(Received for publication, October 19, 1994; and in revised form, January 12, 1995)
From the
Earlier we reported the generation of a conformation-specific
antibody (Ab P2) to the -type platelet-derived growth factor
receptor (Bishayee, S., Majumdar, S., Scher, C. D., and Khan, S.(1988) Mol. Cell. Biol. 8, 3696-3702). Ab P2 is directed to a
16-amino acid peptide
(GluGly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-PheLeu-Arg) of the
cytoplasmic domain of the receptor, and it recognizes the
phosphorylated platelet-derived growth factor receptor but not the
unphosphorylated receptor. We now report that Ab P2 also interacts with
the epidermal growth factor receptor and that the recognition is
specific for a conformation induced by phosphorylation of the receptor;
however, Ab P2 is not directed to phosphotyrosine. Studies conducted
with P2-derived peptides suggest that the conformation-specific
antibody is directed to an acidic tripeptide, Asp-Glu-Glu, and this
sequence is also present in the cytoplasmic domain of the epidermal
growth factor receptor. With respect to the C terminus amino acid or
ATP-binding site, Asp-Glu-Glu is located in different regions in these
receptors; nevertheless, this tripeptide along with the surrounding
amino acids is cryptic in the unphosphorylated receptor, and tyrosine
phosphorylation uncovers this site. This suggests that the Asp-Glu-Glu
sequence may be important in receptor functions.
The earliest consequences of the interaction of a growth factor,
such as platelet-derived growth factor (PDGF), ()with its
receptor are the dimerization of the receptor followed by the
activation of its intrinsic tyrosine kinase
site(1, 2) . We have previously reported the
generation of a conformation-specific antibody to the
-type PDGF
receptor. This anti-peptide antibody (Ab P2), directed to amino acid
residues 964-979
(GluGly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-LeuArg) of the
cytoplasmic domain of the human
-receptor recognizes specifically
the phosphorylated receptor but not the unphosphorylated
receptor(3) . A similar finding has also been reported by
others(4) . This indicates that receptor phosphorylation
uncovers this peptide epitope and suggests a phosphorylation-induced
conformational change of the receptor. This is further supported by the
recent observations that only the phosphorylated receptors bind very
tightly with intracellular target molecules containing src homology 2 motifs and that such interactions culminate in DNA
synthesis and cell division (reviewed in (5) ).
Since autophosphorylation is important in receptor-mediated signal transduction and we have identified a peptide epitope in the PDGF receptor whose conformation is highly susceptible to the phosphorylation state of the receptor, we have extended our studies with Ab P2. We now report that this conformation-specific antibody also recognizes the EGF receptor and, interestingly, that the recognition is also phosphorylation-dependent. In addition, we present evidence to indicate that the antigenic determinant for this conformation-specific antibody is a tripeptide, Asp-Glu-Glu. This suggests that the peptide epitope surrounding this intracellular tripeptide is always cryptic in the PDGF and EGF receptor kinases and that receptor phosphorylation uncovers this sequence. Because of its specificity toward phosphorylated receptors, this conformation-specific antibody may be used in clinical settings to detect activated receptors in tumor samples.
We previously described an anti-peptide antibody (Ab P2)
directed to a cytoplasmic domain of the human -type PDGF receptor
(amino acid residues 964-979) that had very high affinity for the
tyrosine-phosphorylated form of the receptor (M
180,000) but not for the unphosphorylated receptor(3) .
We also showed that immunoprecipitation of the phosphorylated receptor
is not blocked by phenyl phosphate, an analog of phosphotyrosine,
indicating that the antibody is not directed to phosphotyrosine.
Figure 1:
Immunoprecipitation of a
170-kDa protein from A431 cells by Ab P2. Detergent-solubilized A431
membrane preparation (2 µg of protein) was phosphorylated with
[-
P]ATP in the presence of 1 µM EGF for 15 min at 4 °C under conditions as described under
``Experimental Procedures.'' After termination of the
reaction with EDTA, the
P-labeled proteins were purified
using anti-phosphotyrosine antibody-Sepharose and then subjected to
immunoprecipitation with indicated antiserum or nonimmune serum (Ni). Immunoprecipitation was also carried out with Ab P2 in
the presence of 100 ng of cognate peptide. The results of the
electrophoresis and autoradiography are
depicted.
