THE JOURNAL
`OF BIOLOGICAL CHEMISTRY
`0 1991 by The American Society for Biochemistry and Molecular Biology. Inc
`
`Val. 266, No. 3, Issue of January 25, pp. 1716-1720, 1991
`Printed in (i S A .
`
`The Extracellular Domain of p185/neu Is Released from the Surface of
`Human Breast Carcinoma Cells, SK-BR-3*
`
`James R. ZabreckyS, Terence Lam, Sara J. McKenzie, and Walter Carney5
`Amlied bioTechnolom. Inc.. Cambridge. Massachusetts 02142 and $E. I. Du Pont deNemours and Co. Inc.,
`”.
`.
`Ndrth Billerica, Massachusetts 01862-
`
`(Received for publication, August 31, 1990)
`
`The human breast carcinoma cell line SK-BR-3, ex-
`presses the neu oncogene product, p185, which is a
`receptor tyrosine kinase. Using a double monoclonal
`antibody capture enzyme-linked immunosorbent assay
`for p185, activity was detected in conditioned media
`from cultures of SK-BR-3 cells. Two monoclonal anti-
`bodies specific for the extracellular domain of p185/
`neu immunoprecipitated a protein with a molecular
`mass of approximately 105 kDa. p105 was further
`shown to compete with p185 for binding to monoclonal
`antibodies and pulse-chase experiments indicate that
`it was generated by post-translational processing. Pep-
`tide maps showed that p105 and p185 are related
`polypeptides. Since p105 is close to the predicted size
`for the extracellular domain of p185/neu, we propose
`that SK-BR-3 cells specifically process and release this
`portion of the receptor into the medium. The release of
`the extracellular domain may have implications in on-
`cogenesis and its detection could prove useful as a
`cancer diagnostic.
`
`The neu oncogene (HER-2, c-erbB-2) is a member of the
`tyrosine protein kinase class
`of oncogenes which possess
`tumorigenic or transforming activity (1-3). The gene product
`shows structural and functional homology to growth factor
`receptors particularly the epidermal
`growth factor (EGF)’
`receptor. The most extensive
`homology is in the tyrosine
`kinase domain and it is this activity that is
`believed to be
`responsible for signal transduction and growth regulation.
`The neu oncogene product (p185) is a 185-kDa transmem-
`brane glycoprotein. From the deduced amino acid sequence it
`believed to
`possesses a cysteine-rich extracellular domain
`function in ligand binding, a single membrane-spanning do-
`main of about 23 amino acids and a cytoplasmic domain that
`possesses tyrosine protein kinase activity. The putative en-
`dogenous ligand has not been identified. p185/neu presumably
`exerts its effects on cell growth through activation of the
`tyrosine kinase activity; however, the mechanism by which
`this activity is regulated is not well understood. A single point
`mutation in the transmembrane region (4, 5) or simply over-
`expression of the neu gene (6) leads to full oncogenic activa-
`tion. Receptor dimerization has also
`been discussed
`as a
`
`* The costs of publication of this article were defrayed in part by
`the payment of page charges. This article must therefore be hereby
`marked “aduertisement” in accordance with 18 U.S.C. Section 1734
`solely to indicate this fact.
`$To whom correspondence should be addressed Applied bio-
`Technology, Inc., 80 Rogers St., Cambridge, MA 02142. Tel.: 617-
`492-7289.
`’ The abbreviations used are: EGF, epidermal growth factor;
`ELISA, enzyme-linked immunosorbent assay; SDS-PAGE, sodium
`dodecyl sulfate-polyacrylamide gel electrophoresis.
`
`mechanism for enzymatic, and hence oncogenic, activation
`(7, 8).
`There is mounting evidence that alterations in the structure
`and/or expression of the neu gene product play a role in
`human malignancies (9-11). Analysis of a series of human
`mammary tumor cell lines showed overexpression of the neu
`gene as well as elevated levels of the gene product, p185 (12).
`Gene amplification of neu was found in a primary mammary
`carcinoma (13) as well. The neu gene was amplified in 25-
`30% of human breast and ovarian cancers and this correlated
`with poor prognosis
`(10). Overexpression of this gene was
`found to be the second most predictive independent variable
`for patient survival after nodal status. The neu gene product
`thus appears to have a significant influence on cellular growth
`control and its quantitation could have important prognostic
`value.
