(Shiang et al., 1994; Webster and Donoghue, 1996). We have exploited the ...... Mike DiGiovanna during manuscript preparation. We are grateful to Kari Alitalo ...
Oncogene (1997) 14, 687 ± 696 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
Dimerization of the p185neu transmembrane domain is necessary but not sucient for transformation Christine L Burke1, Mark A Lemmon3, Barbara A Coren1, Donald M Engelman2 and David F Stern1 1
Departments of Pathology and Biology, Yale University School of Medicine, 310 Cedar Street, BML 342, New Haven, Connecticut 06510; 2Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, New Haven, Connecticut 06520; 3Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6089, USA
The neu proto-oncogene encodes a receptor tyrosine kinase (RTK). The oncogenic allele neu* (p185*) bears a glutamic acid for valine substitution at position 664 within the predicted transmembrane domain. We have used this mutant to explore the role of the transmembrane domain in signal transduction by RTKs. Analysis of a panel of neu* proteins with second-site mutations in the transmembrane domain revealed a strong correlation of dimerization with transformation. Both dimerization and transformation are dependent on a domain formed by the amino acids Val663-Glu664-Gly665 (VEG). However, movement of the VEG elsewhere within the transmembrane domain promoted weak dimerization but not transformation. Epidermal growth factor receptor (EGFR)/neu chimeras were used to determine if mutations that disrupt activation by Glu664 aect hormone-regulated signal transduction as well. These mutations (of Val663 and Gly665) did not aect regulation by EGF. Introduction of the known transmembrane dimerization domain from Glycophorin A (GpA) stimulated dimerization, but was not sucient for transformation. These results indicate that dimerization is necessary but not sucient for transforming activity. The homologous wild-type domain, VVG, is not required for hormone-regulated signaling. Keywords: receptor tyrosine kinase; dimerization; transmembrane domain; neu/HER2/erbB-2
Introduction Most peptide growth factors regulate cellular proliferation and dierentiation by binding to receptor tyrosine kinases (RTKs). Hormone-regulated receptor dimerization is evidently a necessary component of signal transduction by many of these receptors: hormone binding stimulates receptor dimerization (Cochet et al., 1988), reagents that induce dimerization can activate receptor signaling (Yarden and Schlessinger, 1987), oligomeric receptors have higher associated kinase activity than monomers (Yarden and Schlessinger, 1987) and signaling-defective mutant receptors act as dominant negative mutants (Redemann et al., 1992; Ueno et al., 1991). Dimerization is believed to enable transphosphorylation within receptor dimers, which in Correspondence: DF Stern Received 16 May 1996; revised 7 October 1996; accepted 9 October 1996
turn can catalytically activate the receptor (Hubbard et al., 1994) and permit recruitment of SH2-containing signaling proteins (Songyang et al., 1993). Despite the crucial role of receptor dimerization in coupling hormone binding to signaling by RTKs, little is known about the means by which hormones induce dimerization. The crystal structure of the human growth hormone receptor (hGHR), a non-tyrosine kinase receptor with only a short intracellular domain, provides the most explicit model for any single membrane-spanning receptor: one monomeric hGH molecule bridges two receptor molecules. This is accompanied by additional inter-receptor contacts (De Vos et al., 1992). An analogous model, with both hormone-mediated and hormone-independent contacts, is also likely to apply to RTK dimerization. RTK extracellular domains are clearly sucient for hormonal regulation of dimerization, since epidermal growth factor (EGF) regulates dimerization of soluble EGFR ecto-domains (Watowich et al., 1994). The ®rst evidence for RTK receptor-receptor interactions was that activated forms of c-erbB, the avian EGFR homologue, bear amino terminal truncations that delete the hormone-binding domain. Similarly, hormone-independent transforming alleles of neu, kit, ros, met, ret and trk genes can be created by truncation of regions encoding the extracellular domains (Downward et al., 1984; Martin-Zanca et al., 1986; Matsushime et al., 1986; Takahsi and Cooper, 1987; Yarden and Schlessinger, 1987; Bargmann and Weinberg, 1988a; Giordiano et al., 1989). Thus, dimerization may involve the transmembrane, cytoplasmic or both domains. Genetic evidence suggests the existence of interreceptor contacts within the membrane-spanning domain of the RTK encoded by the neu/erbB-2/ HER-2 gene. neu is a member of the EGFR subfamily of RTKs (Coussens et al., 1985; Bargmann et al., 1986; Yamamoto et al., 1986) and encodes a protein denoted p185 or (non-italicized) neu (Akiyama et al., 1986; Stern et al., 1986), reviewed, (Hynes and Stern, 1994). neu is an orphan receptor, but several candidate ligands have been identi®ed (Samanta et al., 1994; Tarakhovsky et al., 1991; Huang and Huang, 1992). neu can also be activated by interaction with other EGFR family members (Stern and Kamps, 1988; Kokai et al., 1989), reviewed, (Hynes and Stern, 1994; Dougall et al., 1994). The ®rst oncogenic allele of neu discovered, neu*, contains a point mutation in the predicted transmembrane domain of its product neu* (Bargmann et al., 1986), replacing Val with Glu at position 664 (Figure 1).
