©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Conformation-specific Anti-peptide Antibody to the -Type Platelet-derived Growth Factor Receptor Also Recognizes the Activated Epidermal Growth Factor Receptor (*)

(Received for publication, October 19, 1994; and in revised form, January 12, 1995)

Krishnasamy Panneerselvam (1)(§) Hugh Reitz (1) Shabbir A. Khan (2) Subal Bishayee (1)(¶)

From the  (1)Coriell Institute for Medical Research, Camden, New Jersey 08103 and (2)Infinity Biotech Research and Resource, Upland, Pennsylvania 19015

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Earlier we reported the generation of a conformation-specific antibody (Ab P2) to the beta-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.


INTRODUCTION

The earliest consequences of the interaction of a growth factor, such as platelet-derived growth factor (PDGF), (^1)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 beta-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 beta-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.


EXPERIMENTAL PROCEDURES

Materials

3,3`-Bis(sulfosuccinimido)suberate was obtained from Pierce. The B-B homodimeric form of PDGF was purchased from Amgen Biologicals (Thousand Oaks, CA). EGF was purified from mouse submaxillary glands and radiolabeled with I by the chloramine-T procedure(6) . Labeled ATP was prepared with P(i) and -Prep A kit (Promega, Madison, WI) according to the manufacturer's directions. Specific activity of [-P]ATP was adjusted to 40-60 Ci/mmol by adding unlabeled ATP(7) .

Cell Culture

Human MG-63 osteosarcoma, A431 epidermoid carcinoma cells, and murine NR-6 fibroblasts (EGF receptorless variants of 3T3) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Human SK-BR-3 breast adenocarcinoma cells overexpressing neu were grown in McCoy 5A modified medium containing 15% fetal bovine serum. Plasma membranes from these cells were prepared as described(7) .

Antibodies

Anti-peptide antibodies Ab P1, Ab P2, and Ab P3 are directed, respectively, to amino acid residues 1013-1024 (peptide 1), 964-979 (peptide 2), and 740-762 (peptide 3) of the cytoplasmic domain of the human beta-type PDGF receptor(8) . Ab Palpha1 and Ab Palpha2 are directed to amino acid residues 993-1009 (peptide alpha1) and 956-971 (peptide alpha2) of the cytoplasmic domain of the human alpha-type PDGF receptor, respectively(9) . All of these antibodies recognize human and murine PDGF receptors in immunoprecipitation and Western blotting(3, 10, 11) . These antibodies were generated in rabbits using high pressure liquid chromatography-purified peptides according to the method described previously(3) . Monoclonal antibody (mAb 425), raised against human A431 carcinoma cells and polyclonal antibody to denatured EGF receptor were gifts from Dr. M. Das and were developed as described (12, 13) . mAb 425 is directed to a peptide chain of the extracellular domain of the human EGF receptor and recognizes only the native receptor. The mouse monoclonal anti-phosphotyrosine antibody, 1G2, used for purification of tyrosine-phosphorylated proteins, was generated as described and coupled to activated Sepharose(7) .

Purification of Ab P2

The conformation-specific antibody, Ab P2, which binds specifically to tyrosine-phosphorylated receptors for PDGF and EGF was affinity-purified using isolated membranes. For this purpose, plasma membranes from A431 cells, which lack PDGF receptor but express 1-2 times 10^6 EGF receptors/cell were phosphorylated with unlabeled ATP in the absence of EGF for 2 h at 4 °C under autophosphorylation conditions(10) . After centrifugation to remove free ATP, the membranes containing phosphorylated EGF receptors were incubated for 4 h at 4 °C with Ab P2 antiserum in 20 mM HEPES, pH 7.4, 0.15 M NaCl, 1 mM vanadate, protease inhibitors (aprotinin, leupeptin, and phenylmethylsulfonyl fluoride). After centrifugation, the membrane pellet was washed three times to remove unbound antibody, and then the bound antibody was eluted with 0.1 M glycine HCl, pH 3.0, neutralized immediately with 3 M Tris, pH 8.8, and stored at -70 °C. The antibody was also purified using plasma membranes from NR-6 cells that express PDGF receptors (both alpha- and beta-type) but lack EGF receptor(14) . The purification procedure was the same as described above except that the membranes were phosphorylated with unlabeled ATP in the presence of PDGF BB.

