for 20 min at 37
°C contained fragments of 60-65 kDa that were reactive with
AC1-M11 and 100/1 antibodies against the head of - and
-adaptins, respectively (15) (Fig. 4, b and d, lanes 5), and 40-kDa fragments reactive
with 100/2 and Ab32 antibodies against the ears of - and
-adaptins, respectively (10, 15) (Fig. 4, a and c, lanes 5). When proteolysis was
further prolonged, there was a decrease in the amount of immunoreactive
60-65-kDa fragments, indicating that these fragments were
proteolyzed to smaller peptides (not shown). Therefore, control
undigested lysates or lysates treated with trypsin for 5, 10, or 20 min
were precipitated with GST-DIII (lane 1, 2, 3, and 4, respectively). As shown in Fig. 4, a and b, GST-DIII precipitated undigested
-adaptin visible in control precipitates (lanes 1) and in
precipitates obtained after a digestion for 5 min (lanes 2) or
10 min (Fig. 4b, lane 3). In addition,
GST-DIII precipitated a fragment with a molecular mass of 40 kDa, which
was reactive with mAb 100/2 (Fig. 4a, lanes
2-4) and therefore corresponded to the ear of -adaptin.
In contrast, GST-DIII failed to precipitate the other proteolyzed
fragments of AP-2. Thus, it precipitated neither the
NH-terminal domain of -adaptin recognized by mAb
AC1M11 (Fig. 4b, lanes 2-4), nor that of
-adaptin, recognized by mAb 100/1 (Fig. 4d, lanes 3 and 4). Some intact -adaptin and a light
band of 60 kDa that reacted with the 100/1 antibody were observed in
the GST-DIII precipitate after a 5-min digestion (Fig. 4d, lane 2) but were not detectable
after longer times of digestion (Fig. 4d, lanes 3 and 4). Since intact -adaptin remained detectable at
5 and 10 min of digestion (Fig. 4b, lanes 2 and 3), these bands most likely correspond to digested or
undigested -adaptin that is still associated with undigested
-subunit. Finally, GST-DIII did not precipitate the ear of
-adaptin detected by the Ab32 antiserum (Fig. 4c).
These results indicate that Eps15 specifically binds the ear of
-adaptin.
-adaptin. MOLT16 cell lysates, treated with trypsin for 5 min (lanes 2), 10 min (lanes 3), and 20 min (lane
4) or left undigested as controls (lane 1) were
precipitated with GST-DIII (lanes 1-4). Precipitates (pp) and aliquots of the cell lysate after a 20-min digestion (lane 5) were analyzed by Western blotting (WB) using
mouse mAb 100/2 against the -adaptin ear (a), mouse mAb
AC1M11 against the -adaptin NH-terminal domain (head) (b), rabbit-anti-mouse antiserum Ab32 against the
-adaptin ear (c), or mAb 100/1 (d) against the
-adaptin NH-terminal domain (head), as indicated under
``Experimental Procedures.''
GST Fusion Proteins Derived from the COOH-terminal Domain
of
To confirm the results of the
proteolysis studies, a GST-fusion protein encompassing the entire COOH
terminus of -Adaptin Bind Eps15-adaptin and including the hinge region was derived
from mouse -adaptin (Fig. 5A). We observed that a
large fraction of the -adaptin ear was cleaved from the GST moiety
during purification of the GST fusion protein and released in the
bacterial lysate (not shown). This proteolytic cleavage was probably
due to the presence of bacterial proteases and could not be prevented
by the use of exogenous protease inhibitors. Therefore, to demonstrate
the association of Eps15 with the -adaptin ear, bacterial lysates
containing the -adaptin ear were precipitated with Eps15-derived
GST fusion proteins. After three clearing cycles with
glutathione-Sepharose 4B beads to remove free GST, the supernatant was
analyzed by Western blotting using the anti--adaptin ear antibody
100/2. This antibody detected a large band with a molecular mass of 40
kDa, corresponding to the -adaptin ear, and lighter bands of 37
and 67 kDa, corresponding, respectively, to a proteolyzed fragment of
the -adaptin ear (15) and to the intact GST -adaptin
ear fusion protein (GST- ear) which had not been entirely removed
by the clearing procedure (Fig. 5B, lane 1).