p185neu dimerization CL Burke et al
688
This substitution causes increased neu* dimerization, neu* tyrosine kinase activity, and neu* turnover rate (Bargmann and Weinberg, 1988b; Weiner et al., 1989a; Stern et al., 1988), suggesting that this mutation mimics normal activation by the yetunidenti®ed ligand. Intramembrane mutations resembling the neu activating mutation have been identi®ed or produced in other receptors as well, including cytokine receptors (Jenkins et al., 1995), the EGFR (Miloso et al., 1995) and the insulin receptor (Longo et al., 1992). A dominant mutation in the ®broblast growth factor receptor 3 transmembrane domain leads to the predominant genetic cause of human dwar®sm (Shiang et al., 1994; Webster and Donoghue, 1996). We have exploited the activating amino acid substitution within the neu* transmembrane domain for the genetic investigation of inter-receptor contacts and of intramembrane protein structure. In an eort to identify additional amino acids necessary for functioning of the E664 mutation, we produced a series of second-site mutations in cis with E664 (Cao et al., 1992b). E664 mutants with amino acid substitutions for V663 or G665 displayed reduced focus-inducing activity, kinase activity, and phosphorylations of characteristic substrate proteins. Substitutions more distal to position 664 failed to interfere with transforming activity of E664 (with the exception of the mutant substituting L661 for Ala, which had an intermediate phenotype) (Cao et al., 1992b). These data suggested that the Val, Glu and Gly residues at positions 663 ± 5 comprise a domain (`VEG domain') responsible for transforming activity. This domain is not sucient for transformation, however, since a VEG tripeptide at positions 670 ± 672 did not activate transformation. The results of this genetic analysis led us to propose that the VEG domain is a discrete domain necessary for transformation, and led to the hypotheses that the VEG domain nucleates dimerization within the transmembrane domain and that the homologous trio of amino acids (VVG 663 ± 665) functions in signal transduction by the wild-type receptor. In the current report, we use several approaches to examine the relationship between neu dimerization and signaling. First we determined whether mutations that interfere with the function of the E664 activating mutation block neu* dimerization. Second, we determine whether the VEG tripeptide is itself sucient to induce neu dimerization. Third, we introduce a known dimerization motif from glycophorin A (GpA) into the neu transmembrane domain to determine whether dimerization is indispensably coupled to signaling. Finally, we determine whether the VXG tripeptide functions normally in neu-regulated signal transduction. The results support an active role for transmembrane and/or juxtamembrane sequences in RTK dimerization, and demonstrate that dimerization is necessary, but not sucient for signaling.
Results Dimerization of neu mutants Do mutations that block transformation by E664 interfere with dimerization? We had previously
evaluated a panel of mutant neu and neu* proteins with amino acid substitutions, insertions, and deletions in the transmembrane domains for transforming activity and tyrosine phosphorylation (Figure 1; Cao et al., 1992b). We now sought to determine the relationship between transforming ability and neu dimerization. We chose to assay dimerization by measuring the recovery of neu dimers relative to monomers using gel electrophoresis without reducing agents. In our experience, this assay yields data that are similar to those obtained with chemical crosslinking reagents, but they are much more quantitatively reproducible and have higher quantitative resolution. Oligomers induced by V664E comigrate with those induced by cysteine substitutions, which produce speci®c disul®de-linked dimers (Cao et al., 1992a). Previous work has shown that dimerization assayed in this way is stimulated by the activating V664E mutation (Weiner et al., 1989b) and that the extent of neu dimerization increases as a function of neu concentration. We have found in experiments with a subset of mutants that the rank order of dimerization is maintained whether expressed at low levels in NIH3T3 cells, or at much higher levels in COS-7 cells, but that the greater recovery of dimers from COS-7 cells enhances quantitative comparison of dimerization (Cao et al., 1992a; and H. Cao and DFS, unpublished data). COS-7 cells were transiently transfected with expression plasmids containing mutated neu cDNAs, and were metabolically labeled with [35S]Cys. Anti-neu immunoprecipitates were
Figure 1 Mutant p185 transmembrane domains. The wild-type neu transmembrane domain sequence (Bargmann et al., 1986) is shown at the top using the one-letter amino acid code. Amino acid number within the neu polypeptide is indicated. Mutant neu proteins, numbered according to previous work (Cao et al., 1992b), are shown below, beginning with neu*. Amino acid substitution and insertions are shown in upper case and wild-type amino acids in lower case. Parentheses show the sites of amino acid deletions. Results of focus assays of numbered mutants (except for mutant 19) were published previously (Cao et al., 1992b)
p185neu dimerization CL Burke et al
this transforming mutant was not always distinguishable from nontransforming mutants 15 and 16, but the inherent lack of resolution in these assays may mask a threshold for transformation. Regardless, overall these data establish a clear correlation between neu dimerization and transforming activity. Moreover, all of the non-transforming second site mutants have reduced levels of dimerization despite the presence of the glutamic acid.
