Department of Chemistry and Biochemistry and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0367
The receptor tyrosine kinase p185c-neu can be
constitutively activated by the transmembrane domain
mutation Val664 Glu, found in the oncogenic mutant
p185neu. This mutation is predicted to allow intermolecular hydrogen bonding and receptor dimerization. Understanding the activation of p185c-neu has assumed
greater relevance with the recent observation that achondroplasia, the most common genetic form of human dwarfism, is caused by a similar transmembrane
domain mutation that activates fibroblast growth factor
receptor (FGFR) 3. We have isolated novel transforming derivatives of p185c-neu using a large pool of degenerate oligonucleotides encoding variants of the transmembrane domain. Several of the transforming isolates identified were unusual in that they lacked a Glu at residue 664, and others were unique in that they contained
multiple Glu residues within the transmembrane domain. The Glu residues in the transforming isolates often exhibited a spacing of seven residues or occurred in
positions likely to represent the helical interface. However, the distinction between the sequences of the
transforming clones and the nontransforming clones
did not suggest clear rules for predicting which specific
sequences would result in receptor activation and transformation. To investigate these requirements further, entirely novel transmembrane sequences were constructed based on tandem repeats of simple heptad sequences. Activation was achieved by transmembrane
sequences such as [VVVEVVA]n or [VVVEVVV]n,
whereas activation was not achieved by a transmembrane domain consisting only of Val residues. In the
context of these transmembrane domains, Glu or Gln
were equally activating, while Lys, Ser, and Asp were not. Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad
spacing resulting in receptor activation. These results
are discussed in the context of activating mutations in
the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.
The neu oncogene was initially isolated from a rat
ethylnitrosourea-induced neuro/glioblastoma and encodes a receptor tyrosine kinase belonging to the
epidermal growth factor receptor (EGFR)1 family; the oncogenic mutant, referred to as p185neu, is closely related to
its wild-type cellular homologue, referred to as p185c-neu
(Shih et al., 1981 Activation of p185c-neu arises from structural changes,
such as the Val664 Prior mutagenesis studies have suggested that the primary structure in the vicinity of Glu664 must play a significant role in activation of p185neu. For example, substitution of Glu at the neighboring positions 663 or 665 does
not result in activation (Bargmann and Weinberg, 1988b A subdomain containing a loosely defined sequence motif has been noted in the transmembrane domains of many
other receptor tyrosine kinases (Sternberg and Gullick,
1990 For many years, p185neu represented the sole example of
a receptor tyrosine kinase that is activated by mutation
within its transmembrane domain. Recently, however, it
has become clear that some human developmental abnormalities are due to transmembrane domain mutations in
fibroblast growth factor receptor (FGFR) 3. Achondroplasia, the most common genetic form of human dwarfism,
results from a single amino acid change in the transmembrane domain of FGFR3, Gly380 In this study, we have used pools of long oligonucleotides encoding the p185c-neu transmembrane domain to
target degeneracies to specific positions. This represents a
powerful approach for the simultaneous mutation of multiple residues, facilitating the isolation of transforming and
nontransforming clones from a large pool of different mutants. In an effort to understand the differences between
the transforming and nontransforming clones, we constructed completely novel transmembrane domains and
demonstrated that some of these, such as a tandem repeat
of [VVVEVVV]n, are able to induce efficient receptor activation as evidenced by transformation of NIH3T3 cells.
These results demonstrate that an apparently complex
transmembrane domain can be substituted by simple sequences of repeated residues, provided that amino acids
capable of hydrogen bonding, such as Glu or Gln, are included at appropriate positions. Significantly, receptor activation by the tandem repeats is accomplished without
any specific sequence contribution from the native p185c-neu
transmembrane domain. The experiments described here
provide a basis for understanding the role of the transmembrane domain in the activation of p185neu, or for the
activation of other receptor tyrosine kinases such as
FGFR3.
Construction of Degenerate Pools and
Additional Mutants
Oligonucleotides were synthesized encoding the p185c-neu transmembrane
domain with degenerate codons targeted to the presumptive
The synthetic oligonucleotide sequence used to generate the degenerate pools is shown below. Silent restriction sites for NheI (GCTAGC) and
SacI (GAGCTC) embedded within the sequence are shown in bold. Extra
residues were added at both ends to facilitate restriction digestion of the
PCR-amplified double-stranded product. The first and last triplet codons
shown correspond to Ala653 (GCT of NheI site) and Leu701 (CTC of SacI
site), respectively, in the published amino acid sequence of p185c-neu
(Bargmann et al., 1986a Additional mutants described in the text were constructed by synthesizing pairs of single-stranded oligonucleotides to create double-stranded
DNA fragments with NheI and SacI cohesive overhangs, for ligation into
pSV2neuNheI/SacI (Webster and Donoghue, 1996 Focus Assays
NIH3T3 cells were transfected using a modified calcium phosphate transfection protocol (Chen and Okayama, 1987 Isolation of Transmembrane Domains from
Degenerate Pools
At 9 d after transfection of degenerate Pool 1 into NIH3T3 cells, individual foci of transformed cells were expanded and RNA was isolated.
cDNA was prepared using the following primer: 5 Indirect Immunofluorescence
Transiently transfected Cos-1 cells (Chen and Okayama, 1987 For double-label experiments to detect both intracellular and cell surface expression, cells were fixed as described above and then treated with
mouse mAb 7.16.4 (1:50 dilution) against an extracellular epitope of
p185c-neu, followed by FITC-conjugated goat anti-mouse antiserum (Boehringer Mannheim Corp.) at 1:1,500 dilution. The same cells were then permeabilized with 1% Triton X-100/PBS and treated with a rabbit polyclonal p185c-neu C-18 antibody (Santa Cruz Biotechnology, Santa Cruz,
CA) at 1:3,000 dilution, which was detected with rhodamine-conjugated
goat anti-rabbit antiserum (Boehringer Mannheim Corp.) at 1:4,000 dilution.