The same supernatant was then precipitated with GST-DIII (lane
3), or GST-DI as a control (lane 2). The complete
GST- ear was precipitated nonspecifically by glutathione-Sepharose
4B beads and was present in both the GST-DIII precipitate and the
control GST-DI precipitate. In contrast, the 40-kDa -adaptin ear
fragment bound to GST-DIII (lane 3) but not to GST-DI (lane 2), confirming the specific interaction between the
COOH-terminal domain of Eps15 and the ear of -adaptin. GST-DIII
did not precipitate the 37-kDa fragment derived from the ear,
suggesting that this smaller fragment did not contain the binding site
for Eps15. To avoid proteolytic cleavage of GST- ear, smaller GST
fusion proteins were designed. In the absence of the hinge region, the
GST fusion proteins were not sensitive to protein degradation and could
therefore be used to precipitate Eps15 from cell lysates. Eps15 could
be precipitated by a GST fusion protein containing the -adaptin
ear without the hinge region (amino acids 706-938) (Fig. 5C, lane 2). In contrast, it was not
precipitated by fusion proteins with an additional deletion of 50 amino
acids or more on the COOH-terminal side (Fig. 5C, lane 1 and not shown). Together with the lack of precipitation
of the 37-kDa fragment derived from the GST- ear, this result
indicates that Eps15 binding requires the proximal part of the
-adaptin ear.
-adaptin. A,
description of the -adaptin-derived GST fusion proteins. B, lysates of sonicated bacteria transformed with the
GST- ear construct were cleared three times with
glutathione-Sepharose 4B beads to eliminate the excess of cleaved GST,
and the supernatant was precipitated with fusion proteins derived from
Eps15 (lane 1), and precipitates by GST-DI (lane 2)
or GST-DIII (lane 3) were analyzed by Western blotting using
the -adaptin ear specific mAb 100/2. C, two different
constructs encoding residues 756-938 (lane 1) and
residues 706-938 (lane 2) of mouse -adaptin C were
used to precipitate MOLT16 cell lysates (PP). The presence of
Eps15 in the precipitates was revealed by Western blotting (WB) using the 6G4 mAb (upper panel). Coomassie Blue
staining of the membranes revealed that similar amounts of fusion
proteins were used in the two precipitations (lower
panel).
-adaptin as the
component of AP-2 that binds Eps15 and have localized the binding site
within its ear domain. In addition, we have provided preliminary
evidence that Eps15 can associate with a fraction of AP-2 bound to
clathrin. terminus of
Eps15 is observed in End3p(1) , a yeast protein involved in
internalization of the -factor(5) , and the study of
deletion mutants suggests that the EH domain of End3p is required for
normal endocytosis(5) . To define which domain of Eps15
interacts with AP-2, GST fusion proteins derived from each of the three
domains of Eps15 were tested for their ability to precipitate AP-2 from
cell lysates. No interaction could be demonstrated between AP-2 and the
central domain of Eps15 or its NH terminus. This result
does not exclude a role for the EH domains of Eps15 in endocytosis, but
indicates that they do not bind AP-2. In contrast, the GST fusion
protein encoding the COOH-terminal domain of Eps15 precipitated -
and 2-adaptins very efficiently, indicating that this domain
contains the binding site for AP-2. The minimal region required for
this interaction was defined using a series of GST fusion proteins
derived from the COOH terminus of Eps15. The smallest fusion protein
able to precipitate AP-2 comprised amino acids 667-739 and
included only four of the DPF repeats among the 15 present in human
Eps15. Its sequence is shown in Fig. 6A. The binding
site of AP-2 was thus close but distinct from the binding site of Crk
(residues 765-771)(1) , indicating that these proteins
bind Eps15 independently. A search of the data banks using the Blast
program revealed 90% identity with the corresponding sequence in the
murine Eps15 protein, which also interacts with AP-2(6) . In
contrast, the sequence was poorly conserved in Eps15r, the homology
being largely related to the presence of DPF repeats. Furthermore,
there was no significant homology with other known proteins, suggesting
that the association of Eps15 with AP-2 has a very specific function.