analysed by gel electrophoresis under both reducing and non-reducing conditions. Typical results obtained for the mutants described in Figure 1 are illustrated in Figure 2, with the results of four independent experiments tabulated in Table 1. As observed previously under non-reducing conditions, neu migrates primarily as monomers, whereas neu* forms both monomers and homodimers (Cao et al., 1992a; Weiner et al., 1989a) (Figure 2a, lanes 1 and 3, respectively). By contrast, all forms of neu migrate as monomers under reducing conditions (Figure 2b, lanes 1 and 3). In one series of experiments, (mutants 7, 9, 11, 12, 14, 15, 16) second-site mutations in cis with E664 were generated to identify additional amino acids required for transformation. Within this series, every mutation that interfered with transformation (mutants 1, 9, 15, 16) showed signi®cantly reduced dimerization relative to neu*. Moreover, all but one of the transforming mutants consistently dimerized significantly better than the non-transforming mutants (Table 1). The reduced dimerization of mutant 7 suggests that the I659 deleted in this mutant may be part of an inter-receptor contact site. Dimerization of
a
Dimerization induced by intramembrane Glu residues Assuming that V664 is in the transmembrane a-helix, and that V664E does not change the secondary structure, it has been proposed that the mechanism via which the V664E mutation activates neu* is that dimerization of the transmembrane domain itself is induced, leading to the consequent activation of the receptor. One hypothesis for this mechanism is that the protonated carboxylic acid group of E664 directly hydrogen bonds to its equivalent in the second receptor. If the glutamic acid binds across the carboxylic acid groups, then the required valine and glycine may stabilize dimerization in the original 663 ± 5
non-reducing neu 1 2
neu* 3 4
11 5
7 6
7
8
9
12 9 1 10 11 12 13 14
14 16 15 15 16 17 18 19 20
oligomers —
monomers —
b
reducing
1
neu 2
neu* 3 4
11 5
7 6
7
8
9
12 10
9 1 11 12 13 14
14 15 16
16 17 18
15 19 20
monomers —
Figure 2 Dimerization of neu mutants. COS-7 cells in 100 mm dishes were transfected with 10 mg of plasmid DNA harboring neu* mutants described in Figure 1, then labeled in 2 ml of 100 Ci/ml [35S]Cys-Met for 24 h. Cells were washed in 10 ml PBS containing 0.9 mM CaCl2, 0.5 mM MgCl2 and 10 mM iodoacetamide, then lysed in 1.5 ml phosphate-buered RIPA containing iodoacetamide (10 mM), phenylmethyl-Sulfonyl Fluoride (PMSF) (1 mM), Na3VO4 (1 mM) and aprotinin (1%). Iodoacetamide was included in buers to prevent formation of disul®de bonds after lysis. Equivalent portions of lysate were immunoiprecipitated with the rat neu-speci®c 7.16.4 antibody (odd numbered lanes), or normal mouse serum (even numbered lanes). Samples were analysed under non-reducing (a) and reducing (b) conditions on 4 ± 12% acrylamide/0.19 ± 0.56% bis-acrylamide gradient gels. The ¯uorographed gels were exposed to pre¯ashed ®lm at 7808C for 3 days. The numeric values are listed under Experiment 4 in Table 1.
689
p185neu dimerization CL Burke et al
690
Table 1
position, but may not be required for interhelical hydrogen bonding mediated by Glu when present at other positions in the transmembrane domain. Alternatively, it has also been proposed that the glutamic acid of one transmembrane a-helix is involved in a bifurcated hydrogen bond with the backbone of the adjacent helix in a dimer (Sternberg and Gullick, 1989). As discussed above, E664 neu induces strong dimerization provided that the ¯anking amino acids are not altered. However, the poor dimerization of mutants 15 and 16 show that E664 is not sucient for dimerization. Similarly, mutant 1, with E663 and a compensating deletion downstream, also dimerizes poorly (Table 1). The non-transforming mutant 3, with a Glu at position 671 ± two helical turns down from position 664 ± induces dimerization better than wild-type neu or mutant 15, but not nearly as well as neu* (Figure 3). Hence intramembrane Glu residues can only induce moderate dimerization in most contexts. These proteins, which dimerize poorly, also fail to transform.
Relative dimerization Focus a
Exp.
Exp.
Exp.
Exp.
1
2
3
4
b
formation
Mutant
5
+
1.79
2.70
neu*(p185*)
+
(1.00)
(1.00)
(1.00)
11
+
0.64
ND
12
+
0.44
14
+
7
Consensus c
rank d
ND
1
(1.00)
2
1.3
0.86
3
0.63
0.50
1.24
4
ND
ND
ND
0.64
5
+
0.12
0.11
0.20
0.43
6
9
+/±
ND
0.085
ND
0.49
7
1
±
0.06
0.068
0.23
0.31
8
15
±
0.12
0.034
0.01
0.46
9
16
±
0.12
0.034
0
0.35
10
WT (p185)
±
0.057
0.023
0
0.31
11
1.4
35
COS-7 cells were transfected with neu plasmids, labeled with [ Cys,
and
immunoprecipitated
with
anti-neu
antibody
7.16.4
for experiment 4 are shown in Figure 2a. The ¯uorographs were quanti®ed by scanning with a Molecular Dynamics Calculating densitometer using Image Quant Software v. 3.0. Intensities of monomeric
and
dimeric
bands
were
determined
from
dierent
exposures in order to remain in the linear response range of ®lm. oligomeric
and
monomeric
bands
were
quanti®ed
and the subtraction of local background, as determined by the area of the same sized rectangle in a part of the lane with no visible bands.
The
ratios
of
oligomeric
and
monomeric
areas
neu 2
neu* 3 4
4 5
neu*. Typical actual values for these ratios are 0.052 and 0.33 for neu and neu* in experiment 1.
a
Mutants are described in Figure 1.
b
Focus activities from (Cao et al., 1992b).
c
Consensus rank order
from Experiments 1, 2, 3 and 4 from highest to lowest relative dimerization.
d
Not determined.
19
5 6
7
9
8
3 10
11
15 12
13
14
mock 15 16
oligomers —
monomers —
reducing neu 2
neu* 3 4
4 5
19
5 6
7
were
calculated, then normalized using an arbitrary value of 1.0 for
non-reducing
1
by
measurement of the area of a rectangle encompassing the band,
Despite genetic data implicating VEG as a functional unit required for transformation, we found that this tripeptide does not induce transformation when relocated two turns deeper in the transmembrane domain (Mutant 4, VEG670 ± 672) (Cao et al., 1992b), or just two amino acids C-terminal to its position in neu* (Mutant 19, VEG 666 ± 668) (CB
b
as
electrophoresis on non-reducing SDS-PAGE gradient gels. Results
The
1
S]-
described in the legend to Figure 2. Proteins were resolved by
Dimerization of VEG domain
a
Dimerization of mutant neu proteins
8
9
3 10
11
15 12
13
14
mock 15 16
monomers —
Figure 3 Dimerization of shifted VEG mutants. COS-7 cells in 100 mm dishes were transfected with 10 mg of plasmid DNA harboring neu* mutants described in Figure 1, then labeled in 2 ml of 100 mCi/ml [35S]Cys-Met for 24 h. Cells were processed identically to those in Figure 2. The ¯uorographed gels were exposed to pre¯ashed ®lm at 7808C for 3 days. The numeric values are listed under experiment 1 in Table 2.