Figure 2:
Phosphorylation of the 170-kDa protein
recognized by Ab P2 is EGF-dependent. Detergent-solubilized A431
membrane preparations were phosphorylated with
[-
P]ATP for 10 min at 4 °C in the
absence or presence of the indicated concentrations (Conc.) of
EGF. Following termination of the reaction with a solution containing
EDTA (10 mM) and unlabeled ATP (1 mM), the labeled
proteins were immunoprecipitated with Ab P2 antiserum or mAb 425 and
then subjected to electrophoresis and autoradiography. The region
containing the 170-kDa band was densitometrically
scanned.
Figure 3: The 170-kDa EGF receptor is recognized by Ab P2 in Western blot analysis. Wheat germ agglutinin-agarose-purified A431 membrane proteins were phosphorylated with unlabeled ATP for 30 min at room temperature or left untreated. The phosphorylated receptor preparation ((P)EGFR) (2 µg/lane) was subjected to Western blotting with a 1:25 dilution of Ab P2 antiserum either in the presence (lane1) or absence (lane2) of cognate peptide P2 according to procedures described under ``Experimental Procedures.'' Unphosphorylated receptor preparation (EGFR) (2 µg/lane) was also probed with Ab P2 antiserum (1:25 dilution) (lane3) and polyclonal antibody to denatured EGF receptor (1:1000 dilution) (lane4). The autoradiogram was exposed at -70 °C for 1 h for lane4 and 20 h for lanes1-3.
Figure 4:
Only phosphorylated EGF receptor is
recognized by Ab P2. A, immunoprecipitation of the P-labeled EGF receptor by Ab P2 is competed out by
phosphorylated and not by unphosphorylated EGF receptor. B,
phosphorylated EGF receptor ((P)EGFR) chemically cross-linked
with
I-EGF is immunoprecipitated by Ab P2. A,
for this experiment, wheat germ agglutinin-purified EGF receptor from
A431 membrane preparation was phosphorylated with unlabeled ATP or left
untreated as described in legend to Fig. 3.
P-Labeled A431 EGF receptor, purified with 1G2-Sepharose,
was then immunoprecipitated with Ab P2 antiserum in the absence (Control) or presence of the indicated amount of
phosphorylated or unphosphorylated receptor in a total volume of 20
µl and subjected to SDS-PAGE/autoradiography. B, for this
experiment, isolated membranes from A431 cells were phosphorylated with
unlabeled ATP for 2 h at 4 °C or left untreated. Following
centrifugation, the membrane pellets were incubated with 50 nM
I-EGF (3.5
10
cpm/ng) for 1 h at
4 °C in 20 µl of 20 mM HEPES, pH 7.4, 0.15 M NaCl, 1 mM vanadate, 1 mg/ml bovine serum albumin. After
centrifugation to remove free EGF,
I-EGF bound to the
receptor was covalently cross-linked by incubating the membranes with 1
mM 3,3`-bis(sulfosuccinimido)suberate as
described(2) . The Nonidet P-40-solubilized membranes (60
10
cpm for both phosphorylated and unphosphorylated
covalent complexes) were then subjected to immunoprecipitation with mAb
425 and also with Ab P2 antiserum either in the absence or presence of
cognate peptide P2 and then analyzed by 3.5-10% SDS-PAGE. The
170- and 330-kDa receptor-
I-EGF cross-linked complexes
are indicated by arrows.