`We have used a series of neu-specific monoclonal antibodies
`(14) to examine expression of the neu gene product in the
`human mammary adenocarcinoma cell line, SK-BR-3. Using
`a capture ELISA, we were able to detect soluble p185/neu
`activity in clarified, conditioned media from these cells. Fur-
`ther analysis revealed that this activity is associated with a
`neu-related protein having a molecular mass of about 105
`kDa, a value that closely matches the predicted size of the
`extracellular domain of p185/neu (-118 kDa). Several lines
`of evidence are presented which demonstrate that this is in
`fact the extracellular domain and that it is released from the
`surface of SK-BR-3 cells. We will discuss the possible signif-
`icance of this finding in cell transformation and potential
`applications in cancer diagnostics.
`
`EXPERIMENTAL PROCEDURES
`Materials-SK-BR-3 cells were grown in Dulbecco’s modified Ea-
`gle’s medium containing 10% fetal calf serum. PB3, NB3, and TA1
`monoclonal antibodies were prepared as described (14). TA1 was
`biotinylated using NHS-biotin from Sigma. Biotinylated c-neu (Ab-
`1) rabbit polyclonal antibody was purchased from Oncogene Sciences,
`[%]Cysteine was from Du Pont-New England Nuclear.
`Radioimmunoprecipitation-A subconfluent monolayer of cells in
`a IO-cm Petri dish was labeled overnight with 500 pCi of [35S]cysteine
`in 10 ml of cysteine-free Dulbecco’s modified Eagle’s medium plus
`10% fetal calf serum. The supernatant was collected and clarified by
`centrifugation at 15,000 X g for 15 min. The cells were scraped from
`the plate, washed 2 X with phosphate-buffered saline (10 mM phos-
`phate, 150 mM NaC1, pH 7.4), and lysed with IP (immunoprecipita-
`tion) buffer (1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate,
`10 mM Tris, 0.65 M NaC1, pH 7.2) containing the protease inhibitors
`phenylmethylsulfonyl fluoride (1 mM) and soybean trypsin inhibitor
`(100 pg/ml). For immunoprecipitation, samples were incubated over-
`night at 4 “C with 5 pg of antibody. Each sample was then incubated
`for 2 h at 4 “C with 50 pl of a 1:l slurry of protein A-Sepharose
`(Pharmacia LKB Biotechnology Inc.). In the case of TA1, which is
`an IgG,, the protein A-Sepharose was first mixed with 20 pg of a
`capture antibody, rabbit anti-mouse IgG, (Organon Teknika). The
`protein A-Sepharose was pelleted by centrifugation, washed 4 X with
`
`1716
`
`IMMUNOGEN 2050, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Lysate
`
`1717
`
`condilbned
`Media -
`
`- 2 0 0
`
`'"I - 9 7
`
`- 6 9
`
`- 4 5
`
`Anllbody 2
`I-
`FIG. 1. Immunoprecipitation of lysate and conditioned me-
`dia. [~'SSjCysteine-labeled whole cell lysate from neu-transfected NIH
`3T3 cells and clarified conditioned medium from SK-RR-3 cells were
`prepared and immunoprecipitated as described under "Experimental
`Procedures." Two monoclonal antibodies (TAl and PH3) which are
`specific for the extracellular domain of plR5/nru were used. Immu-
`noprecipitated samples were separated by SDS-PAGE and fluoro-
`graphed.
`
`p185/neu Extracellular Domain from SK-BR-3 Cells
`IP buffer and once with Tris-buffered saline (10 mM Tris, 150 mM
`NaCI, pH 8.2). The pelleted Sepharose was then boiled for 5 min in
`SDS sample buffer and run on SDS-polyacrylamide gels (15). The
`gels were fixed, treated with ENI'HANCE (Du Pont-New England
`Nuclear), dried, and fluorographed.