Immunoprecipitation Technique

This was carried out as described(2) . Briefly, P-, S-, or I-labeled receptor preparation was incubated with the indicated antibody either in the absence or presence of cognate peptide for 4 h at 4 °C in 20 µl (unless otherwise indicated) of 20 mM HEPES, pH 7.4, 0.15 M NaCl, 0.2% Nonidet P-40, 1 mg/ml bovine serum albumin, 1 mM vanadate, protease inhibitors, and 40 mM phenyl phosphate. The immune complexes were isolated by incubating the mixture at 4 °C for 1 h with formaldehyde-fixed Staphylococcus aureus (The Enzyme Center, Boston, MA). After washing the bacterial pellets to remove unbound radioactivity, the bound radioactivity was eluted by boiling the pellets with SDS sample buffer and then subjected to SDS-PAGE (7.5% gel unless otherwise indicated). The gels containing I- or P-labeled receptors were dried and subjected to autoradiography at -70 °C with Kodak X-Omat AR film and DuPont intensifying screens. The gels containing S-labeled proteins were prepared for fluorography by immersion in acetic acid containing 2,5-diphenyloxazole, washed in water, dried, and exposed to x-ray films(3) .

Western Blot Analysis

This was carried out essentially as described previously except that the electrophoretically separated proteins were transferred to polyvinylidene difluoride membrane (Millipore Corp.) by electrophoresis at 4 °C overnight at 0.2 mA (3) .


RESULTS

We previously described an anti-peptide antibody (Ab P2) directed to a cytoplasmic domain of the human beta-type PDGF receptor (amino acid residues 964-979) that had very high affinity for the tyrosine-phosphorylated form of the receptor (M(r) 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.

Ab P2 Also Recognizes the EGF Receptor

In studies utilizing the human epidermoid carcinoma cell (A431), which is known to express 1-2 times 10^6 EGF receptors/cell, we were able to immunoprecipitate a P-labeled 170-kDa protein with Ab P2 (Fig. 1). The 170-kDa protein is not immunoprecipitated by any other anti-peptide antibodies to the alpha- or beta-type PDGF receptor; however, it is recognized by a monoclonal antibody (mAb 425) to an external peptide epitope of the human EGF receptor (Fig. 1). Furthermore, Ab P2 failed to immunoprecipitate the 170-kDa protein from the phosphorylated A431 receptor preparation from which the EGF receptor was removed by pretreatment with mAb 425 (not shown). As shown in Fig. 2, phosphorylation of A431 membranes with [-P]ATP in the presence of increasing concentrations of EGF resulted in increased immunoprecipitation of the 170-kDa protein by both Ab P2 and mAb 425. These data suggest that the 170-kDa protein recognized by Ab P2 is indeed the EGF receptor and not a truncated form of the PDGF receptor. In addition, the 170-kDa protein was also recognized in Western blot analysis by both Ab P2 and by an EGF receptor-specific antibody (Fig. 3). This suggests that there is direct interaction between Ab P2 and the EGF receptor and eliminates the possibility that the receptor precipitation is due to its tight association with an Ab P2-immunoreactive protein. The slightly higher molecular weight of the receptor in Fig. 3, lane2, is probably due to its phosphorylation, since phosphorylated protein has slower mobility than unphosphorylated protein.


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.