Immunoprecipitation, In Vitro Kinase, and
Dimerization Assays
Cos-1 cells were split at a density of ~2 × 105 cells per 60-mm plate and
transfected with 10 µg of DNA the next day (Chen and Okayama, 1987 To examine dimerization, transfected cells were lysed in RIPA (10 mM
sodium phosphate, pH 7.0, 1% Triton X-100, 0.1% SDS, 1% DOC, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% aprotinin, 1 mM sodium orthovanadate, 100 µM PMSF, and 10 mM iodoacetamide) and immunoprecipitated as described above. 2× nonreducing sample buffer (4% SDS, 10 mM
sodium phosphate, pH 7.0, 20% glycerol, 0.08% bromophenol blue) was
added to the protein A-Sepharose beads and then boiled. For the reducing samples, an aliquot was removed and 2-mercaptoethanol was added to
0.7 M and DTT was added to 20 mM. Samples were boiled again and run
on a 4-12% gradient gel. Proteins were then transferred to nitrocellulose,
blotted with a rabbit polyclonal p185c-neu C-18 antiserum, and detected by
enhanced chemiluminescence (ECL).
To examine phosphotyrosine incorporation in immunoprecipitated receptors, reduced samples prepared as above were run on a 4-12% gradient gel, transferred to nitrocellulose, blotted with mouse antiphosphotyrosine mAb 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY) and
detected by ECL. The membrane was then reprobed with polyclonal
p185c-neu C-18 antiserum to examine receptor expression, which was again detected by ECL.
Pools of Mutants with Degenerate
Transmembrane Domains
The model proposed by Sternberg and Gullick (1989) postulated the transmembrane domain of p185neu as an In degenerate Pool 1 (Fig. 1 B), we randomly targeted
codons for Val, Ala, Gly, or Glu to heptad positions "a,"
"d," and "e." This choice allowed us to examine the potential effects of multiple Glu residues targeted to a variety of
positions. The pool of degenerate oligonucleotides was
amplified by PCR and then ligated into a pSV2-derived
vector encoding p185c-neu (pSV2neuNheI/SacI), replacing
the wild-type transmembrane domain. The DNA was transfected into NIH3T3 cells, and the resulting foci were expanded. Subsequently, to recover individual transmembrane domains from foci of transformed cells, RNA was
prepared from expanded cells and subjected to reverse
transcriptase-PCR as described in Materials and Methods,
and the resulting sequences were then subcloned as unique
plasmids. Individual transmembrane domains recovered
in this fashion were next ligated back into pSV2neuNheI/ SacI and reassayed for transformation of NIH3T3 cells.
This ensured that transformation would be due to a
unique sequence, rather than a mixture of transmembrane
sequences.
The sequences of the transforming mutants isolated
from this pool, designated DEG.1 through DEG.5 (Fig.
1 C), were completely distinct in comparison with other
mutants previously described. Interestingly, none of the
activated mutants except DEG.1 exhibited a Glu at residue 664. Moreover, all of these mutants, except for DEG.2, exhibited multiple Glu residues spaced at intervals of seven residues, or multiples of seven residues.
These results were intriguing considering that Glu residues are rare in hydrophobic transmembrane domains.
These transforming isolates provided clear evidence that
multiple Glu residues do not disrupt the ability of the
transmembrane domain to insert properly into the membrane.
Approximately 15 nontransforming mutants were also
isolated from Pool 1, five of which, DEG.6 through
DEG.10, are described in Fig. 1 D. The distinctions between the transforming and nontransforming isolates were
not entirely obvious. Double-label indirect immunofluorescence was used to determine whether the lack of transforming activity of these mutants might reflect an abnormal subcellular localization. This was accomplished by using
an antibody against an extracellular epitope to detect surface expression in cells before permeabilization, followed
by a second antibody against the COOH terminus to detect intracellular expression in the same cell after permeabilization (Fig. 2). Fig. 2 B shows the immunofluorescence observed for control cells under identical conditions,
with mock-transfected cells negative for both intracellular
and surface staining (Fig. 2 B, A and B), whereas cells expressing p185neu exhibit readily detectable staining both
intracellularly and also at the cell surface (Fig. 2 B, C and
D). As shown in Fig. 2 A, the mutants DEG.7, DEG.9, and
DEG.10 were expressed intracellularly but failed to reach
the cell surface (Fig. 2 A, D, H, and J), suggesting that their
lack of biological activity might be due to aggregation or
interaction with some component of the ER/Golgi. In contrast, the mutants DEG.6 and DEG.8 were expressed at
the cell surface (Fig. 2 A, B and F), suggesting that their
transmembrane domains fail to activate these receptors,
despite localizing normally to the plasma membrane.
Importantly, no transforming mutants were isolated
from degenerate Pool 2 (Fig. 1 B), in which Val, Ala, or
Gly, but not Glu, were targeted to heptad positions "a,"
"d," and "e." Clearly, the presence of Glu residues, which
can potentially satisfy a requirement for hydrogen bonding, is a key element in receptor activation.
Design of Transmembrane Domains with Consensus
Heptad Repeats
The transmembrane domains of the transforming mutants
DEG.1-DEG.5 suggested that there are many allowed
positions for Glu residues, yet the distinction between
these sequences and those of the nontransforming mutants
DEG.6-DEG.10 did not suggest clear rules for predicting
which specific sequences will result in receptor activation
and transformation. We therefore designed simple consensus repeats, containing a centrally located Glu residue, with
most of the other residues substituted by Val, such as
[VVVEVVA]n, [VVVEVVG]n, [VVVEVVV]n, or [AVVEGVL]n (designated CONS.A, CONS.B, CONS.C, and
CONS.D, respectively). Mutants were then constructed with the transmembrane domain composed entirely of
these consensus sequences, repeated over the entire transmembrane domain of ~25 residues. Surprisingly, these
constructs were transforming, with the CONS.A and
CONS.D constructs exhibiting the greatest activity (Fig.
3 B). These results suggest that these simple repeating sequences contain all the information necessary for receptor
activation. Evidently, there is little sequence specificity
necessary for constitutive activation within the p185neu
transmembrane domain itself.
It was also important to demonstrate that transformation was due to specific interactions involving Glu residues. Towards this end, we constructed a derivative of
the CONS.C sequence in which the central Glu of each repeat was changed to Val. This mutant transmembrane domain, consisting entirely of Val residues, was designated
CONS.CE Design of Derivative Transmembrane Domains Based
on Consensus Heptad Repeats
We also tested whether other residues could substitute for
Glu in the context of the CONS.A heptad repeat (Fig. 3 C).