`neu Captuw ELISA-96-Well microtiter plates (Nunc) were coated
`with NR3 (20 pg/ml in 50 mM sodium carbonate, pH 9.6; 100 pl/well)
`for 2 h at 37 "C. The plate was washed 3 X with wash buffer (10 mM
`phosphate, pH 7.4, 150 mM NaCI, 0.05% Tween). 100 pl of sample to
`be assayed, diluted in 50% normal goat serum, was added to each well
`then incubated overnight at 4 "C. The plate was again washed 3 X
`with wash buffer, and 100 pl of biotinylated TA1 (0.88 pg/ml in 50%
`normal goat serum) added to each well and incubated for 2 h at 37 "C.
`Following another wash, 100 pl of avidin-horseradish peroxidase (1
`pg/ml in 10% normal goat serum) was added incubated for 1 h
`at
`37 "C, then the plate
`was developed for 5 min
`using tetra-
`methylbenzidine (Sigma) and stopped with H2SOI. The absorbance
`was measured at 450 nm. A whole cell lysate prepared from a neu-
`transfected NIH 3T3
`to
`cell line designated 17-3-1 (14) was used
`generate a standard curve using an arbitrary definition for units.
`Pub-chase-A nearly confluent 10-cm plate of SK-BR-3 cells was
`labeled overnight as described above. Aliquots were collected over
`time and stored at -80 "C. Samples were then thawed, clarified by
`centrifugation, and immunoprecipitated with the PB3
`monoclonal
`antibody. These were divided and counted for ""S or separated by
`SDS-PAGE and fluorographed.
`Antihody Competition-An ELISA format similar to that described
`ahove was used to demonstrate competition
`for antibody binding
`between the two antigens p105 and ~18.5. p185 was prepared as a cell
`lysate from 17-3-1 cells. p105 was prepared from SK-BR-3 culture
`supernatants that had
`been clarified
`then concentrated 10-fold in
`Amicon concentrator using a YM-10 membrane (Amicon). Units of
`new activity were determined by capture ELISA. Plates were coated
`with NR3 as described above. A constant, near-saturating amount of
`plA5 was added to each well. This was followed by the addition of
`increasing amounts of p105 antigen up to a 10-fold excess over p185.
`Duplicate samples were developed using one of two biotinylated
`detection antibodies: TA1 which recognizes the extracellular domain
`or c-neu which recognizes the cytoplasmic domain.
`Peptide Map-""S-Labeled SK-RR-3 culture supernatants and 17-
`3-1 cell lysates were prepared as described, immunoprecipitated with
`
`the PR3 antibody, and separated on a 6% SDS-PAGE. Using Rainbow
`M, markers (Amersham Corp.) as a guide, hands were cut from the
`gel at the appropriate molecular weight for p105 and p185. The gel
`slices were minced with a razor blade, placed
`in a microcentrifuge
`tube along with 2.5 pg of Endoproteinase Glu-C (Boehringer Mann-
`heim) in 40 pI of 25 mM ammonium bicarbonate, pH
`7.8, and
`incubated for 1 h a t 37 "C. SDS sample buffer was added, the mixture
`a 15% SDS gel. The gel was
`boiled for 5 min then applied to
`fluorographed as described above.
`
`TABLE I
`plRfi/neu ELlSA actioity in SK-RR-.? crl1.q
`ELISA activity from one IO-cm plate ( 3 X lofi cells) of SK-RR-3
`cells after 3 days in culture. Activity is expressed as arbitrary units
`relative to a whole cell lvsate standard.
`I:nits ( x 10-9
`4.3
`3.9
`0.42
`
`Total activity
`Cell-associated
`Media
`
`'; of total
`100
`90
`10
`
`115 kDa and hence will be referred to as plO5. This value is
`close to that predicted for the extracellular domain of pl8Fi.
`RESULTS
`about 118 kDa. The three neu-specific monoclonals used in
`It has been previously demonstrated that SK-BR-3 cells
`these experiments (TA1 and NB.3 in the capture ELISA; TA1
`
`overexpress pl85/neu. We have confirmed this result by meas-
`and PB3 in immunoprecipitation) have been shown to rec-
`uring p185 activity in whole cell lysates of SK-BR-3 cells by
`ognize independent epitopes on the extracellular domain of
`means of a capture ELISA. Immunoprecipitation using a neu-
`pl85/neu (14).