Only the Phosphorylated EGF Receptor Is Recognized by Ab P2

Ab P2 has very high affinity for the tyrosine-phosphorylated beta-type PDGF receptor and not for the unphosphorylated receptor(3, 4) . Furthermore, we demonstrated earlier that PDGF alone has no effect on the recognition of the receptor by Ab P2(3) , suggesting that ligand-induced receptor dimerization does not expose the antigenic epitope. We investigated whether this antibody also has similar specificity with respect to the EGF receptor. To test this, we studied the effect of unphosphorylated and phosphorylated EGF receptors on the immunoprecipitation of the P-labeled receptor by Ab P2. In this experiment, like the previous one (Western blotting, Fig. 3), EGF receptor in A431 cell membranes was phosphorylated in the absence of EGF by prolonged incubation at room temperature with unlabeled ATP. As shown in Fig. 4A, although more than 90% of the immunoprecipitation was blocked by 0.2 µg of the phosphorylated receptor preparation, no such effect could be seen with the unphosphorylated receptor preparation, indicating that the phosphorylated EGF receptor has very high affinity for Ab P2 compared with the unphosphorylated receptor. This was further confirmed by studies conducted with the EGF receptor covalently complexed with I-EGF (Fig. 4B). In this experiment, isolated membranes from A431 cells were either phosphorylated with unlabeled ATP in the absence of EGF or left untreated. Both the phosphorylated and control membranes were then incubated with I-EGF, chemically cross-linked with 3,3`-bis(sulfosuccinimido)suberate, and solubilized with detergent. The receptor-I-EGF complexes were then immunoprecipitated with Ab P2 in the presence of phenyl phosphate and subjected to SDS-PAGE and autoradiography. The results showed that when phosphorylated receptor was complexed with I-EGF, a 170-kDa (monomeric) and a 330-kDa (dimeric) complex were immunoprecipitated by Ab P2, and such immunoprecipitation was blocked by the cognate peptide, P2 (Fig. 4B, lanes1 and 2). However, when unphosphorylated receptor was used, the extent of immunoprecipitation of the receptor complex by Ab P2 was less than that seen with phosphorylated receptor (Fig. 4B, lane5). It should be noted that the extent of immunoprecipitation of the phosphorylated and unphosphorylated receptor complexes was the same when mAb 425 was used (Fig. 4B, lanes3 and 6), indicating that phosphorylation per se has no effect on the binding or covalent cross-linking of I-EGF to its receptor. Furthermore, the immunoprecipitation of the phosphorylated receptor complex but not the unphosphorylated receptor complex by Ab P2 also suggests that the phosphorylated receptor rather than the dimeric receptor is recognized by the antibody. We also conducted experiments with [S]methionine-labeled receptor. Extracts from [S]methionine-labeled A431 cells were purified with wheat germ agglutinin and then left untreated, incubated with 1 µM EGF alone, or subjected to phosphorylation by prolonged incubation with unlabeled ATP. Both the control and treated samples were immunoprecipitated with Ab P2 and then analyzed by SDS-PAGE/fluorography. Ab P2 immunoprecipitated the receptor only from the sample that had been phosphorylated with ATP and not from the untreated sample or from the sample that had been incubated with EGF (data not shown). These results confirm that, as with the PDGF receptor, Ab P2 recognizes the EGF receptor only after it is phosphorylated, and receptor phosphorylation rather than ligand-induced receptor dimerization unfolds the antigenic site.


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 nMI-EGF (3.5 times 10^5 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 times 10^3 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.

The Conformation-specific Antibody Is Directed to a Tripeptide Sequence, Asp-Glu-Glu

