(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 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 .
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) ()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) .
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 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.
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 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.
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 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
.
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--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).
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--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.
Figure 4:
Binding of Eps15 with the ear domain of
-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.''
Figure 5:
Binding of the COOH-terminal domain of
Eps15 to fusion proteins derived from -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).
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 -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.
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 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.
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 ( 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 and
2 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.
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. ()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.