`specific monoclonal antibody also revealed the presence of a
`T o determine if p105 was generated by de nouo synthesis
`band at 185 kDa (Fig. 1; lane I ). neu immunoactivity was also
`or by post-translational processing of p185, a pulse-chase
`experiment was performed. A confluent 10-cm plate of SK-
`detected in the conditioned media from cultures of SK-BR-3
`cells. Table I shows the relative activity from the neu capture
`BR-3 cells was labeled with ["S]Cys for 24 h. The medium
`ELISA which is cell associated compared to that detected in
`was replaced with unlabeled medium, and small aliquots were
`the conditioned media. We found that 10% of the total activity
`collected over time. Each sample was clarified by centrifuga-
`produced in a
`confluent 10-cm plate of SK-BR-3 cells is
`tion, immunoprecipitated with the PR3 monoclonal then
`present in the media following three days in culture. Prior to
`either counted for :'"S or separated by SDS-PAGE and fluo-
`analysis, the conditioned media was always clarified by cen-
`rographed. Fig. 2 shows neu-specific immunoprecipitable
`trifugation at 15,000 X g for 15 min. This suggests that the
`counts increased over the course of 54 h and this corresponded
`activity is in soluble form and not simply derived from mem-
`to an increase in the intensity of the bands at 105 kDa (see
`brane fragments of damaged or dead cells.
`inset). If this material was synthesized from an alternate
`To further characterize the activity, 1 ml of clarified con-
`message, there should be no increase in incorporation since
`ditioned media was immunoprecipitated with two monoclonal
`the labeled amino acid had been removed. On the contrary,
`antibodies, TA1 and PB3. Fig. 1, lanes 2 and 3, shows that
`p105 that had been labeled during the pulse continued to
`using either antibody the predominant immunoprecipitable
`accumulate in the media over time. Moreover, Northern
`bands migrate with an apparent molecular mass range of 97-
`analysis reported by others (12) and confirmed by our own
`
`IMMUNOGEN 2050, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`1718
`
`p185/neu Extracellular Domain from SK-BR-3 Celk;
`4000 1
`
`3000 -
`
`FIG. 2. Pulse-chase experiment
`showing release of p105 from SK-
`BR-3 cells. A near-confluent culture of
`SK-BR-3 cells was labeled overnight
`E 2ooo-
`with [""Sjcysteine. The medium was re-
`moved and replaced with unlabeled me- o
`dium (time = 0). Aliquots were collected
`over time and immunoprecipitated with
`the monoclonal antibody PR3. Duplicate
`samples were counted for ""S or sepa-
`rated by SDS-PAGE and
`fluorographed
`( inset ) .
`
`-
`
`1000
`
`0
`
`0
`
`2 4
`
`
`
`1
`1 2
`
`o 3 a
`26 3 2 5 4
`Time (hr)
`
`I
`
`I
`3 6
`Time (hr)
`
`4 8
`
`1 2 3
`I -., m
`
`- 2 0 0
`
`- 9 7
`
`- 6 9
`
`- 4 5
`
`p 1 0 5 .
`
`w
`
`sized
`experiments (data not shown) detected no aberrantly
`neu mRNA. Therefore, p105 appears to be generated as the
`result of post-translational processing, presumably proteolysis
`of cell surface p185/neu.
`Immunoprecipitation of this putative extracellular domain
`from SK-BR-3 conditioned medium generally yields a rather
`diffuse band at around 105 kDa (see Figs. 1-3). In fact, this
`of the
`often appears as a broad doublet with the intensity
`lower band varying from one preparation to the next. The
`same result is sometimes seen with the intact receptor, p185,
`immunoprecipitated from cell lysates. This heterogeneity
`could be due either to differences in glycosylation, since the
`extracellular domain is thought to be heavily glycosylated or
`to proteolytic processing or degradation.