p185neu dimerization CL Burke et al
Table 2
691
Dimerization of Shifted VEG domain mutants
a
α-PTyr
Focus a
Mutant
b
formation
Exp. 1
Exp. 2
Exp. 3
1.96
5
+
1.53
1.72
neu*
+
[1]
[1]
[1]
±
0.71
0.44
0.40
19
±
0.52
0.34
0.35
4
±
0.51
0.32
0.31
15
±
0.36
0.24
0.22
neu
±
0.36
0.18
0.20
3 c
COS-7 cells were transfected with neu plasmids, labeled with [
35
+EGF
–EGF
15 3T3 E/neu- WT 14 16 15 3T3 WT 14 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
S]-
b
Cys-Met (Invitro labeling mix, Amersham), and immunoprecipitated
[35S]-Cys 16 15 3T3 15 3T3 WT 14 E/neu- WT 14 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
with anti-neu antibody 7.16.4 as described in the legend to Figure 2. Proteins were resolved by electrophoresis on non-reducing SDSPAGE gradient gels. Results for experiment 1 are shown in Figure 3a. The ¯uorographs were quanti®ed by scanning with a Molecular Dynamics PhosphorImager SI using ImageQuant Software v. 4.0. The
oligomeric
and
monomeric
bands
were
quanti®ed
by
measurement of the area of a rectangle encompassing the band, and the subtraction of local background. The ratios of oligomeric and monomeric areas were calculated, then normalized using an arbitrary value of 1.0 for neu*.
a
c
Mutants are described in Figure 1.
b
Focus activities from (Cao et al., 1992b).
c
Focus assay negative (C
Burke, data not shown)
and DFS, unpublished data). These results suggest either that VEG is not sucient for dimerization, or that dimerization itself is not sucient for transformation. We therefore evaluated the dimerization of three dierent neu proteins with VEG domains at alternative locations (mutants neu*, mutant 4, mutant 19). Mutants 4 and 19 consistently dimerized slightly better than neu, and much worse than neu* (Figure 3, Table 2). Mutant 4 (VEG670 ± 2) dimerized no better than mutant 3 (E671 only), indicating that the tripeptide does not enhance dimerization relative to the Glu-only substitution. Interestingly, VEG670 ± 2, which only modestly induces dimerization on its own, greatly enhances dimerization in cis with E664, suggesting that VEG670 ± 2 may provide additional energy for transmembrane helix association. Role of VVG in normal signaling Since amino acid substitutions at positions 663 or 665 interfere with transformation induced by E664, we determined whether these amino acids are also important for hormone-regulated signaling by wildtype neu. Since a neu-speci®c ligand has not been de®nitively identi®ed, chimeric proteins consisting of the EGFR extracellular domain and the neu transmembrane plus intracellular domains (E/neu) were used to permit EGF-regulated signaling by neu (Lee et al., 1989; LehvaÈslaiho et al., 1989) (Substitution of Glu at neu position 664 activates transformation by identical chimerae to those described here; Goldman et al., 1990). Amino acid substitutions at position 665 or both positions 663 and 665 were engineered into the E/ neu transmembrane domain. These mutants correspond to transforming mutant 14, and the nontransforming mutants 15 and 16, (with Val at position 664 instead of Glu) (E/neu-14, E/neu-15 and E/neu-16; Figure 4c). Stable NIH3T3 derivatives expressing each of the wild-type E/neu chimera and the three mutant chimerae were established (Figure 4). The cells were analysed for neu protein expression (Figure 4b) and
Figure 4 Regulation of E/neu chimerae in NIH3T3 cells. 100 mm cultures of NIH3T3 cell lines stably transfected with EGFR/neu chimeric genes were metabolically labeled in 2 mls 100 mCi/ml [35S]Cys for 24 h. Cells were mock-treated with 0.1% Calf Serum (CS) (lanes 11 ± 20), or incubated with 100 ng/ml EGF (mouse, Receptor Grade, Sigma, St. Louis, MO) for 10 min at room temperature (lanes 1 ± 10) and lysed. Immunoprecipitates were prepared with either the EGFR-speci®c antibody 528 (oddnumbered lanes), or a non-speci®c goat anti-mouse antibody (even-numbered lanes). Proteins were resolved under reducing conditions on 7.5% acrylamide/0.175% bis-acrylamide gels and immunoblotted with an anti-phosphotyrosine antiserum (a) or detected by ¯uorography (b). (a) Proteins were immunoblotted with a rabbit anti-phosphotyrosine antiserum (8589C) and detected with [125I] Protein A. The ®lter was exposed to pre¯ashed ®lm for 4 days at 7808C. (b) Duplicate samples were resolved by gel electrophoresis, ¯uorographed and exposed to pre¯ashed ®lm for 14 days at 7808C. (c) depicts the E/neu mutant constructs. They correspond to mutant transmembrane domains in Figure 1, but with V664. Wild-type amino acids are shown in solid type, substitutions are in shadow type
EGF-regulated Tyr phosphorylation (Figure 4a). Despite slight variations in receptor expression, EGF activated Tyr phosphorylation of all four chimerae to a similar extent (Figure 4a). As a further measure for function of the chimerae, we assayed EGF-stimulated thymidine incorporation. EGF stimulated [3H]thymidine incorporation only approximately twofold in the NIH3T3 cells, but eight- to 16-fold in the NIH3T3 derivatives expressing the E/neu chimerae (Table 3). Since mutations at Val663 and Gly665 failed to interfere with the function of these E/neu chimerae, we conclude that these residues are not essential for receptor-receptor interactions in normal signaling. To ensure that neu signaling was not due to endogenous EGFR cross-talk, we repeated these experiments in NR6 cells, which do not produce the EGFR (Pruss and Herschman, 1977). We obtained consistent results (data not shown).