The experiments described above were carried out with rabbit polyclonal antiserum that may contain antibodies directed to different epitopes within the antigenic peptide. To test whether the same antibody recognizes the phosphorylation-induced conformations of both the PDGF receptor and the EGF receptor, the antibody was affinity-purified using phosphorylated membranes from A431 cells that do not express the PDGF receptor (see Fig. 1). Like the antiserum, the affinity-purified Ab P2 also recognizes the tyrosine-phosphorylated form of both receptors (data not shown). In addition, the immunoprecipitation of the EGF receptor was competed out by excess phosphorylated PDGF receptor from murine NR-6 cells (an EGF receptor-negative mutant of 3T3 cells) and vice versa, suggesting that the same antibody interacts with both receptors.
Figure 5:
Immunoprecipitation of the EGF receptor by
Ab P2 is inhibited by a peptide containing the Asp-Glu-Glu sequence.
The P-labeled EGF receptor was immunoprecipitated with an
affinity-purified Ab P2 in the absence (Control) or presence
of 1.0 µg of N-terminal (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val)
or C-terminal (Val-Asp-Glu-Glu-Phe-Leu-Arg) peptide in a total volume
of 10 µl and then analyzed by
SDS-PAGE/autoradiography.
It is not unusual for an anti-peptide antibody to interact with different proteins. Such cross-reactivity is due to partial sequence identity among related proteins. However, the interesting fact about Ab P2 is that it not only recognizes the PDGF receptor in a phosphorylation-dependent fashion, it also displays similar binding characteristics with the EGF receptor, i.e. it recognizes only the phosphorylated EGF receptor and not the unphosphorylated receptor. It should be mentioned here that all of our immunological studies were carried out in the presence of phenyl phosphate, suggesting that the antibody recognizes the phosphorylated protein and not phosphotyrosine. In addition, the lack of an effect of EGF on the binding of the unphosphorylated receptor to the antibody eliminates the possibility that ligand-induced EGF receptor dimerization uncovers the antigenic epitope. A similar finding has also been demonstrated with the PDGF receptor(3) .
In P2, there are two tripeptide sequences
(Tyr-Gln-Gln and Asp-Glu-Glu) that are also present in the EGF
receptor. Our studies conducted with peptides containing either the
Tyr-Gln-Gln or the Asp-Glu-Glu sequence suggest that the
conformation-specific antibody is directed to Asp-Glu-Glu (Fig. 5). In the human EGF receptors and in the PDGF receptors,
the Asp-Glu-Glu sequence is present in the cytoplasmic domain, and it
is located 279 (amino acid residues 979-981) and 130 (amino acid
residues 974-976) amino acids from the C terminus, respectively.
However, irrespective of the location of Asp-Glu-Glu with respect to C
terminus amino acids in these receptor molecules, this tripeptide is
cryptic in unphosphorylated receptors, and phosphorylation uncovers
this sequence. This suggests that tyrosine phosphorylation induces
similar conformational changes in the PDGF and EGF receptor that result
in unmasking of the Asp-Glu-Glu epitope. It will be of interest to
identify the tyrosine residues that must be phosphorylated to bring
about such structural changes in the receptors. In the EGF receptors,
five autophosphorylation sites have been identified(16) . Out
of these sites, Tyr is nearest to the tripeptide
sequence, and it is 11 amino acids downstream of this sequence. In
addition, a putative acceptor site (Tyr
), which is 27
amino acids upstream of the tripeptide sequence, has recently been
reported(17) . In the
-type PDGF receptor, eight
autophosphorylation sites have been
identified(18, 19) . However, the two tyrosine
residues located within the P2 peptide at amino acids 966 and 970 are
not among these sites. The tyrosine residues nearest to the Asp-Glu-Glu
sequence that undergo autophosphorylation are 35 amino acids downstream
(Tyr
) and 137 amino acids upstream (Tyr
),
respectively, of the tripeptide sequence. Working with an anti-peptide
antibody (Ab 83) similar to our Ab P2, Fantl et al.(20) demonstrated that unlike the wild type receptor, a
murine
-type PDGF receptor mutant, Y825F, in which Tyr
(in humans, Tyr
) was converted to Phe, was not
recognized by the antibody. Based on this and related studies, it was
concluded that phosphorylation of Tyr
is essential for
the conformational change and for signal transduction as well. However,
since Y825F mutant receptor has considerably lower kinase activity
compared with the wild type receptor, it is possible that not all of
the acceptor tyrosine sites in the receptor undergo
autophosphorylation. Thus, it is an open question whether the failure
of the mutant receptor to bind to the antibody is due to lack of
receptor phosphorylation at Tyr
or at some other acceptor
site(s). This question can be resolved by using other Tyr
Phe
receptor mutants. In addition, since the conformation surrounding the
Asp-Glu-Glu sequence is highly susceptible to the phosphorylation state
of the receptor, it will be of interest to investigate whether
Asp-Glu-Glu or other amino acids surrounding this sequence play any
role in biological signal transduction. It should be mentioned in this
context that a PDGF receptor mutant with a truncation of 141 amino
acids (CT141) from the C terminus displays almost no kinase activity,
whereas a mutant with a deletion of 98 amino acids (CT98) retains its
kinase activity(21) . Since the Asp-Glu-Glu sequence is located
between 141 and 98 amino acids from the C terminus, it is retained in
the CT98 mutant (kinase-active) but not in the CT141 mutant
(kinase-inactive).