and for the plasma membrane, and for the
trans-Golgi) complexed with two smaller proteins of 48-50 and
16-17 kDa (µ1, µ2 and 1, 2
respectively)(7, 8) . In agreement with our previous
observations(6) , Eps15-derived fusion proteins precipitated
four proteins with molecular masses consistent with the four components
of the adaptor complexes. The - and -adaptins, but not
-adaptin, were found in the GST-Eps15 precipitate, a result that
confirms the specific association of Eps15 with AP-2 but does not allow
us to determine which subunit of AP-2 interacts with Eps15. Several
protein associations with components of the adaptor complexes have
already been described. The 1 and2 subunits, which are 85%
identical(14, 16) , mediate binding of both AP-1 and
AP-2 adaptors to clathrin and promote clathrin coat assembly (17, 18, 19) . The µ and
µ subunits, which share 40%
identity(20, 21) , interact with tyrosine-based
signals of several integral membrane proteins(22) . The -
and -adaptins have an overall identity of only 25%, mainly
restricted to the NH domain(23) . This domain
contains sequences which simultaneously determine coassembly with the
correct µ and subunits and targeting to the appropriate
membrane(24, 25) . In addition, the
NH-terminal domain of -adaptin binds to clathrin cages (26) and contains a binding site for polyphosphoinositols
which, in vitro, inhibit AP-2 self-association, binding of
AP-2 to clathrin, and clathrin coat assembly(27, 28) .
In contrast to the other components of adaptor complexes, the ears of
- and -adaptins show no homology, suggesting that they have
distinct functions(23) . Precipitation of the fragments of AP-2
released by limited proteolysis indicates that the Eps15-derived fusion
proteins bound to the ear of -adaptin. This result was confirmed
by precipitation of Eps15 with a fusion protein encompassing the ear of
-adaptin C. Furthermore, the ear of -adaptin C released in
the lysate of transformed bacteria could be precipitated by a GST-Eps15
fusion protein, demonstrating that Eps15 and -adaptin interact
directly. These data are consistent with the specificity of Eps15 for
AP-2 and supports the hypothesis that the ear of -adaptin is
endowed with a specific function. A model summarizing the interaction
of Eps15 with AP-2 is shown in Fig. 6B.
)Eps15 might thus be related to the unidentified 150-kDa
protein observed by Beck and Keen (18) in AP-2 aggregates and
in coated vesicles and/or to the 140-kDa protein observed by Lindner
and Ungewickell (29) among the components of bovine
clathrin-coated vesicles. The putative function of Eps15 in endocytosis
remains to be defined. Our study, which delineates the domain of Eps15
involved in AP-2 binding, provides the basis for the design of mutated
proteins that could elucidate the in vivo function of Eps15.
)))
We thank Dr. A. Sorkin for the kind gift of Ab32
antibody and Dr. M. S. Robinson for the generous gift of mouse
-adaptin C cDNA, AC1-M11, and AC2-M15 antibodies, and helpful
advice; Drs. A. Fischer, J. P. Di Santo, J. P. De Villartay, and C.