p185neu dimerization CL Burke et al
692
Table 3 E-neu-WT
EGF-stimulated [3H]Thymidine incorporation E-neu-14
E-neu-16
E-neu-15
NIH3T3
3409+638 6773+3088 3371+2081 2659+1503 mock 47743+6408 33380+3698 54412+13881 56222+5177 5773+1655 EGF 86792+15029 61013+7752 113460+8372 57190+6677 60851+6971 FCS 9.1 9.8 8.0 16.7 2.2 Fold Inductionb EGF to mock NIH3T3 cells stably transfected with genes encoding the E/neu-mut chimerae (described in Figure 4c) were plated in 96well dishes, serum-starved in 0.1% Calf Serum (CS) for 72h, then treated with 10ng/ml EGF in 0.1% CS or with 10% fetal calf serum (FCS) for 4h. The cells were labeled with 1uCi [3H]thymidine in 200ul of 0.1% CS for 24h, then lysed by freezing at ±808C overnight. The lysates were transferred to ®lters by vacuum ®ltration, then the ®lters were rinsed with water, dried, immersed in scintillation ¯uid and counted in a liquid scintillation counter (Wallac Oy 1450 Microbeta). Eight replicate wellsa were assayed per cell line stimulated with bEGF, and four replicate wells were assayed for mock and FCS stimulations. Values are averages+standard deviation. The ratio of activities of EGF-treated relative to mocktreated lysates 5224+762a
Dimerization of neu/GpA chimerae The data presented above establish a strong correlation between dimerization and transformation, since mutations that interfered with transformation also all interfered with dimerization, and since non-transforming mutants all dimerized poorly. However, other data suggest that dimerization alone may not be sucient for transformation. For example, substitution of Cys at position 653 induces stable in vivo dimerization of neu, but not transformation (Cao et al., 1992a). However, it is not certain be certain that the induced disul®de bond in the extracellular domain actually forces dimerization of the transmembrane domains. Since we wanted to study the role of dimerization in the transmembrane domain, we took another approach to this problem. Glycophorin A (GpA) is a membrane spanning protein that forms constitutive dimers as a result of transmembrane a-helix interactions. Extensive in vitro mutagenesis and dimerization analysis of a chimeric fusion protein with the transmembrane domain of GpA fused to the carboxyl terminus of Staphyloccus aureus nuclease (SN) has identi®ed a putative interhelical contact face. Single substitutions at only seven of 23 transmembrane positions greatly aect dimerization. This GpA motif, LIxxGVxxGVxxT (Lemmon et al., 1992) is sucient for dimerization when expressed in a featureless poly(leu) context, or when introduced into the neu or EGFR transmembrane domains (Lemmon et al., 1994). Surprisingly, an SN/neu mutant (SN/neuGpA1) which contains just ®ve of the seven residues comprising the GpA motif dimerized more strongly than an SN/neu mutant (SN/neu-GpA2) containing the entire motif, indicating that the N-terminal region of the neu transmembrane domain itself may participate in a transmembrane a-helix interaction (Lemmon et al., 1994). To establish whether the GpA dimerization motif would behave identically in a full-length receptor expressed in mammalian cells, and whether GpAdriven dimerization would induce transforming activity, we created a series of neu chimeras with transmembrane domains derived from this data set (described, Figure 1b). neu* showed greater relative dimerization than any of the neu/GpA chimerae (Figure 5, Table 4). Introduction of the GpA motif (LIxxGVxxGVxxT) at a central position in the neu transmembrane domain (neu/GpA2), caused signi®cant dimerization. Consistent with the SN/neu results, neu/GpA1 dimerized more strongly than neu/GpA2. neu/GpA3, in which
the GpA motif is displaced four residues upstream compared with neu/GpA2, barely dimerized. Its low level of dimerization may result from the positioning of the GpA dimerization motif too close to the extracellular border of the transmembrane domain. These results indicate that the GpA binding motif does increase neu dimerization and they are completely consistent with in vitro SN-GpA results, wherein GpA1 and GpA2 dimerize substantially better than GpA3. The V664E mutation did not aect dimerization greatly in the context of neu/GpA2 ± both neu/GpA2 and neu*/GpA2 dimerize to similar extents (although it may do so via dierent sets of interactions). neu*/ GpA3 dimerized strongly, showing that E664 functions independently in promoting dimerization. Transformation by neu/GpA chimerae Since several of the neu/GpA chimerae dimerized well, we next sought to determine whether dimerization mediated by the GpA transmembrane domain induces transformation. Both chimerae that contained E664 (neu* GpA2 and neu* GpA3), showed strong transforming activity (Table 4), with the former equivalent to neu* itself. In contrast, none of the neu/GpA chimerae had transforming activity, even though neu/GpA1, and neu/ GpA2 dimerized signi®cantly better than neu*/GpA3, and at similar levels to neu*/GpA2. These data extend the previous correlation establishing that dimerization is necessary for transformation, and that E664 induces transformation even with multiple amino acid substitutions elsewhere. However, these results also clearly demonstrate that neu dimerization is not itself sufficient for transformation since several dimerizing mutants had no transforming activity. Analysis of receptor expression and tyrosine phosphorylation To determine whether introduction of the GpA dimerization motif caused weak activation of neu that was insucient to render it transforming, we also analysed the ability of the neu/GpA mutants to autophosphorylate on tyrosine. NIH3T3 cells stably expressing these mutated proteins were immunoprecipitated with an anti-neu antibody, then immunoblotted with an anti-neu or an anti-phosphotyrosine antibody (Figure 6a and b respectively). Three distinct patterns of expression and neu tyrosine phosphorylation were evident. We have
p185neu dimerization CL Burke et al
display intermediate phosphorylation relative to neu and neu*. These data verify that dimerization by these chimerae does result in some degree of enhanced receptor phosphorylation. It presumably occurs via cross-phosphorylation, but is not sucient to activate the receptor for transformation of cells.