We investigated why an antibody directed to a peptide epitope of the PDGF receptor recognizes the EGF receptor in a conformation-specific fashion. A computer search revealed that in P2 peptide, there are two tripeptide sequences (Tyr-Gln-Gln and Asp-Glu-Glu) that are also present in the cytoplasmic domain of the EGF receptor. Tyr-Gln-Gln and Asp-Glu-Glu are located in amino acid residues 1148-1150 and 979-981 of the human EGF receptor, respectively(15) . A dipeptide sequence, Glu-Phe, of the P2 peptide is also present in the EGF receptor (amino acid residues 943-944). To investigate which of these peptide sequences generates the phosphorylation-induced conformation-specific antibody, we synthesized two short forms of P2, one having the sequence Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val (N-terminal peptide) and the other one with Val-Asp-Glu-Glu-Phe-Leu-Arg (C-terminal peptide). In addition, we also synthesized a 16-amino acid-long peptide with a Glu-Phe sequence; this peptide corresponds to amino acid residues 931-946 of the human EGF receptor(15) . Of these three peptides, only the C-terminal peptide with Asp-Glu-Glu blocks the immunoprecipitation of the P-labeled EGF receptor by the A431 membrane affinity-purified Ab P2 (Fig. 5). The inhibitory effect of this peptide is highly specific for Ab P2 since it had no effect on unrelated antibodies such as mAb 425 (data not shown). These results suggest that the conformation-specific antibody is directed to Asp-Glu-Glu. It should be mentioned in this connection that the alpha-type PDGF receptor, which is structurally related to the beta receptor but lacks the Asp-Glu-Glu sequence, is not recognized by Ab P2 (10, 11) .


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.



Affinity of Ab P2 for Asp-Glu-Glu Is Influenced by Its Chain Length

Since a phosphorylation-induced conformation of the receptors for PDGF and EGF is recognized by an antibody (Ab P2) directed to Asp-Glu-Glu, we investigated the role of the conformation of the antigenic peptide in Ab P2 recognition. For this purpose, we determined the affinities of the full-length P2 and two short forms of P2 for the antibody. The two truncated peptides are Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg (K-R peptide) and Val-Asp-Glu-Glu-Phe-Leu-Arg (V-R peptide; same as the C-terminal peptide of Fig. 5), which lack 5 and 9 amino acids, respectively, from the N terminus of P2. However, both contain the antigenic determinant, Asp-Glu-Glu. To determine the affinities of these peptides for Ab P2, the P-labeled EGF receptor from A431 cells or the PDGF receptor from NR-6 cells was immunoprecipitated with purified Ab P2 in the presence of increasing concentrations of a peptide. The immune complex was analyzed by SDS-PAGE/autoradiography, and the 170-kDa EGF receptor or the 180-kDa PDGF receptor band was densitometrically scanned. There was a gradual decrease in the intensity of the respective receptor bands with increasing peptide concentrations. A plot of the data revealed that a similar concentration of a peptide is required for 50% inhibition of both receptors. For full-length P2, a 50% inhibition of immunoprecipitation of the EGF and PDGF receptors was achieved at 3 and 4 ng/ml, respectively. However, for two truncated peptides, relatively high concentrations were required for 50% inhibition (Table 1). For K-R peptide, which lacks 5 amino acids from the N terminus, 6 µ/ml was required for 50% inhibition of both receptors, whereas 40 and 20 µ/ml of V-R peptide were needed for the EGF and PDGF receptors, respectively (Table 1). Since the antibody is directed to Asp-Glu-Glu, which is present in all three peptides, the drastic reduction of the affinity of peptides for Ab P2 with decreasing chain length strongly suggests that the initial interaction between the antibody and the antigenic determinant is further stabilized through the participation of neighboring amino acids.




DISCUSSION

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 beta-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 beta-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.


FOOTNOTES

*
This work was supported by Grant C9304 from the W. W. Smith Charitable Trust (to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

S. B. dedicates this paper to the memory of his long standing collaborator, friend, and wife, Dr. (Prof.) Manjusri Das.

§
Partially supported by a Scott fellowship.

To whom correspondence should be addressed: Coriell Inst. for Medical Research, 401 Haddon Ave., Camden, NJ 08103. Tel.: 609-757-9701; Fax: 609-964-0254.

(^1)
The abbreviations used are: PDGF, platelet-derived growth factor; EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; Ab, antibody; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We thank Drs. Gary Butler and Bruce Byrne for help with computer analyses.


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