It should be mentioned that the Asp-Glu-Glu
sequence is also present in neu, a receptor tyrosine kinase
that is related to but distinct from the EGF receptor(22) .
Unlike the receptors for PDGF and EGF in which the Asp-Glu-Glu sequence
is intracellular, in neu it is extracellular (amino acid
residues 623-625) and near the transmembrane domain(22) .
However, the P-labeled 185-kDa neu from human
SK-BR-3 breast adenocarcinoma cells that overexpress neu(23, 24) is not recognized by Ab P2 (data not
shown). Lack of recognition of neu by Ab P2 is probably due to
the steric hindrance contributed by the oligosaccharide chains. In
fact, there are a number of potential N-linked glycosylation
sites in neu including a site 8 amino acids downstream of the
Asp-Glu-Glu sequence (22) . Alternatively, such lack of
recognition may be due to the absence of certain amino acids in neu that play an important role in stabilizing the interaction between
Asp-Glu-Glu and Ab P2. This is supported by our finding that truncation
of 5 amino acids (Glu-Gly-Tyr-Lys-Lys) from P2 peptide drastically
reduces its affinity for Ab P2; however, removal of 4 more amino acids
(Lys-Tyr-Gln-Gln) does not further significantly reduce the affinity of
the truncated V-R peptide for the antibody (Table 1). Thus, it
will be of interest to identify the amino acids within the pentapeptide
that influence the complex interaction between Asp-Glu-Glu and Ab P2.
In addition, studies using different EGF receptor mutants with or
without the tripeptide will give us an insight into this
antigen-antibody interaction.
Apart from its use as a biological tool in studying the structure-function relationship of receptors, this antibody can be of use in clinical settings. Since autophosphorylation is a consequence of receptor activation and Ab P2 recognizes only the phosphorylated receptor, it is capable of discriminating between activated and dormant kinases. This characteristic of Ab P2 may be exploited in detecting activated EGF receptor in biopsy samples derived from adenocarcinomas and gliomas. At least two other anti-peptide antibodies, one directed to the insulin receptor and the other one to neu, that recognize specifically the phosphorylated receptors have been reported (24, 25) . In the later case, the antibody is directed to a phosphotyrosine-containing peptide of neu and the EGF receptor. Although both Ab P2 and neu antibody recognize activated EGF receptor, there are certain differences between these two antibodies. Ab P2 is directed to a peptide epitope, whereas neu antibody is directed to a phosphopeptide. Second, Ab P2 recognizes both the activated PDGF and EGF receptors, whereas neu antibody interacts with activated EGF receptor and neu. Thus, Ab P2 in combination with neu antibody may be useful in clinical settings to detect specifically the activated EGF receptor in biopsy samples.
S. B. dedicates this paper to the memory of his long standing collaborator, friend, and wife, Dr. (Prof.) Manjusri Das.