`Immunological analysis was used to confirm the relation-
`ship of p105 to the neu gene product. If p105 is truly derived
`from the extracellular domain of p185/neu it should be com-
`
`petitive with the intact receptor for binding to the neu-specific
`antibodies. This was tested in two ways. First, immunoprecip-
`itation was used to show that a 10-fold excess of p185 could
`compete with radiolabeled p105 for binding to the monoclonal
`antibody, PB3. p185 as part of a whole cell lysate from 17-3-
`1 cells (see "Experimental Procedures") and :'%-labeled p105
`from SK-BR-3 cell conditioned medium were prepared and
`FIG. 3. Competitive immunoprecipitation of plOS. SK-RR-3
`assayed for activity by ELISA. Antibody was titrated to ensure
`conditioned medium was labeled overnight with ["Slcysteine. Unla-
`that its concentration was limiting in the immunoprecipita-
`beled p18.5 was prepared as a whole cell lysate from 15-3- 1 cella. Unit-q
`tion. Fig. 3, lane 1, shows an immunoprecipitation of the
`of activity were determined by capture EIJSA. The concentration of
`labeled conditioned media. Lane 2 shows that when the anti-
`monoclonal antibody PR3 was titered SO that it would he limiting
`body/antigen mixture is co-incubated with a 10-fold excess of
`with respect to antigen. Imne I shows a straight immunoprecipitation
`of labeled conditioned media. Lune 2 was immunoprecipitated under
`unlabeled p185, the p105 band disappears. A comparable
`the same conditions except that a
`IO-fold excess of unlaheled p185
`amount of nonrecombinant NIH 3T3 cell lysate had no effect
`was added. To control for nonspecific inhihition, the immunoprecip-
`(lane 3). This experiment indicates that an excess of p185
`itation was also done in the presence of a comparahle amount of cell
`specifically competes with the p105 antigen for binding to the
`lysate from nontransfected NIH 3T3 cells (lane 3 ) .
`mu-specific monoclonal.
`The narrowest interpretation of the above experiment is
`that the monoclonal antibody has a greater affinity for p185
`than for p105. This is not surprising since
`p185 was
`the
`original antigen to which the monoclonal was raised. There-
`fore, it was important to demonstrate the
`reverse competition,
`that is, that an excess of p105 effectively competes off p185.
`However, it was difficult to prepare sufficient quantities of
`labeled p105 a t high enough concentration to perform this
`experiment, so a capture ELISA format was used
`instead.
`Antigen was captured with NB3 as in the standard assay. T o
`
`- 3 0
`"
`
`distinguish between captured p105 and p1t35, a different de-
`tector antibody, c-mu, which is specific for the C terminus of
`p185/neu was used. The specificity was confirmed by testing
`17-3-1 cell lysates (p185) and SK-RR-3 media ( ~ 1 0 5 ) samples
`in this assay. c-neu gave a positive signal
`for p185 but was
`negative for p105, whereas TA1 produced a positive signal for
`both antigens (data not shown). For the competition experi-
`ment, a constant amount of p185 antigen was added to each
`well. Increasing levels of p105 antigen up to a 10-fold excess
`was then added and the ELISA was developed in the standard
`
`IMMUNOGEN 2050, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`a0
`
`0
`8
`
`0
`A
`
`1
`
`.01
`
`. . ....
`10
`
`I,
`
`. . "4
`
`100
`
`SK-BR-3 Celb
`p185/neu Extracellular Domain from
`
`
`1719
`way using either biotinylated c-mu or TA1. Using the signal
`the bound antigens. The signal remained near the maximal
`in the absence of any added p105 as loo%, the results are
`level (triangles). These results indicated that p105 can effec-
`plotted as the percent of p185 bound versus the ratio of p105
`tively compete with p185 with near equal affinity.
`to p185 in each sample well (Fig. 4). As the level of competing
`Finally, both antigens were subjected to peptide mapping
`antigen was increased the signal decreased to as low as 30%
`analysis. "S-Labeled SK-BR-3 conditioned medium and 17-
`of control at a 10-fold excess of competitor (open squares).