neu*
neu/GpA3
neu*/GpA3
neu/GpA2
neu*/GpA2
neu/GpA1
mock
α-neu
1
2
3
4
5
6
7
8
1
6
7
— p185 mock
neu/GpA1
neu*/GpA2
neu/GpA2
neu/GpA3
neu* 4 5
2 3
neu*/GpA3
non-reducing
neu
a
a
neu
observed previously that neu* and other transforming mutants generally accumulate to low steady-state levels (owing to rapid turnover), but that they are highly phosphorylated (Cao et al., 1992b; Stern et al., 1988). The strongest transforming mutants, neu* and neu*/ GpA2, conform to this pattern, showing low accumulation and high phosphorylation relative to neu. neu*/ GpA3 is intermediate, with higher accumulation and strong phosphorylation (When neu*/GpA3 is expressed at a lower level, the ratio of expression to phosphorylation is still approximately equal (data not shown). neu/GpA3, which does not dimerize well, closely resembles neu. Intriguingly, both neu/GpA1, and neu/ GpA2, which dimerize well but do not transform,
b
9 10 11 12 13 14 15 16
8
α-P-Tyr, neu 1
2
3
4
5
6
7
8
— p185 oligomers —
Figure 6 Expression and tyrosine phosphorylation of neu/GpA constructs. 150 cm2 ¯asks containing NIH3T3 cells stably expressing the neu/GpA constructs were starved for 2 days with 0.1% CS, then lysed, 400 mg of protein from each cell line was incubated with either 1 mg 7.16.4 or 10 mg Py20 for 2 h. The immunoprecipitated material was analysed by SDS ± PAGE on 10% acrylamide/0.13% bis-acrylamide (anti-phosphotyrosine IPs) or 7.5% acrylamide/0.173% bis-acrylamide (anti-neu IPs) gels, at 10 mAmp per gel overnight. The gels were transferred to nitrocellulose and blocked for 1 h in: 5% BSA-1% ovalbumin for anti-phosphotyrosine blots; 5% milk in TBS-T for anti-neu blots. TBS-T consists of: 10 mM Tris-HCl pH 7.4, 0.9% NaCl, 0.01% Tween-20. The blots were incubated for 3 h in primary antibody (8589C or SC284 respectively) and rinsed 5610 min with 200 mL TBS-T. They were then incubated with secondary antibody (anti-rabbit HRP conjugated, 1:2000, in 5% milk in TBS-T) for 1 h and washed 7610 min with 200 mL TBS-T. The blots were visualized with ECL reagents (Amersham, Arlington Heights, IL) and exposed to ®lm. (a) is a one minute exposure of an anti-neu IP and anti-neu blot. (b) is a 6 min exposure of an anti-neu IP and anti-phosphotyrosine blot
monomers —
b
reducing 1
2
3
4
5
6 7
8
9 10 11 12 13 14
monomers —
Figure 5 Dimerization of neu/GpA Mutants. COS-7 cells in 100 mm dishes were transfected with 10 mg of plasmid DNA harboring neu/GpA mutants described in Figure 1. Transfection conditions and sample analysis were identical to Figure 2. The ¯uorographed gels were exposed to pre¯ashed ®lm at 7808C for 1 day. The numeric values are listed in Table 4
Table 4 a
Mutant
Dimerization and focus formation of neu/GpA mutants
Dimerization
neu*
foci
Experiment 1 colonies
1800 245
Focus formation
Experiment 2 colonies
ratio
foci
ratio
5000
0.36
1480
3800
0.39
2600
0.09
100
3700
0.03
2200
4800
0.46
1500
4300
0.35
10
800
0.01
5
1500
0
1.40
+
[1]
±
neu*/GpA2
0.87
+
neu/GpA2
0.79
±
neu*/GpA3
0.71
+
920
3500
0.26
140
700
0.20
neu (WT)
0.44
±
140
2600
0.05
350
5000
0.07
neu/GpA3
0.35
±
14
1200
0.01
15
500
0.03
neu/GpA1
In
dimerization
experiment,
COS-7
cells
were
transfected
with
neu
plasmids,
labeled
with
35
[
S]Cys-Met,
and
immunoprecipitated with anti-neu antibody 7.16.4 as described in the legend to Figure 2. Proteins were resolved by electrophoresis on non-reducing SDS±PAGE gradient gels. The ¯uorographs were quanti®ed by scanning with a Molecular Dynamics PhosphorImager SI using ImageQuant Software v. 4.0, and analysed as described in Table 2. Focus assays were performed as described in Materials and methods.
a
Mutants described in Figure 1
693
p185neu dimerization CL Burke et al
694
Discussion The present data provide a strong genetic argument in favor of the model that RTK dimerization is necessary for transforming activity. Mutations that interfere with transforming ability of neu* also impede its dimerization. The consensus rank order for the extent of dimerization is remarkably consistent with the simple model that receptor dimerization is necessary for transforming activity. The data also demonstrate that dimerization itself is not sucient for RTK signaling. In spite of the wide acceptance of the dimerization model, to our knowledge this is the ®rst extensive genetic analysis that compares dimerization to signaling by a RTK. The activating E664 mutation stimulates dimerization of neu* (Weiner et al., 1989a). The present data extend this observation to show that neu* mutations that interfere with transformation also disrupt dimerization. Our previous data suggested that the E664 mutation acts in a discrete dimerization site, rather than as a component of an extended helical interface as seen for GpA (Lemmon et al., 1992), since only mutations at adjacent amino acids strongly interfered with transformation. The present data con®rm this suggestion. E664 is present in a VEG tripeptide, and mutation of either V663 or G665 reduces neu* dimerization to baseline levels. Despite the role of VEG in dimerization as it is located in neu*, it does not signi®cantly enhance dimerization when introduced at two alternative positions in the neu transmembrane domain. At one position, the VEG tripeptide is no better than a Glu substitution only. Apparently, additional sequence context, packing, or positional constraints are required for this tripeptide to function properly in enhancing neu dimerization. The enhanced dimerization of mutant 5, which contains two VEG domains, suggests that, once the appropriate alignment is nucleated by neu*, the second VEG domain can function to further stabilize transmembrane a-helix interactions. The occurrence of intramembrane Glu residues in other activated receptors has led to the belief that they may be sucient for inducing dimerization or transformation (Jenkins et al., 1995; Miloso et al., 1995; Longo et al., 1992; Shiang et al., 1994; Webster and Donoghue, 1996). However, of the 15 mutant transmembrane domains discussed here that contain intramembrane Glu, only those associated with VEG663 ± 665 have strong transforming or dimerizing activity, indicating that Glu alone can only weakly stabilize dimerization in other contexts. The explanation for this paradox is probably selection bias since in screens for transforming mutants, only positive results are reported. Nonetheless, they may be involved in promoting dimerization when present in the context of another dimerization site ± perhaps working in concert with other distributed inter-receptor contacts with which they must be in register. In particular, the strong inhibition of dimerization by deletion of I659 (mutant 7) suggests the existence of an additional site of interaction, as does the fact that neu/GpA1 dimerizes more eciently than neu/GpA2. Although the VEG tripeptide is clearly important in dimerization of mutant neu*, mutations in V663 and G665 fail to interfere with activation and signaling of
E/neu chimeras. We conclude that the spontaneous dimerization induced by E664 must occur via a dierent process than ligand-induced dimerization. This would be possible if VEG663 ± 5 induces a high anity inter-receptor contact point, which then nucleates secondary interactions and receptor dimerization. In the normal protein however, hormonestabilized dimerization is thought to cooperate with many distributed inter-receptor contacts. Thus, if V663 and G665 are contained in one of these many contact sites, they would have a less critical role in the initiation of dimerization. Although dimerization is necessary for transformation, the robust dimerization of the transformationdefective neu/GpA mutants shows that dimerization is not sucient for transformation. neu/GpA1 and neu/GpA2 both dimerized extremely well, and higher basal receptor phosphorylation followed dimerization, indicating that dimerization is sucient for some inter-receptor communication, but not for transformation. Only those mutants that contained E664 were transforming. These results indicate that the position of the GpA motif aects the ability of the receptor to dimerize, and consequently to autophosphorylate. Why do these molecules lack transforming activity? The general resilience of RTK signaling to mutations in the transmembrane domains makes it dicult to explain the existence of transformation-defective/ dimerization-competent mutants. However, the relatively non-speci®c or even promiscuous nature of transmembrane alpha helix packing (Lemmon and Engelman, 1994) permits a number of inter-receptor geometries, with variables including the angle of interaction, and the exact contact faces. This packing degeneracy suggests the possibility that most deletions and insertions have only minor eects on the helix registration or packing faces, and transformationdefective mutants may be those that induce radically dierent packing angles or represent other metastable con®gurations. Ultimately the packing geometry might in¯uence the availability of cross-phosphorylation sites to dimerization partners, with concomitant changes in the binding of substrates or other interacting proteins. The predicted dimerization face induced by GpA is opposite that for E664, if the latter works through face-to-face carboxylic acid group hydrogen bonding. It has further been suggested that the GpA motif promotes a right-handed interaction of helices (Adams et al., in press), while left-handed crossing angles predominate in a-helical supercoils of polytopic proteins of known transmembrane structure (Lemmon and Engelman, 1994). A better understanding of the in¯uences regulating transmembrane a-helix packing is an important constituent of a mechanistic understanding of RTK signal transduction, and will aid in the development of receptor-speci®c therapeutic agents. Deciphering receptor dimerization is also crucial to the understanding of signal transduction by erbB family receptors for an entirely dierent reason. Although all RTKs dimerize as a constituent of signal transduction, the signaling activity of neu is further in¯uenced by its ability to form heterodimers with other erbB family receptors, which confer responsiveness to at least six sets of peptide hormones (Hynes
p185neu dimerization CL Burke et al
and Stern, 1994). Thus homo- and hetero-dimerization will likely in¯uence the quality, as well as the extent, of signals emitted by neu.