We replaced Glu with Lys, Gln, Ser, and Asp, creating the
mutants CONS.AE As shown in Fig. 4, A-L, indirect immunofluorescence
demonstrated that most of the mutants exhibited cell surface expression. The ability of these altered receptors to
reach the surface indicates that the mutant transmembrane domains did not interfere with the proper localization of the protein. The only exception was CONS.AE
Spacing and Number of Glu Residues
The design of the degenerate pools of transmembrane domains, as well as the design of the subsequent consensus
repeat clones described above, assumed without any real
evidence that there would exist a fundamental heptad
structural motif resulting from the
Most of the transforming DEG mutants described earlier contained multiple Glu residues (Fig. 1 C), as did the
transforming mutants CONS.A-CONS.D, which all contained three Glu residues (Fig. 3 B). The occurrence of
multiple Glu residues is in contrast to p185neu, which contains only a single Glu residue in the transmembrane domain at position 664. We therefore examined whether
multiple Glu residues are in fact required for activity.
This issue was addressed through two different sets of
mutants, one constructed in the background of CONS.A,
[VVVEVVA], and another set in the background of
CONS.C, [VVVEVVV]. These two sets of mutants yielded
somewhat different results. As shown in Fig. 6 A, "singleGlu" derivatives of the CONS.A sequences were designed, each possessing only a single Glu residue located at different positions, and designated CONS.AE2, CONS.AE3,
and CONS.AE4. None of these mutants was transforming,
indicating that within the context of the CONS.A heptad
repeat, a single-Glu residue is not sufficient to provide activation. This result is in contrast to the single Glu664 residue that leads to activation of p185neu, suggesting that
there may be sequence information within the transmembrane domain, in addition to the Val664
Several mutants were also designed in the CONS.C
background that had either one, two, three, or four Glu
residues, designated CONS.C1xE-CONS.C4xE. The clone
containing three Glu residues, CONS.C3xE, is the original
CONS.C clone. As shown in Fig. 6 B , the mutants with either two or four Glu residues, designated CONS.C2xE and
CONS.C4xE, exhibited the greatest activity, almost twice as
much as the mutant with three Glu residues. However, in
this series of mutants, even the presence of a single Glu
residue, as in CONS.C1xE, led to significant transformation
above background.
It is curious that a single Glu residue is activating in the
mutant CONS.C1xE (Fig. 6 B), but not in the very similar
mutant CONS.AE2 (Fig. 6 A). The active mutant has a background CONS.C motif of [VVVEVVV], whereas the inactive mutant has a background CONS.A motif of [VVVEVVA]; these sequences differ only by the Val Kinase Activity of Mutants
Transformation by the neu oncogene is dependent upon
p185neu having a functional kinase domain, and p185neu exhibits increased turnover, autophosphorylation, tyrosine
phosphorylation of intracellular substrates, and activation
of PLC- To confirm kinase activation of the mutants described
here, selected mutants were examined for receptor activation using an immunoprecipitation/kinase assay of transfected Cos-1 cells. As shown in Fig. 7 A, p185neu exhibited
approximately threefold greater autophosphorylation than
p185c-neu (lanes 3 and 2, respectively). The mutants
CONS.CE
Immunoprecipitated samples were also examined for
the presence of phosphotyrosine by immunoblotting. Fig.
8 A demonstrates that two transforming mutants, CONS.C
(lane 5) and CONS.AE
These same mutants were also examined for receptor
dimerization using gradient gel electrophoresis under nonreducing conditions (Weiner et al., 1989b
Function of the Transmembrane Domain
The results presented here demonstrate that activation of
p185c-neu can be achieved by a transmembrane domain
with very little sequence specificity. Glu residues in the appropriate positions of a simplified transmembrane domain, such as [VVVEVVV]n, lead to receptor activation as
demonstrated by biological transformation (Fig. 3), increased kinase activity (Fig. 7), increased incorporation of
phosphotyrosine (Fig. 8), and receptor dimerization (Fig.
9). Receptor activation is conferred by as few as one or as
many as four Glu residues within some of these repeats.
Moreover, a periodic heptad spacing seems to be critical
for activation; this was demonstrated by systematically
varying the spacing between two Glu residues from two to
eight, with only the heptad spacing resulting in activation (Fig. 5 B). Thus, an apparently complex system can be
reduced to a repeated sequence motif. Furthermore, in
these simplified transmembrane domains, Gln is able to
substitute efficiently for Glu, as shown by the mutant
CONS.AE From the transforming isolates of degenerate Pool 1, it
is clear that the location of Glu at position 664 is not critical for activation (Fig. 1), in contrast to prior results suggesting that the primary structure around Glu664 plays an
important role. For instance, mutants with Glu663 or Glu665
were inactive (Bargmann and Weinberg, 1988b Our isolation of the transforming mutants DEG.2, DEG.3,
DEG.4, and DEG.5, all of which lack a Glu at position
664, would suggest that there exists considerable flexibility
with regard to the allowed placement of the Glu residue(s)
that may mediate activation. The isolation of these transforming mutants also demonstrates that the construction
of a degenerate pool of oligonucleotides encoding transmembrane domains provides a powerful approach to identifying and isolating those rare sequences that may allow receptor activation.
Our data show that activation of p185c-neu can be mediated by Glu residues spaced seven amino acids apart in a
repeating heptad. Analysis of the sequences of the isolates
from Pool 1 (Fig. 1) is consistent with this proposal. Most
of the transforming isolates contain Glu residues separated by multiples of seven amino acids. In the nontransforming isolate, DEG.6, which was expressed at the cell
surface (in contrast to DEG.7 and DEG.10), there are Glu
residues spaced seven amino acids apart, but there is also a
Glu that does not follow that pattern. This may suggest
that the presence of a Glu in a nonheptad motif may cause
a disruption in the interaction between receptor molecules
that prevents transforming ability, although it is difficult to
infer absolute rules because of the other sequence variations exhibited by the DEG mutants.
The Val The hydrophobic environment of the membrane creates
an energetic need to shield polar side chains such as that of
Glu (even if uncharged), which can be accomplished by
appropriate hydrogen bonding. Previously, it was proposed that there may be a requirement for Ala661 in
p185neu to allow hydrogen bonding between the Glu664 side
chain of one receptor with the carbonyl oxygen of Ala661 in
the other. There is clearly no strict requirement for Ala at
position 661, however, as shown by the fact that Val is tolerated at position 661 in many of the biologically active
mutants described here. The presence of the branched side
chain at Val661, which might interfere with side chain-
backbone hydrogen bonding, argues against this model
but does not exclude it. More likely, however, would be a
model of side chain-side chain hydrogen bonding between
the Glu of one subunit with the corresponding Glu of the other subunit. The choice between these two models will
require further biophysical characterization. While the results presented in this work are certainly consistent with a
coiled-coil arrangement for the interacting transmembrane domains of p185neu, other structural models cannot
be excluded at the present time, such as one proposed for
the homodimerizing transmembrane domain of glycophorin A (Lemmon et al., 1992 Relevance of p185neu Activation to Other Systems
As summarized in Fig. 10, prior studies have demonstrated
that substitutions at position 664 of p185neu exhibit the following pattern of biological activity: Glu, Gln > Asp, Tyr
The transmembrane domain of the T cell receptor Transmembrane Domain Mutations in Human
Developmental Syndromes
Transmembrane-mediated receptor activation clearly plays
a fundamental role in several human developmental syndromes. Achondroplasia, the most common genetic form
of human dwarfism, results from a single amino acid change
in the transmembrane domain of FGFR3, Gly380 It is clear that transmembrane domains play a more substantial role than serving as structural elements for membrane anchoring. The examples discussed above underscore the importance of understanding the molecular
details of activating mutations in the transmembrane domains of receptors. The principles elucidated here are
likely to be generally relevant to understanding activation
and signal transduction by other receptor tyrosine kinases
in addition to p185neu.
; Schechter et al., 1984
; Bargmann et al., 1986a
, b; Dougall et al., 1994
). Like other members of the
EGFR family, p185c-neu consists of an extracellular ligandbinding domain, a transmembrane domain, and an intracellular tyrosine kinase domain (Drebin et al., 1984
; Ullrich and Schlessinger, 1990
). As for other receptor tyrosine
kinases, ligand binding to the wild-type p185c-neu receptor
induces receptor dimerization, leading to tyrosine kinase activation and subsequent downstream signaling events
(Peles et al., 1991
; Dougall et al., 1994
; Stein et al., 1994
;
for review see Heldin, 1995
).
Glu mutation in the transmembrane
domain (Bargmann et al., 1986b
) or deletions in the extracellular juxtamembrane region (Siegel et al., 1994
). Activated p185neu resembles a ligand-stimulated receptor, as evidenced by its increased tyrosine phosphorylation of other
proteins, as well as elevated levels of autophosphorylation
(Bargmann and Weinberg, 1988a
; Segatto et al., 1988
;
Stern et al., 1988
; Weiner et al., 1989a
; Peles et al., 1991
;
Ben-Levy et al., 1992
; Cao et al., 1992
; Qian et al., 1995
).
In addition, p185neu is primarily found in an aggregated
form while wild-type p185c-neu is monomeric (Weiner et
al., 1989b
). The mutation Val664
Glu may facilitate
dimerization by allowing intermolecular hydrogen bonding between the side chain of Glu664 in one receptor and
the carbonyl oxygen of Ala661 in another receptor, thereby
stabilizing the interaction between the two
-helical transmembrane domains (Sternberg and Gullick, 1989
). In the
initial characterization of p185neu, various mutations at
Val664 were examined. Both Glu and Gln were found to be
equally activating for transformation, with other potential
hydrogen bond-forming residues such as Asp and Tyr exhibiting low activity (Bargmann and Weinberg, 1988b
).
).
The importance of the subdomain surrounding Glu664 has
been further examined by the mutations Val663
Gly and
Gly665
Val, which both abolished transforming activity
(Cao et al., 1992
). This study also found that the lateral position and rotational orientation of Glu in the transmembrane domain did not correlate with transformation.
). This motif consists of a five-residue segment: position 0 (P0), corresponding to Ala661 in p185c-neu, exhibits a
small side chain such as Gly, Ala, Ser, or Thr; position P3
displays an aliphatic side chain like Ala, Val, Leu, or Ile, but is occupied by Glu664 in activated p185neu; and position
P4 exhibits either Gly or Ala. The presence of this motif in
many different receptor tyrosine kinases suggests an important role in facilitating ligand-induced receptor dimerization.
Arg (Rousseau et al.,
1994
; Shiang et al., 1994
). As shown in work from this laboratory, this mutation results in constitutive activation of
FGFR3, not unlike the constitutive activation of p185neu
by the mutation Val664
Glu (Webster and Donoghue,
1996
). Achondroplasia is not the only developmental syndrome arising from transmembrane domain mutations in
FGFR3, as Meyers et al. (1995)
recently identified a novel
substitution mutation in FGFR3 that is responsible for two
distinct developmental syndromes: acanthosis nigricans in association with Crouzon Syndrome. The existence of these
human developmental abnormalities arising from activating mutations in the transmembrane domain of a receptor
tyrosine kinase suggests that the paradigm of receptor activation provided by p185neu is relevant to human disease
and well worth probing in molecular detail.
Materials and Methods
-helical positions designated "a," "d," and "e," shown in Fig. 1. In Pool 1, codons for
Val, Ala, Gly, and Glu were targeted to each of these positions. In Pool 2, the codons for Val, Ala, or Gly, but not Glu, were targeted to these positions. The degenerate oligonucleotides were synthesized on an oligonucleotide synthesizer (Appiled Biosystems Inc., Foster City, CA) and then
amplified by PCR. The degenerate pools were ligated into a pSV2-derived vector encoding p185c-neu (pSV2neuNheI/SacI), replacing the wild-type
transmembrane domain at silent NheI and SacI restriction sites (Webster
and Donoghue, 1996
). The NheI site corresponds to bases 1973-1978, and
the SacI site corresponds to bases 2114-2119, in the published nucleotide sequence encoding p185c-neu (Bargmann et al., 1986a
).
Fig. 1.
Isolates recovered from pools with degenerate transmembrane domains. (A) The sequences of wild-type p185c-neu
and oncogenic p185neu (with the mutation Val664 Glu) are
shown. The locations of the presumptive heptad repeats of the
transmembrane region are shown, along with the letters indicating the heptad positions. Vertical lines designate the probable
borders of the transmembrane domain. The activating Glu residue of p185neu is boldfaced. The five-residue sequence motif
found in many receptor tyrosine kinase transmembrane domains
(Sternberg and Gullick, 1990
), referred to as P0-P4, corresponds
to residues 661-665. The location of P0 and P3 are shown. (B)
Oligonucleotides coding for degenerate transmembrane domains
were synthesized, amplified, and cloned into pSV2neuNheI/SacI
as described in Materials and Methods. In the first pool of degenerate oligos, codons for either Ala, Val, Gly, or Glu were randomly targeted to the "a," "d," and "e" positions. In Pool 2, codons for Ala, Val, or Gly, but not Glu, were targeted to the
same positions. Asterisks denote the positions at which these degeneracies were targeted. (C) Pool 1 was transfected into
NIH3T3 cells; individual foci were expanded and used for PCR-
mediated recovery of the unique transmembrane domain present
in each focus. Each recovered transmembrane domain was ligated into pSV2neuNheI/SacI to ensure that transformation was
due to a unique transmembrane domain rather than a mixture of
sequences. The sequences of these transforming isolates are shown, with dashes designating the residues that remained unchanged from the parent p185c-neu. Glu residues are boldfaced.
(D) For comparison, nontransforming isolates from Pool 1 were
also characterized. The sequences of some of these isolates are
shown. For A, C, and D, transformation by each isolate was
quantitated as a percentage of p185neu. Results represent the average values from three independent experiments, normalized by
cotransfection with pSV2neo, and presented as follows:
, 0-5%
of p185neu; +, 6-40% of p185neu; ++ , 41-100% of p185neu. For B,
transformation by DNA representing the entire pool was recorded as follows:
, 0 foci per 10 µg of DNA; ++, ~35 foci per
10 µg of DNA.
[View Larger Version of this Image (28K GIF file)]
): 5
-GCAGAGA.GCT.AGC.CCG.GTG.ACA. TTC.ATC.ATT.GXA.ACT.GTA .GXA.GXA .GTC.CTG.GXA .TTC.CTG.
GXA.GXA.GTG.GTG.GXA.GTT.GGA .GXA.GXA. ATC. AAA.CGA. AGG. AGA.CAG. AAG. ATC.CGG. AAG.TAT.ACG.ATG.CGT. AGG.
CTG.CTG.CAG.GAA.ACT.GAG.CTC.GTGGAGCC
3
. Degeneracies
were encoded as follows: for Pool 1, X = A, G, C, or T; and for Pool 2, X
= G, C, or T. The pools of single-stranded degenerate oligonucleotides
were PCR amplified using standard conditions with the following primers:
a sense strand primer, corresponding to the 5
-end of the degenerate oligonucleotide sequence shown above and spanning the NheI site, 5
-GCAGAGAGCTAGCCCGGTGAC-3
; and an antisense strand primer,
corresponding to the complement of the 3
-end of the degenerate oligonucleotide sequence shown above and spanning the SacI site, 5
-GGCTCCACGAGCTCAGTTTCC-3
.
).
), as described previously (Maher et al., 1993
). Transfection frequencies were determined by cotransfecting with 0.1 µg of pSV2neo, and half of the cells were subsequently split
into media containing G418 (Maher et al., 1993
). For each DNA sample,
the number of foci was normalized with respect to the number of Neoresistant colonies and then expressed as a percentage of the transformation efficiency obtained with pSV2neuNT, encoding p185neu with the mutation Val664
Glu. All mutants were assayed at least three times in independent experiments for transformation efficiency.
-ATACGCTTCATCTAGAATTTCTTTG-3
, complementary to bases 2323-2347 in the
published nucleotide sequence encoding p185c-neu (Bargmann et al., 1986a
).
This cDNA was used for PCR with primers described above, and the
products digested with NheI and SacI. For the mutants designated DEG.1
through DEG.5, each recovered transmembrane domain was ligated into
pSV2neuNheI/SacI and sequenced, and its ability to activate p185c-neu to a
transforming phenotype was reconfirmed by transfection into NIH3T3
cells. This protocol ensured that transformation was due to a unique sequence, containing a single transmembrane domain, rather than a mixture
of sequences. The nontransforming isolates, DEG.6 through DEG.10,
were recovered in the same way but were negative for NIH3T3 transformation when assayed as individual clones.
) were fixed
in 3% paraformaldehyde/PBS for 10 min and then permeabilized in 0.5%
Triton X-100/PBS for 5 min (Lee and Donoghue, 1992
). Cells were incubated with mouse monoclonal antibody 7.16.4 to an extracellular epitope
of p185c-neu (c-neu [AB-4] clone 7.16.4 from Oncogene Science Inc., Manhasset, NY) followed by FITC-conjugated goat anti-mouse antiserum
(Boehringer Mannheim Corp., Indianapolis, IN). To detect cell surface
expression of p185c-neu and derivatives, cells were fixed with paraformaldehyde and incubated without permeabilization, as described previously
(Hannink and Donoghue, 1986
; Lee and Donoghue, 1992
).
).
After 2 d, the transfected cells were labeled for ~7 h with 100 µCi of
[35S]Cys and [35S]Met each per ml in DME lacking Cys and Met. The cells
were rinsed with TS buffer and lysed in 0.5 ml NP-40 lysis buffer (20 mM
Tris, pH 7.5, 137 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, 5 mM EDTA, 10 µg/ml aprotinin, 10% glycerol), and clarified lysates were prepared (Maher et al., 1993
). Immunoprecipitation was carried out using
mAb 7.16.4, and immune complexes were collected with protein A-Sepharose beads. After immunoprecipitation, the Sepharose beads were washed
in NP-40 lysis buffer, and the samples were split in half. Samples for the
kinase assay were washed twice in kinase buffer (20 mM Tris, pH 7.5, 10 mM
MnCl2, 5 mM MgCl2), resuspended in 20 µl kinase buffer containing 10 µCi
-[32P]ATP, incubated at room temperature for 10 min, washed twice with NP-40 lysis buffer, and analyzed by 7.5% SDS-PAGE and autoradiography. The other half of the samples was analyzed by 7.5% SDSPAGE, and the 35S-labeled proteins were visualized by fluorography.
Results
-helical sequence in which Glu664 promotes activation by creating a hydrogen bond between two receptor molecules. To
further characterize these interactions, we constructed pools of degenerate oligonucleotides that permitted variability in the transmembrane domain at positions P0, P3,
and P4 in the Sternberg and Gullick notation, which are
predicted to be of greatest structural importance based on
a comparison of transmembrane domains from many
growth factor receptors (Sternberg and Gullick, 1990
). The
repeating heptad motif present in interacting
-helical domains can also be designated (abcdefg)n, in which the "a"
and "d" positions are occupied by the residues at the interface where the two
-helical domains interact.
Fig. 2.
Immunofluorescence of nontransforming isolates
DEG.6-DEG.10. Double-label indirect immunofluorescence was
used to detect either cell surface expression of p185c-neu-related
proteins (right) or, after permeabilization of cells, intracellular
expression (left). Conditions for the double-label immunofluorescence are described in Materials and Methods. (A) (A and B)
mutant DEG.6; (C and D) mutant DEG.7; (E and F) mutant DEG.8; (G and H) mutant DEG.9; (I and J) mutant DEG.10. (B)
(A and B) Mock transfected cells; (C and D) p185neu.
[View Larger Versions of these Images (40 + 76K GIF file)]
Fig. 3.
Consensus sequences and derivatives. (A) The p185c-neu and p185neu sequences
are shown. The locations of the presumptive
heptad repeats, along with the letters indicating the heptad positions, are specified. Vertical lines designate the probable borders of
the transmembrane domain. Glu664 is in boldface. (B) Consensus sequence mutants were
constructed as described in the text. The
placement of the Glu residues in each mutant
is identical. Based on the variations found in
the transforming isolates from degenerate
Pool 1, the heptad position g in CONS.B and
CONS.C was mutated from Ala in CONS.A
to Gly and Val, respectively. The repeating
heptad in CONS.D is heptad 2 from p185neu
with Thr662 changed to Val. The mutant
CONS.CE V has a transmembrane domain
composed entirely of Val residues and was
constructed as a negative control. Glu residues are boldfaced. (C) Derivatives of
CONS.A mutant. Derivatives were designed
in which the Glu residues were substituted by
other residues capable of hydrogen bonding,
such as Lys, Gln, Ser, or Asp. For example, CONS.AE
Q is identical to CONS.A except that the Glu residues are substituted by Gln. Transformation by each isolate was quantitated as a percentage of p185neu. Results represent the average values from three independent experiments, normalized by cotransfection with pSV2neo, and presented as
, +, or ++ as described in Fig. 1. Surface expression was determined by indirect immunofluorescence, as described in text.
[View Larger Version of this Image (28K GIF file)]
V, and was completely devoid of transforming
activity (Fig. 3 B).
K, CONS.AE
Q, CONS.AE
S, and
CONS.AE
D. These mutants were designed to test whether
other hydrogen-bonding residues could substitute in place
of Glu. Only substitution with Gln allowed significant
transforming activity, whereas Lys, Ser, and Asp were unable to activate the receptor. The observation that Gln can
substitute for Glu in the mutant CONS.AE
Q is consistent
with the original characterization of p185neu showing that
Val664
Glu and Val664
Gln were equally effective at activation (Bargmann and Weinberg, 1988b
).
D,
which exhibited little or no cell surface staining, even
though the protein is clearly being translated, as shown by
the intracellular staining of permeabilized cells (Fig. 4, O).
However, its reduced surface expression may be due to aggregation or interaction with another protein that is retained in the ER and/or Golgi, as discussed previously for
some of the nontransforming mutants, DEG.7, DEG.9, and DEG.10.
Fig. 4.
Indirect immunofluorescence of consensus mutants and derivatives. Indirect
immunofluorescence using the
monoclonal antibody 7.16.4 directed against the extracellular
region of p185c-neu and a fluorescein-conjugated goat anti-
mouse secondary antibody revealed both cell surface and
intracellular protein expression. A-L show nonpermeabilized cells to examine cell surface expression. (A) p185c-neu;
(B) p185neu; (C) CONS.A; (D)
CONS.B; (E) CONS.C; (F)
CONS.D; (G) CONS.AE2; (H)
CONS.AE4; (I) CONS.AE K;
(J) CONS.AE
Q; (K) CONS.
AE
S; (L) CONS.CE
V. M-O
show cells after permeabilization to examine intracellular expression. (M) p185c-neu; (N)
p185neu; (O) CONS.AE
D.
[View Larger Version of this Image (67K GIF file)]
-helical transmembrane domain. To directly examine this premise, we constructed a series of clones in which we varied the spacing between Glu residues. As the parental clone for this series,
we chose a clone with two Glu residues spaced seven residues apart in a transmembrane domain composed otherwise of only Val residues, designated CONS.C2xE. The
spacing between the two Glu residues was then varied
from two to eight residues, as shown in Fig. 5 A. The results of transformation assays with these clones revealed a
dramatic effect of spacing, with the heptad spacing yielding significantly greater transforming activity than any of
the other clones (Fig. 5 B). When cells expressing these
constructs were examined by indirect immunofluorescence, all constructs exhibited cell surface expression (data
not shown), indicating that the lack of transformation was
not due to a defect in surface localization. This experiment
provides compelling evidence for a basic heptad structural
motif in the transmembrane domain of activated forms of
p185c-neu.
Fig. 5.
Importance of the spacing of Glu residues. (A) A series
of clones, based on the mutant CONS.C2xE, were constructed
varying the spacing between two Glu residues, from a minimum
spacing of two residues (diad mutant) to a maximum spacing of
eight residues (octad mutant). (B) Transformation by each isolate shown in (A) was quantitated as a percentage of p185neu. Numerical results of transformation assays are presented graphically and represent the average values from two independent experiments, normalized by cotransfection with pSV2neo.
[View Larger Versions of these Images (18 + 16K GIF file)]
Glu mutation,
that stabilizes dimerization (Cao et al., 1992
). We considered the possibility that these mutant proteins might fail to
reach the cell surface and that this defect might explain their inactivity in transformation assays. However, all of
the single-Glu proteins were expressed at the cell surface,
as shown by indirect immunofluorescence in Fig. 4, G and
H, for CONS.AE2 and CONS.AE4.
Fig. 6.
Importance of the number of Glu
residues. (A) Derivatives of CONS.A. Three
derivatives were constructed, each having a
single Glu residue. Depending upon the position of the Glu residue, these mutants were
designated CONS.AE2, CONS.AE3, and
CONS.AE4. (B) Derivatives of CONS.C. Derivatives were constructed in the background
of CONS.C having from one to four Glu residues. These clones are designated CONS.C1xE,
CONS.C2xE, CONS.C3xE, and CONS.C4xE.
Note that CONS.C3xE is the parental
CONS.C mutant presented in Fig. 3. Transformation by each isolate was quantitated as
a percentage of p185neu. Results represent the
average values from three independent experiments, normalized by cotransfection with pSV2neo, and presented as , +, or ++ as described in Fig. 1. For transformation results shown in B, numerical values are also shown in parentheses as the percentage of p185neu.
[View Larger Version of this Image (19K GIF file)]
Ala substitution in the last position of the repeat motif. This
suggests that the ability of a single Glu residue to activate
a particular transmembrane domain, as occurs in the case
of p185neu, may be critically dependent upon other specific
details of the transmembrane domain that are not immediately obvious. These results may help to explain the negative results of earlier studies in which repositioning of the
Glu664 residue to other locations in the transmembrane domain did not result in transformation (Cao et al., 1992
).
(Bargmann and Weinberg, 1988a
; Stern et al.,
1988
; Weiner et al., 1989a
,b; Peles et al., 1991
; Cao et al.,
1992
; Brown et al., 1994
). Moreover, transformation and
SH2-dependent signaling by p185neu requires intermolecular receptor association that is mediated by the transmembrane domain, as demonstrated by functional complementation between truncated kinase-active p185neu and full-length
kinase-inactive p185neu (Qian et al., 1994
, 1995
). Another
indication of receptor dimerization and aggregation is provided by measurement of ligand-binding using chimeras
consisting of the ligand-binding domain of EGFR substituted into either p185c-neu or p185neu; the Val
Glu mutation in the transmembrane domain of such EGFR/Neu chimeras results in the conversion of low-affinity ligandbinding sites into high-affinity binding sites, consistent
with an oligomerized state of the oncogenic receptor
(Ben-Levy et al., 1992
).
V and CONS.C were examined, as they represent an interesting pair of closely related mutants. The first
of these is inactive in transformation assays, whereas the
latter mutant is active. In the immunoprecipitation/kinase
assay, the biologically active mutant CONS.C exhibited a
similar increase in autophosphorylation compared with
the inactive receptor CONS.CE
V (lanes 5 and 4, respectively). We also examined another pair of closely related
mutants, CONS.AE
S and CONS.AE
Q, where once again
the first mutant is inactive in transformation assays, but
the latter mutant is active. Once again, in the immunoprecipitation/kinase assay, the biologically active mutant
CONS.AE
Q exhibited an increase in autophosphorylation compared with the inactive mutant CONS.AE
S
(lanes 7 and 6, respectively). In this experiment, similar
levels of protein expression were achieved for p185c-neu,
p185neu, and the various mutants examined, as demonstrated by 35S-metabolically labeled/immunoprecipitated
proteins from the same lysates (Fig. 7 B). Thus, consistent
with prior experimental results from other laboratories
(Bargmann and Weinberg, 1988a
; Stern et al., 1988
; Weiner
et al., 1989a
,b; Cao et al., 1992
), biologically active derivatives constructed in this work exhibited increased levels of
kinase activity, as determined by receptor autophosphorylation in immunoprecipitation/kinase assays.
Fig. 7.
Immunoprecipitation/kinase assay of consensus mutants. Lysates from transfected cells, labeled metabolically with
[35S]Cys and [35S]Met, were subjected to immunoprecipitation using monoclonal antibody 7.16.4, as described in Materials and
Methods. (A) Kinase assay. Immunoprecipitated lysates were
subjected to in vitro kinase reactions using -[32P]ATP. 32P-labeled
proteins were detected by SDS-PAGE and autoradiography. Exposure time was 21 h. (B) Expression. To demonstrate equivalent levels of protein expression for different mutants, identical aliquots of immunoprecipitated lysates as used in A were analyzed by SDS-PAGE followed by fluorography to detect 35S-labeled
proteins. Exposure time was 3 d. The samples shown in lanes 2, 4,
and 6 represent nontransforming clones, while the samples shown
in lanes 3, 5, and 7 represent transforming derivatives. Lane 1,
mock; lane 2, p185c-neu; lane 3, p185neu; lane 4, CONS.CE
V; lane
5, CONS.C; lane 6, CONS.AE
S; lane 7, CONS.AE
Q.
[View Larger Version of this Image (41K GIF file)]
Q (lane 7), exhibit significant incorporation of phosphotyrosine into immunoprecipitated
receptors, as did the positive control, p185neu (lane 3).
Little or no phosphotyrosine was associated with the nontransforming mutants, CONS.CE
V (lane 4) and
CONS.AE
S (lane 6), or with the nontransforming control, p185c-neu (lane 2). As a control for receptor expression
levels, B demonstrates approximately equivalent levels of
receptor expression, determined by immunoblotting using
polyclonal p185c-neu C-18 antiserum.
Fig. 8.
Detection of phosphotyrosine in immunoprecipitated
receptors. Lysates were prepared from transfected cells and were
subjected to immunoprecipitation using monoclonal antibody
7.16.4, as described in Materials and Methods. Immunoprecipitated lysates were analyzed on a 4-12% gradient gel under reducing conditions, transferred to nitrocellulose, probed with monoclonal phosphotyrosine antiserum 4G10 (A) or with polyclonal
p185c-neu C-18 antiserum (B), and specific proteins were detected
by ECL. The samples shown in lanes 2, 4, and 6 represent nontransforming clones, while the samples shown in lanes 3, 5, and 7 represent transforming derivatives. A shows detection of phosphotyrosine, and B shows detection of receptor expression. Samples are: lane 1, mock; lane 2, p185c-neu; lane 3, p185neu; lane 4,
CONS.CE V; lane 5, CONS.C; lane 6, CONS.AE
S; lane 7,
CONS.AE
Q.
[View Larger Version of this Image (36K GIF file)]
; Burke et al.,
1997
). Fig. 9 B presents these results, while the same lysates were also analyzed under reducing conditions as a
control, shown in A. Two transforming mutants, CONS.C
(lane 5) and CONS.AE
Q (lane 7), exhibited significant
bands of dimeric receptors, as did the positive control,
p185neu (lane 3). Importantly, no dimerization was observed for the nontransforming mutants analyzed here,
CONS.CE
V (lane 4) and CONS.AE
S (lane 6), or for the
nontransforming control, p185c-neu (lane 2). The ability to
detect receptor dimerization for the transforming mutants,
but not for the biologically inactive mutants, indicates that
the consensus sequences described here promote receptor
dimerization, and that in this respect they are similar to
the parental transforming clone, p185neu.
Fig. 9.
Dimerization assay of consensus mutants.
Lysates were prepared from
transfected cells in RIPA
containing 10 mM iodoacetamide and were subjected to
immunoprecipitation using
monoclonal antibody 7.16.4, as described in Materials and
Methods. Immunoprecipitated lysates were analyzed
on a 4-12% gradient gel and
transferred to nitrocellulose,
probed with a polyclonal
p185c-neu C-18 antiserum, and
specific proteins were detected by ECL. The samples
shown in lanes 2, 4, and 6 represent nontransforming
clones, while the samples
shown in lanes 3, 5, and 7 represent transforming derivatives. A shows samples
analyzed under reducing gel
electrophoresis conditions.
B shows samples analyzed under nonreducing gel electrophoresis conditions. Samples are: Lane 1, mock; lane 2, p185c-neu; lane 3,
p185neu; lane 4, CONS.CE V; lane 5, CONS.C; lane 6, CONS.AE
S; lane 7, CONS.AE
Q.
[View Larger Version of this Image (38K GIF file)]
Discussion
Q. This is consistent with previous observations
(Bargmann and Weinberg, 1988b
) that Gln, but not Ser,
Lys, or Asp, was able to substitute for Glu at position 664 in receptor activation (Fig. 3).
). More recently, Cao et al. (1992)
analyzed the requirements for activation of the p185neu transmembrane domain; all of their
transforming mutants retained Glu at position 664, and a
mutant (designated lil670VEG) with the motif VEG seven
residues COOH-terminal, was nontransforming.
Glu mutation that activates p185neu is also activating when introduced into the corresponding position
of the Drosophila EGFR homologue (DER), resulting in
increased kinase activity (Wides et al., 1990
). Although it
was initially reported that the corresponding Val627
Glu
mutation does not activate the human EGFR transmembrane domain (Kashles et al., 1988
), a more recent study
suggests that this mutation is activating (Beguinot et al.,
1995
). Additionally, a Val
Glu mutation at position 659 in c-erbB-2, the human Neu homologue that is overexpressed in many breast cancers, results in elevated tyrosine
kinase activity (Segatto et al., 1988
). These examples provide further evidence for activation of EGFR family members by appropriate introduction of a strongly polar residue in their transmembrane domains.
; Treutlein et al., 1992
).
Val, Lys, Gly, His (Bargmann and Weinberg, 1988b
). These results are generally consistent with a role of polar
or hydrophilic residues in promoting activation, although
the failure of Lys and His to allow for activation remains
to be explained. The E5 oncoprotein of bovine papilloma
virus (BPV) provides another example of the importance
of the transmembrane domain for activation, since this 44residue integral membrane protein requires dimerization and disulfide bond formation for its biological activity
(Horwitz et al., 1988
, 1989; Goldstein et al., 1992
). A key
requirement of its hydrophobic transmembrane domain is
the presence of Gln17, which appears to participate in interhelical hydrogen bonding and may be substituted by either Glu or Lys, and to a lesser extent by His (Meyer et al.,
1994
). Significantly, the transmembrane domain of E5 can
be largely replaced by the membrane-spanning region
from activated p185neu (Meyer et al., 1994
).
Fig. 10.
Oncoproteins and receptors activated by mutations in
the transmembrane domain. Amino acids are shown that allow
activation of p185c-neu when substituted at residue 664, activation
of BPV-E5 when substituted at residue 17, and activation of
FGFR3 when substituted at residue 380, as discussed in the text.
Those substitutions that allow activation in these three different
systems share the property that they are strongly polar in an otherwise hydrophobic membrane environment, and thus share the
ability to participate in hydrogen bond formation that may stabilize dimer formation.
[View Larger Version of this Image (29K GIF file)]
chain (TCR
) also appears to play a key role in complex
formation between TCR
and the CD3
chain, mediated
by two basic residues in the TCR
transmembrane domain (Cosson et al., 1991
). As another relevant example,
the basic residues within the transmembrane domain of gp41 in human immunodeficiency virus type 1 (HIV-1)
have been suggested to play a role in oligomerization of
the HIV-1 envelope glycoprotein (Owens et al., 1994
).
Arg
(Rousseau et al., 1994
; Shiang et al., 1994
). This mutation results in constitutive activation of FGFR3, leading to abnormal development at the growth plate of long bones
(Webster and Donoghue, 1996
). As depicted in Fig. 10, we
have previously demonstrated that mutations at residue
380 of FGFR3 exhibit the following pattern of activation:
Arg, Glu, Asp > Gln, His
Lys (Webster and Donoghue, 1996
). Recently, Meyers et al. (1995)
characterized an autosomal dominant mutation in FGFR3 that is manifested
as two distinct developmental syndromes presented together: first, acanthosis nigricans, a hyperplastic epithelial
proliferation syndrome resulting in thickened hyperpigmented skin; and second, Crouzon Syndrome, characterized by craniosynostosis or premature fusion of the cranial sutures, resulting in cranial malformation and ocular proptosis. The mutation responsible for these developmental
anomalies maps to the transmembrane domain of FGFR3
and, surprisingly, involves a single substitution mutation
creating a Glu residue (Ala391
Glu). Using FGFR3/Neu
chimeras, we have recently demonstrated that the Ala391
Glu mutation also leads to constitutive receptor activation (Webster, M.K., and D.J. Donoghue, unpublished data).
Received for publication 3 October 1996 and in revised form 18 February 1997.
1. Abbreviations used in this paper: BPV, bovine papilloma virus; ECL, enhanced chemiluminescence; EGFR, epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; TCRWe thank Robert Weinberg (Massachusetts Institute of Technology, Cambridge, MA) for providing the pSV2neuN and pSV2neuNT plasmids. We thank Laura Castrejon for excellent editorial assistance, Brendan Galvin for excellent technical support and advice, and all lab members for their many valuable comments and suggestions concerning experimental design and preparation of the manuscript. We also thank David Stern and Chris Burke (Yale University, New Haven, CT) for advice about the dimerization assay.
This work was supported by grant CA 40573 from the National Institutes of Health, grant 1RB-0318 from the University of California Breast Cancer Research Program, and grants 3RT-0242 and 4FT-0193 (to M.K. Webster) from the University of California Tobacco Related Disease Research Program.