`3-1 cell lysates were immunoprecipitated with PR3 and sep-
`Under ideal conditions where all antigens have equal affinities
`arated by SDS-PAGE. The bands corresponding to the ap-
`for the antibodies, one would expect to see about 10% of the
`propriate molecular weight
`were extracted from the gel,
`p185 remaining bound in the presence of 10-fold excess com-
`minced, digested with Endoproteinase Glu-C (V8 protease)
`petitor. T o control for nonspecific inhibition by other com-
`and then separated on
`a second SDS gel. The results are
`ponents in the p105 preparation, an identical series of samples
`shown in Fig. 5. Many of the peptide bands line up in the two
`was developed with the TA1 antibody which detects either of
`samples suggesting that p105 and p185 are related proteins.
`The extra bands seen
`in the pl85 sample were presumably
`120 -
`derived from the cytoplasmic domain which is predicted to be
`absent in p105.
`100 -
`a 80 -
`3
`DISCUSSION
`.-
`u) 60 -
`We have described a p185/mu immunoreactive polypeptide
`m
`that is found in the media of cultured SK-BR-3 cells. This
`40 -
`protein has an apparent molecular mass of 105 kDa and is
`20 -
`cross-reactive with three monoclonal antibodies that are spe-
`cific for independent epitopes on the extracellular domain of
`TA1 Antibody
`0 ! . .
`, . . . . . . . . I
`p185/mu. By two methods, we have demonstrated that this
`protein is competitive with p185 for binding to mu-specific
`.1
`1
`I P 1 0 5 I I I P 1 8 5 1
`
`monoclonal antibodies and peptide mapping showed that p105
`FIG. 4. Competitive ELISA. SK-BR-3 cell conditioned media
`and p185 are related polypeptides. Pulse-chase results suggest
`and 17-3-1 cell lysate were prepared and assayed for activity by the
`that p105 was generated by post-translational processing. We
`standard neu ELISA (see "Experimental Procedures"). For the com-
`and others have been unable to detect an alternate transcript
`petitive assay, an ELISA plate was coated with monoclonal antibody
`that would account for this form as is true for A431 cells
`NR3 and a constant, near saturating level of p185 antigen was added
`to each well. Each sample was then co-incubated with an increasing
`which secrete high levels of a truncated EGF receptor (16,
`concentration of competing antigen, p105, up to a 10-fold excess. The
`17). On the basis of this evidence, we contend that plO5 is
`detector antibody was either TA1 (detects p105 and p185) or c-mu
`the extracellular domain of p185/neu and is released from the
`(only detects p185). The ELISA was developed as described. The
`surface of SK-BR-3 cells probably as the result of proteolysis.
`signal obtained in the absence of added competing antigen was defined
`The mechanism by which m u and other receptor tyrosine
`as 100% p185 hound and all values were normalized
`to this level.
`kinases induce transformation is not well understood. How-
`Data are plotted as the percent of p185 bound uersuv the ratio of
`p105 to p185 in the sample well. The signal with the TA1 antibody
`of the tyrosine kinase
`ever, it is clear that activation
`is
`stays near the saturating level, indicting that there is no nonspecific
`essential and it now appears that this activity can he induced
`inhihition from the p105 preparation.
`in several different ways. The role of overexpression of recep-
`tor tyrosine kinases has been well documented (18, 19). The
`binding of ligand is certainly fundamental in the activation
`and modulation of kinase activity but, in the case of m u , the
`endogenous ligand has not yet been identified. Mutation, as
`in the replacement of Val with Glu in
`the transmembrane
`domain (4), results in constitutive activation of the tyrosine
`kinase and transforming activity. Finally, many experiments
`have suggested a role for the extracellular domain in receptor
`regulation. The oncogenic form of EGF receptor (v-erhB)
`resembles a truncated receptor lacking i t s extracellular do-
`main (16). This form possesses tyrosine kinase activity that
`may be constitutively active (20). Furthermore, exposure to
`EGF was shown to induce an N-terminal truncation
`in the
`intact EGF receptor in a protease dependent manner and it
`was proposed that this form could possess altered intracellular
`signaling capacity (21). Expression in chicken fibroblasts of a
`i t s ligand binding domain
`truncated EGF receptor lacking
`resulted in a weak but constitutive oncogenic activation (22).
`Similar experiments with m u have produced more dramatic
`results. Expression of N-terminally truncated neu in NIH
`3T3 cells resulted in a 10-fold greater transforming activity
`compared to the full length gene (6). Cells transfected with
`this construct were also the most potent in inducing tumors
`in nude mice.
`that the
`Much of the evidence discussed above suggests
`remaining cell-associated cleavage product, made up of the
`transmembrane and cytoplasmic domains, represents an ac-
`tive and oncogenic form of the tyrosine kinase. Transforma-
`
`FIG. 5. Peptide map of p185 and p105. ("SSjCysteine labeled
`SK-RR-3 supernatant and 17-3-1 cell lysate were prepared as de-
`scribed. Each was immunoprecipitated with monoclonal antibody
`PR3 and separated on a 6% SDS-PAGE. Slices of the gel at the
`appropriate molecular weight for p185 and plOR were cut from the
`gel, minced with a razor blade, and digested with Endoproteinase
`Glu-C as described under "Experimental Procedures." Digestion mix-
`tures were separated on a 15% SDS gel and fluorographed.
`
`p185 p1Os 1
`
`I ' - 1 4
`
`IMMUNOGEN 2050, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`1720
`
`Acknowledgments-We would like to acknowledge Paula Marks
`and Zari Nakhost for their technical assistance. We also thank Arthur
`Bruskin for his helpful suggestions and critical review of this manu-
`script.
`
`Note Added in Proof-During
`the preparation of this manuscript
`we became aware of a similar observation by Lin and Clinton (Lin,
`Y. J. and Clinton, G. M. (1991) Oncogene 6, in press) of a soluble
`form of p185/neu released from breast carcinoma cells.
`
`p185/neu Extracellular Domain from SK-BR-3 Cells
`tion due to overexpression could simply result from a propor-
`tional increase in the absolute number of truncated receptors.
`Ligand binding or mutation in the transmembrane
`region
`could induce a conformational change rendering the receptor
`more susceptible to proteolysis and hence create a transform-
`ing molecule. It seems plausible that proteolytic release of the
`extracellular domain is part of a mechanism by which the
`tyrosine kinase becomes activated. This process could involve
`a specific but as yet unidentified membrane-associated pro-
`REFERENCES
`tease. Such a protease could be subject to its own set of
`1. Bargmann, C. I., Hung, M.-C., and Weinberg, R. A. (1986) Nature
`regulatory constraints and also provide a means for cross-talk
`319,226-229
`between different signal transduction systems. To determine
`2. Coussens, L., Yang-Feng, T. L., Liao, Y.-C., Chen, E., Gray, A.,
`if cleavage of the extracellular domain which we have observed
`McGrath, J., Seeburg, Ph.H., Libermann, T. A., Schlessinger,
`in SK-BR-3 cells is in this way related to their transformed
`J., Franke, U., Levinson, A,, and Ullrich, A. (1985) Science
`230,1132-1139
`state will require further experimentation.
`3. Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K., Normura, N.,
`Proteolysis is likely to be important in the normal process
`K. (1986) Nature
`Miyajima, N., Saito, T., and Toyoshima,
`of receptor down-regulation. All known receptor tyrosine
`319,230-234
`kinases are down-regulated in response to the binding
`of
`4. Bargmann, C. I., Hung, M. C., and Weinberg, R. A. (1986) Cell
`ligand. From studies with receptor mutants, this process has
`45,649-657
`5. Bargmann, C. I., and Weinberg, R. A. (1988) EMBO J. 7, 2043-
`been shown
`to require an active tyrosine kinase
`(23-25).
`2052
`However, in the case of the c-fms proto-oncogene product,
`6. DiFiore, P. P., Pierce, J. H., Kraus, M. H., Segatto, O., King, C.
`which is the CSF-1 receptor, treatment of cells with phorbol
`R., and Aaronson, S. A. (1987) Science 237, 178-182
`ester induced cleavage and release of the extracellular domain
`7. Steinberg, M. J. E., and Gullick, W. J . (1989) Nature 339, 587
`(24). This process, which involves a specific protease activated
`8. Weiner, D. B., Liu, J., Cohen, J. A,, Williams, W. V., and Green,
`M. I. (1989) Nature 339, 230-231
`by protein kinase C, was proposed as an alternate route to
`9. Paik, S., Hazan, R., Risher, E. R., Sass, R. E., Fisher, B., Red-
`ligand induced receptor down-regulation that is independent
`mond, C., Schlessinger, J., Lippman, M. E., and King, C. R.