Hivroz for helpful advice and discussions; and Dr. D. Ojcius for
reading the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Masuyama, T. Kuronita, R. Tanaka, T. Muto, Y. Hirota, A. Takigawa, H. Fujita, Y. Aso, J. Amano, and Y. Tanaka HM1.24 Is Internalized from Lipid Rafts by Clathrin-mediated Endocytosis through Interaction with {alpha}-Adaptin J. Biol. Chem., June 5, 2009; 284(23): 15927 - 15941. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Parachoniak and M. Park Distinct Recruitment of Eps15 via Its Coiled-coil Domain Is Required For Efficient Down-regulation of the Met Receptor Tyrosine Kinase J. Biol. Chem., March 27, 2009; 284(13): 8382 - 8394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Pandey, E. N. Harris, J. A. Weigel, and P. H. Weigel The Cytoplasmic Domain of the Hyaluronan Receptor for Endocytosis (HARE) Contains Multiple Endocytic Motifs Targeting Coated Pit-mediated Internalization J. Biol. Chem., August 1, 2008; 283(31): 21453 - 21461. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Chi, H. Cao, J. Chen, and M. A. McNiven Eps15 Mediates Vesicle Trafficking from the trans-Golgi Network via an Interaction with the Clathrin Adaptor AP-1 Mol. Biol. Cell, August 1, 2008; 19(8): 3564 - 3575. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Roxrud, C. Raiborg, N. M. Pedersen, E. Stang, and H. Stenmark An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor J. Cell Biol., March 24, 2008; 180(6): 1205 - 1218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Brindley and W. Maury Equine Infectious Anemia Virus Entry Occurs through Clathrin-Mediated Endocytosis J. Virol., February 15, 2008; 82(4): 1628 - 1637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Z. Rappoport, S. Kemal, A. Benmerah, and S. M. Simon Dynamics of clathrin and adaptor proteins during endocytosis Am J Physiol Cell Physiol, November 1, 2006; 291(5): C1072 - C1081. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Clement, V. Tiwari, P. M. Scanlan, T. Valyi-Nagy, B. Y.J.T. Yue, and D. Shukla A novel role for phagocytosis-like uptake in herpes simplex virus entry J. Cell Biol., September 25, 2006; 174(7): 1009 - 1021. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. McNiven and H. M. Thompson Vesicle formation at the plasma membrane and trans-Golgi network: the same but different. Science, September 15, 2006; 313(5793): 1591 - 1594. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Toulme, F. Soto, M. Garret, and E. Boue-Grabot Functional Properties of Internalization-Deficient P2X4 Receptors Reveal a Novel Mechanism of Ligand-Gated Channel Facilitation by Ivermectin Mol. Pharmacol., February 1, 2006; 69(2): 576 - 587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Naslavsky and S. Caplan C-terminal EH-domain-containing proteins: consensus for a role in endocytic trafficking, EH? J. Cell Sci., September 15, 2005; 118(18): 4093 - 4101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Puri, D. Tosoni, R. Comai, A. Rabellino, D. Segat, F. Caneva, P. Luzzi, P. P. Di Fiore, and C. Tacchetti Relationships between EGFR Signaling-competent and Endocytosis-competent Membrane Microdomains Mol. Biol. Cell, June 1, 2005; 16(6): 2704 - 2718. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Mattera, B. Ritter, S. S. Sidhu, P. S. McPherson, and J. S. Bonifacino Definition of the Consensus Motif Recognized by {gamma}-Adaptin Ear Domains J. Biol. Chem., February 27, 2004; 279(9): 8018 - 8028. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Hinrichsen, J. Harborth, L. Andrees, K. Weber, and E. J. Ungewickell Effect of Clathrin Heavy Chain- and {alpha}-Adaptin-specific Small Inhibitory RNAs on Endocytic Accessory Proteins and Receptor Trafficking in HeLa Cells J. Biol. Chem., November 14, 2003; 278(46): 45160 - 45170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Morgan, K. Prasad, S. Jin, G. J. Augustine, and E. M. Lafer Eps15 Homology Domain-NPF Motif Interactions Regulate Clathrin Coat Assembly during Synaptic Vesicle Recycling J. Biol. Chem., August 29, 2003; 278(35): 33583 - 33592. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Cobbold, J. Coventry, S. Ponnambalam, and A. P. Monaco The Menkes disease ATPase (ATP7A) is internalized via a Rac1-regulated, clathrin- and caveolae-independent pathway Hum. Mol. Genet., July 1, 2003; 12(13): 1523 - 1533. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Yao, M. Ehrlich, Y. I. Henis, and E. B. Leof Transforming Growth Factor-beta Receptors Interact with AP2 by Direct Binding to beta 2 Subunit Mol. Biol. Cell, November 1, 2002; 13(11): 4001 - 4012. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Sieczkarski and G. R. Whittaker Influenza Virus Can Enter and Infect Cells in the Absence of Clathrin-Mediated Endocytosis J. Virol., September 11, 2002; 76(20): 10455 - 10464. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Klapisz, I. Sorokina, S. Lemeer, M. Pijnenburg, A. J. Verkleij, and P. M. P. van Bergen en Henegouwen A Ubiquitin-interacting Motif (UIM) Is Essential for Eps15 and Eps15R Ubiquitination J. Biol. Chem., August 16, 2002; 277(34): 30746 - 30753. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R. Henry, K. D'Hondt, J. Chang, T. Newpher, K. Huang, R. T. Hudson, H. Riezman, and S. K. Lemmon Scd5p and Clathrin Function Are Important for Cortical Actin Organization, Endocytosis, and Localization of Sla2p in Yeast Mol. Biol. Cell, August 1, 2002; 13(8): 2607 - 2625. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Baillat, S. Gaillard, F. Castets, and A. Monneron Interactions of Phocein with Nucleoside-Diphosphate Kinase, Eps15, and Dynamin I J. Biol. Chem., May 17, 2002; 277(21): 18961 - 18966. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Nishimura, H. Plutner, K. Hahn, and W. E. Balch The delta subunit of AP-3 is required for efficient transport of VSV-G from the trans-Golgi network to the cell surface PNAS, May 14, 2002; 99(10): 6755 - 6760. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Poupon, S. Polo, M. Vecchi, G. Martin, A. Dautry-Varsat, N. Cerf-Bensussan, P. P. Di Fiore, and A. Benmerah Differential Nucleocytoplasmic Trafficking between the Related Endocytic Proteins Eps15 and Eps15R J. Biol. Chem., March 8, 2002; 277(11): 8941 - 8948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-S. Lee, J. Rohrer, R. Kornfeld, and S. Kornfeld Multiple Signals Regulate Trafficking of the Mannose 6-Phosphate-uncovering Enzyme J. Biol. Chem., January 25, 2002; 277(5): 3544 - 3551. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rohrer and R. Kornfeld Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in the trans-Golgi Network Mol. Biol. Cell, June 1, 2001; 12(6): 1623 - 1631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Martina, C. J. Bonangelino, R. C. Aguilar, and J. S. Bonifacino Stonin 2: An Adaptor-like Protein that Interacts with Components of the Endocytic Machinery J. Cell Biol., May 29, 2001; 153(5): 1111 - 1120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. G. Ferguson Evolving Concepts in G Protein-Coupled Receptor Endocytosis: The Role in Receptor Desensitization and Signaling Pharmacol. Rev., March 1, 2001; 53(1): 1 - 24. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M Ehrlich, A Shmuely, and Y. Henis A single internalization signal from the di-leucine family is critical for constitutive endocytosis of the type II TGF-(&bgr;) receptor J. Cell Sci., January 5, 2001; 114(9): 1777 - 1786. [Abstract] [PDF]
|
|
|
|
|
|