Materials and methods Cell culture Cells were grown in Dulbecco-Vogt modi®ed Eagle's medium (DMEM) supplemented with 10% calf serum (CS) under an atmosphere of 5% CO2 at 378C. Antibodies The following antibodies were used in this paper: anti-neu antibodies 7.16.4 (Drebin et al., 1984) (Ab-4, Oncogene Science, Cambridge, MA) and SC284 (Santa Cruz Biotechnology, Santa Cruz, CA); anti-EGFR 528 (Gill et al., 1984) (H Masui, Rockefeller University, New York); anti-phosphotyrosine antibodies 8589C (Kamps and Sefton, 1988; Stern et al., 1988), and Py20 (Transduction Laboratories, Lexington, KY); and secondary antibodies, used against 528, a goat anti-mouse antibody (Pierce, Rockford, IL) and used against SC284, an HRP-linked donkey anti-rabbit immunoglobulin (Amersham, Arlington Heights, IL). The normal mouse serum (NMS) was purchased from Pierce. Plasmids Most wild-type and mutant DOL-neu and SRaneu plasmids were described previously (Bargmann and Weinberg, 1988b; Cao et al., 1992a,b). The EGFR/neu (E/neu) chimera gene was produced as follows. An XbaI fragment (25 ± 2336) of pSV2EGFR/neu (LehvaÈslaiho et al., 1989) was subcloned into the polylinker of pBluescript SK by Pan Zheng to produce SK-EGFR. The sequence encoding the EGFR extracellular domain was released at XhoI and NaeI sites. The fragment was ligated to the SRaneu backbone through a blunt-end ligation of the EGFR XhoI site with the SRaneu EcoRI site, then with an adaptor joining the NaeI and SRaneu NdeI sites. The sequences of the adaptors to join the NaeI and NdeI sites are as follows: 5'-GGCCACATATGC-3' and 5'-GGCCGCATATGTGGCC-3'. Mutations in E/neu transmembrane domains were introduced by PCR ampli®cation. Two outside primers spanned the NdeI and BgII sites in neu, while inside forward and reverse mutagenic primers introduced mutations. Two fragments were ampli®ed with one of each of the outside or inside primers. The ampli®ed fragments were then anealed, extended, then re-ampli®ed to create a full-length fragment containing NdeI and BgII sites. The synthetic oligonucleotides used for the outside primers had the following sequences: 5'-GGAAGTACCCGGATGAGGAGGG-3' and 5'-CCAGCTGTACTGTGGATGTCAGG-3'. The inside primers had the following sequences: E/neu-14: 5'-GTAGTGGGCGTCGGCCTGTTCCTGATC-3' and 5'-GATCAGGAACAGGCCGACGCCCACTAC-3'; E/ neu-16: 5'-CATCATTGCAACCGGTGTGGTCGTCCTGCTG-3' and 5'-CAGCAGGACGACCACACCGGTTGCAATGATG-3'; E/neu-15: 5'-GCAACTGTAGTGGTCGTTCTGCTGTTCC-3' and 5'-GGAACAGCAGAACGACCACTACAGTTGC-3'. Introduction of the GpA binding motif into the transmembrane domain of neu The transmembrane domain of intact neu was replaced with that from chimeric proteins (SN/neuTM) in which the mutated transmembrane a-helix of interest was fused to the carboxy-terminus of the nuclease (SN) from
Staphylococcus aureus (Lemmon et al., 1994). The DNA encoding the altered transmembrane domains was generated using PCR from the plasmids encoding the chimerae. Oligonucleotide primers (sense and antisense) were synthesized for fusing the amino- and carboxytermini of the altered transmembrane portion of neu present in the chimerae to the contiguous sequence of intact neu. In addition, oligonucleotides priming upstream of the NdeI site (sense) at position 1900 of the cDNA for neu and downstream of the BglI site (antisense) at position 2390 were synthesized. These primers were utilized in two rounds of four-primer PCR, using the SN/neuTM plasmids and neu cDNA as templates, to generate an altered NdeI to BglI fragment encoding residues 627 to 790 of neu. After digestion with NdeI and BglI, this fragment was ligated with two fragments of the plasmid pSRaneu DEcoRI which had been constructed (Cao et al., 1992a). The sequences of the altered inserts and their ¯anking regions were con®rmed by dideoxy sequencing of the resulting plasmids. The amino acid sequences of the altered transmembrane domains thus generated are presented in Figure 1. COS cell expression SRa plasmids were introduced into COS-7 cells by transfection with DEAE-dextran and chloroquine as described (Guan and Rose, 1984), except that calcium and magnesium-free phosphate-buered saline (PBS) was used rather than Tris-saline. Cells were labeled beginning 24 h after transfection. Procedures for metabolic labeling with [35S]Cys, immunoprecipitation, gel electrophoresis, and immunoblotting with anti-phosphotyrosine have been described (Stern et al., 1986; Kamps and Sefton, 1988; Cao et al., 1991). Immunoprecipitations were done in RIPA buer consisting of 10 mM NaPO4 pH 7.2, 1% Triton X100, 0.1% Sodium Dodecyl Sulfate (SDS), 1% Sodium deoxycholate, 150 m M NaCl, 1% Aprotinin, 2 mM EDTA, 50 mM NaF, 100 mM NaOrthovanadate and 1 mM ATP. Antibodies were precipitated with Protein A Sepharose CL-4B (Pharmacia, Piscataway, NJ) which was presoaked in PBS, preadsorbed with unlabeled parental cell lysates for one hour, then washed with RIPA. Production of stable cell lines expressing EGFR/neu chimerae NIH3T3 cells were transfected with the pSRaEGFR/neu plasmids and pSV2neo in a 10:1 ratio using the calciumphosphate method. The cells were selected for G418 resistance with 0.5 mg/ml Geneticin (Gibco/BRL, Grand Island, NY). G418-resistant colonies were ring-cloned and screened for expression of E/neu and E/neu mutant derivatives E/neu-14, E/neu-15 and E/neu-16. Cell lines expressing similar levels of the chimeric proteins were selected for further experimentation. E/neu, E/neu-14 and E/neu 15 are derived from pooled polyclonal cell lines, while E/neu-16 is derived from a monoclonal cell line. Analysis of transforming ability by focus assay NIH3T3 cells were transfected with the SRaneu plasmids and SV2neo in a 10:1 ratio using the calcium-phosphate method. Two days after transfection, the cells were divided into two groups: no selection (5% CS), and selection for G418 resistance with 0.5 mg/mL Geneticin (Gibco/BRL, Grand Island, NY) in 10% CS. Both sets of cells were incubated as described above for 14 days, changing the medium every 3 days, and then were stained with crystal violet (Sigma). The foci on the unselected plates, and the colonies on the selected plates, were counted and are tabulated in Table 4.
695
p185neu dimerization CL Burke et al
696
Acknowledgements We would like to thank Ken Hu and Pan Zheng for constructing the initial EGFR/neu chimera. We appreciate the critical comments of Dan DiMaio, David Riese, and Mike DiGiovanna during manuscript preparation. We are grateful to Kari Alitalo for pSV2EGFR/neu and to Hideo
Masui for anti-EGFR antibodies. This work was supported by the National Cancer Institute, PHS grant RO1CA45708 to DFS. DME thanks the National Foundation for Cancer Research and the National Science Foundation MCB 9406983.
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