`of tyrosine kinase activity. In preliminary studies in our
`(1990) J. Clin. Oncol. 8, 103-112
`laboratory, exposure of SK-BR-3 cells to phorbol ester re-
`0. Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J . A., Wong, S.
`1
`G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich,
`sulted in a 3-4-fold increase in neu immuno-reactivity in the
`A., and Press, M. F. (1989) Science 244, 707-712
`cell culture medium (data not shown). The present finding
`1. Tandon, A. K., Clark, G. M., Chamness, G. C., Ullrich, A., and
`that 10% of the extracellular domain is released in the absence
`McGuire, W. L. (1989) J. Clin. Oncol. 7, 1120-1128
`of added mitogen is not well understood. It is possible that
`2. Kraus, M. H., Popescu, N. C., Amsbaugh, S. C., and King, C. R.
`this is part of a ligand independent mechanism for normal
`(1987) EMBO J. 6, 605-610
`receptor turnover.
`3. King, C. R., Kraus, M. H., and Aaronson, S. A. (1985) Science
`The detection of the extracellular domain of p185/neu has
`229.974-976
`14. McKenzie, S. J., Marks, P. J., Lam, T., Morgan, J., Panicali, D.
`potential diagnostic and prognostic value. Amplification and/
`L., Trimpe, K. L., and Carney, W. P. (1989) Oncogene 4, 543-
`or overexpression of the gene has been correlated with human
`584
`.
`, ~
`malignancies. In a recent study by Hayes et ~ l
`the neu
`15. Laemmli, U. K. (1970) Nature 227, 680-685
`capture ELISA was used to detect a neu-related protein in
`16. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A.,
`the plasma of women with breast cancer. Significantly higher
`Tam, A. W., Lee, J., Yarden, Y., Libermann, T. A., Schlessin-
`ger, J., Downward, J., Mayes, E. L. V., Whittle, N., Waterfield,
`circulating levels of this activity were found in patients with
`M. D., and Seeburg, P. H. (1984) Nature 309, 418-425
`metastatic disease as compared to normal control
`subjects.
`17. Weber, W., Gill, G. N., and Spiess, J. (1984) Science 224, 294-
`Biochemical analysis of this circulating activity is underway.
`297
`It is an interesting possibility that this immuno-reactivity
`18. Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203-212
`represents the extracellular domain which is shed from tumors
`19. Yarden, Y., and Ullrich, A. (1988) Annu. Reu. Biochem. 57,443-
`478
`overexpressing neu.
`20. Kris, R. M., Lax, I., Gullick, W., Waterfield, M. D., Ullrich, A,,
`Much additional work is required in order to establish a
`Fridkin, M., and Schlessinger, J. (1985) Cell 40, 619-625
`role for proteolysis in kinase activation versus normal receptor
`21. Decker, S. J. (1989) J. Biol. Chem. 264, 17641-17644
`down-regulation. It will be interesting to identify and explore
`22. Khazaie, K., Dull, T. J., Graf, T., Schlessinger, J., Ullrich, A.,
`Beug, H., and Vennstrom, B. (1988) EMBO J. 7, 3061-3071
`the effect of specific protease inhibitors on kinase activity
`23. Chou, C. K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D.,
`and growth control. Identification and purification
`of the
`Ullrich, A., and Rosen, 0. M. (1987) J. Biol. Chem. 262,1842-
`protease(s) and manipulation
`by recombinant DNA tech-
`1847
`niques would contribute to a better understanding of the role
`24. Downing, J. R., Roussel, M. F., and Sherr, C. J . (1989) Mol. Cell
`of proteolysis in carcinogenesis.
`Biol. 9, 2890-2896
`25. Glenney, J . R., Chen, W. S., Lazar, C. S., Walton, G. M., Zokas,
`' D. F. Hayes, W. Carney, C. Tondini, D. Petit, S. J. McKenzie, C .
`L. M., Rosenfeld, M. G., and Gill, G. N. (1988) Cell 52, 675-
`684
`Henderson, D. W. Kufe, manuscript