E. Santolini, C. Puri, A. E. Salcini, M. C. Gagliani, P. G. Pelicci, C. Tacchetti, and P. P. Di Fiore Numb Is an Endocytic Protein J. Cell Biol., December 11, 2000; 151(6): 1345 - 1352. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Benmerah, V. Poupon, N. Cerf-Bensussan, and A. Dautry-Varsat Mapping of Eps15 Domains Involved in Its Targeting to Clathrin-coated Pits J. Biol. Chem., February 4, 2000; 275(5): 3288 - 3295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J Modregger, B Ritter, B Witter, M Paulsson, and M Plomann All three PACSIN isoforms bind to endocytic proteins and inhibit endocytosis J. Cell Sci., January 12, 2000; 113(24): 4511 - 4521. [Abstract] [PDF]
|
|
|
|
|
|
B Gagny, A Wiederkehr, P Dumoulin, B Winsor, H Riezman, and R Haguenauer-Tsapis A novel EH domain protein of Saccharomyces cerevisiae, Ede1p, involved in endocytosis J. Cell Sci., January 9, 2000; 113(18): 3309 - 3319. [Abstract] [PDF]
|
|
|
|
 |
|
 A. Cadavid, A Ginzel, and J. Fischer The function of the Drosophila fat facets deubiquitinating enzyme in limiting photoreceptor cell number is intimately associated with endocytosis Development, January 4, 2000; 127(8): 1727 - 1736. [Abstract] [PDF]
|
|
|
|
|
|
J. A. Rosenthal, H. Chen, V. I. Slepnev, L. Pellegrini, A. E. Salcini, P. P. Di Fiore, and P. De Camilli The Epsins Define a Family of Proteins That Interact with Components of the Clathrin Coat and Contain a New Protein Module J. Biol. Chem., November 26, 1999; 274(48): 33959 - 33965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. Page, P. J. Sowerby, W. W.Y. Lui, and M. S. Robinson {gamma}-Synergin: An EH Domain–containing Protein that Interacts with {gamma}-Adaptin J. Cell Biol., September 6, 1999; 146(5): 993 - 1004. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Gaidarov and J. H. Keen Phosphoinositide–AP-2 Interactions Required for Targeting to Plasma Membrane Clathrin-coated Pits J. Cell Biol., August 23, 1999; 146(4): 755 - 764. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Ungewickell Wrapping the package PNAS, August 3, 1999; 96(16): 8809 - 8810. [Full Text] [PDF]
|
|
|
|
|
|
L. M. Traub, M. A. Downs, J. L. Westrich, and D. H. Fremont Crystal structure of the alpha appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly PNAS, August 3, 1999; 96(16): 8907 - 8912. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Marsh and H. T. McMahon The Structural Era of Endocytosis Science, July 9, 1999; 285(5425): 215 - 220. [Abstract] [Full Text]
|
|
|
|
|
|
V. Poupon, B. Begue, J. Gagnon, A. Dautry-Varsat, N. Cerf-Bensussan, and A. Benmerah Molecular Cloning and Characterization of MT-ACT48, a Novel Mitochondrial Acyl-CoA Thioesterase J. Biol. Chem., July 2, 1999; 274(27): 19188 - 19194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Okamoto, S. Schoch, and T. C. Sudhof EHSH1/Intersectin, a Protein That Contains EH and SH3 Domains and Binds to Dynamin and SNAP-25. A PROTEIN CONNECTION BETWEEN EXOCYTOSIS AND ENDOCYTOSIS? J. Biol. Chem., June 25, 1999; 274(26): 18446 - 18454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. K. Hussain, M. Yamabhai, A. R. Ramjaun, A. M. Guy, D. Baranes, J. P. O'Bryan, C. J. Der, B. K. Kay, and P. S. McPherson Splice Variants of Intersectin Are Components of the Endocytic Machinery in Neurons and Nonneuronal Cells J. Biol. Chem., May 28, 1999; 274(22): 15671 - 15677. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Laporte, R. H. Oakley, J. Zhang, J. A. Holt, S. S. G. Ferguson, M. G. Caron, and L. S. Barak The beta 2-adrenergic receptor/beta arrestin complex recruits the clathrin adaptor AP-2 during endocytosis PNAS, March 30, 1999; 96(7): 3712 - 3717. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Chen, V. I. Slepnev, P. P. Di Fiore, and P. De Camilli The Interaction of Epsin and Eps15 with the Clathrin Adaptor AP-2 Is Inhibited by Mitotic Phosphorylation and Enhanced by Stimulation-dependent Dephosphorylation in Nerve Terminals J. Biol. Chem., February 5, 1999; 274(6): 3257 - 3260. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Berlioz-Torrent, B. L. Shacklett, L. Erdtmann, L. Delamarre, I. Bouchaert, P. Sonigo, M. C. Dokhelar, and R. Benarous Interactions of the Cytoplasmic Domains of Human and Simian Retroviral Transmembrane Proteins with Components of the Clathrin Adaptor Complexes Modulate Intracellular and Cell Surface Expression of Envelope Glycoproteins J. Virol., February 1, 1999; 73(2): 1350 - 1361. [Abstract] [Full Text]
|
|
|
|
|
|
M. R. Torrisi, L. V. Lotti, F. Belleudi, R. Gradini, A. E. Salcini, S. Confalonieri, P. G. Pelicci, and P. P. Di Fiore Eps15 Is Recruited to the Plasma Membrane upon Epidermal Growth Factor Receptor Activation and Localizes to Components of the Endocytic Pathway during Receptor Internalization Mol. Biol. Cell, February 1, 1999; 10(2): 417 - 434. [Abstract] [Full Text]
|
|
|
|
|
|
A Benmerah, M Bayrou, N Cerf-Bensussan, and A Dautry-Varsat Inhibition of clathrin-coated pit assembly by an Eps15 mutant J. Cell Sci., January 5, 1999; 112(9): 1303 - 1311. [Abstract] [PDF]
|
|
|
|
|
|
H Boleti, A Benmerah, D. Ojcius, N Cerf-Bensussan, and A Dautry-Varsat Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants: clathrin-independent entry into cells and dynamin-dependent productive growth J. Cell Sci., January 5, 1999; 112(10): 1487 - 1496. [Abstract] [PDF]
|
|
|
|
 |
|
 D. T. Stimson, P. S. Estes, M. Smith, L. E. Kelly, and M. Ramaswami A Product of the Drosophila stoned Locus Regulates Neurotransmitter Release J. Neurosci., December 1, 1998; 18(23): 9638 - 9649. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Yamabhai, N. G. Hoffman, N. L. Hardison, P. S. McPherson, L. Castagnoli, G. Cesareni, and B. K. Kay Intersectin, a Novel Adaptor Protein with Two Eps15 Homology and Five Src Homology 3 Domains J. Biol. Chem., November 20, 1998; 273(47): 31401 - 31407. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. de Beer, R. E. Carter, K. E. Lobel-Rice, A. Sorkin, and M. Overduin Structure and Asn-Pro-Phe Binding Pocket of the Eps15 Homology Domain Science, August 28, 1998; 281(5381): 1357 - 1360. [Abstract] [Full Text]
|
|
|
|
|
|
D.-S. Lim, D. G. Kirsch, C. E. Canman, J.-H. Ahn, Y. Ziv, L. S. Newman, R. B. Darnell, Y. Shiloh, and M. B. Kastan ATM binds to beta -adaptin in cytoplasmic vesicles PNAS, August 18, 1998; 95(17): 10146 - 10151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Lohi, A. Poussu, J. Merilainen, S. Kellokumpu, V.-M. Wasenius, and V.-P. Lehto EAST, an Epidermal Growth Factor Receptor- and Eps15-associated Protein with Src Homology 3 and Tyrosine-based Activation Motif Domains J. Biol. Chem., August 14, 1998; 273(33): 21408 - 21415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Warren, F. A. Green, P. E. Stenberg, and C. A. Enns Distinct Saturable Pathways for the Endocytosis of Different Tyrosine Motifs J. Biol. Chem., July 3, 1998; 273(27): 17056 - 17063. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Wendland and S. D. Emr Pan1p, Yeast eps15, Functions as a Multivalent Adaptor That Coordinates Protein-Protein Interactions Essential for Endocytosis J. Cell Biol., April 6, 1998; 141(1): 71 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Benmerah, C. Lamaze, B. Begue, S. L. Schmid, A. Dautry-Varsat, and N. Cerf-Bensussan AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis J. Cell Biol., March 9, 1998; 140(5): 1055 - 1062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. J. Bryant and T. H. Stevens Vacuole Biogenesis in Saccharomyces cerevisiae: Protein Transport Pathways to the Yeast Vacuole Microbiol. Mol. Biol. Rev., March 1, 1998; 62(1): 230 - 247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Coda, A. E. Salcini, S. Confalonieri, G. Pelicci, T. Sorkina, A. Sorkin, P. G. Pelicci, and P. P. Di Fiore Eps15R Is a Tyrosine Kinase Substrate with Characteristics of a Docking Protein Possibly Involved in Coated Pits-mediated Internalization J. Biol. Chem., January 30, 1998; 273(5): 3003 - 3012. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Cupers, A. P. Jadhav, and T. Kirchhausen Assembly of Clathrin Coats Disrupts the Association between Eps15 and AP-2 Adaptors J. Biol. Chem., January 23, 1998; 273(4): 1847 - 1850. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Geli and H Riezman Endocytic internalization in yeast and animal cells: similar and different J. Cell Sci., January 4, 1998; 111(8): 1031 - 1037. [Abstract] [PDF]
|
|
|
|
|
|
P. Cupers, E. ter Haar, W. Boll, and T. Kirchhausen Parallel Dimers and Anti-parallel Tetramers Formed by Epidermal Growth Factor Receptor Pathway Substrate Clone 15 (EPS15) J. Biol. Chem., December 26, 1997; 272(52): 33430 - 33434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Yamaguchi, T. Urano, T. Goi, and L. A. Feig An Eps Homology (EH) Domain Protein That Binds to the Ral-GTPase Target, RalBP1 J. Biol. Chem., December 12, 1997; 272(50): 31230 - 31234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Salcini, S. Confalonieri, M. Doria, E. Santolini, E. Tassi, O. Minenkova, G. Cesareni, P. G. Pelicci, and P. P. Di Fiore Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module Genes & Dev., September 1, 1997; 11(17): 2239 - 2249. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. C. Dell'Angelica, C. E. Ooi, and J. S. Bonifacino beta 3A-adaptin, a Subunit of the Adaptor-like Complex AP-3 J. Biol. Chem., June 13, 1997; 272(24): 15078 - 15084. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Tebar, S. Confalonieri, R. E. Carter, P. P. Di Fiore, and A. Sorkin Eps15 Is Constitutively Oligomerized Due to Homophilic Interaction of Its Coiled-coil Region J. Biol. Chem., June 13, 1997; 272(24): 15413 - 15418. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. v. Delft, C. Schumacher, W. Hage, A. J. Verkleij, and P. M.P. v. B. e. Henegouwen Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin J. Cell Biol., February 24, 1997; 136(4): 811 - 821. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Tebar, T. Sorkina, A. Sorkin, M. Ericsson, and T. Kirchhausen Eps15 Is a Component of Clathrin-coated Pits and Vesicles and Is Located at the Rim of Coated Pits J. Biol. Chem., November 15, 1996; 271(46): 28727 - 28730. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kariya, S. Koyama, S. Nakashima, T. Oshiro, K. Morinaka, and A. Kikuchi Regulation of Complex Formation of POB1/Epsin/Adaptor Protein Complex 2 by Mitotic Phosphorylation J. Biol. Chem., June 9, 2000; 275(24): 18399 - 18406. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Fingerhut, K. von Figura, and S. Honing Binding of AP2 to Sorting Signals Is Modulated by AP2 Phosphorylation J. Biol. Chem., February 16, 2001; 276(8): 5476 - 5482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Rotem-Yehudar, E. Galperin, and M. Horowitz Association of Insulin-like Growth Factor 1 Receptor with EHD1 and SNAP29 J. Biol. Chem., August 24, 2001; 276(35): 33054 - 33060. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Scheele, C. Kalthoff, and E. Ungewickell Multiple Interactions of Auxilin 1 with Clathrin and the AP-2 Adaptor Complex J. Biol. Chem., September 21, 2001; 276(39): 36131 - 36138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. I. Slepnev, G.-C. Ochoa, M. H. Butler, and P. De Camilli Tandem Arrangement of the Clathrin and AP-2 Binding Domains in Amphiphysin 1 and Disruption of Clathrin Coat Function by Amphiphysin Fragments Comprising These Sites J. Biol. Chem., June 2, 2000; 275(23): 17583 - 17589. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |