(Received for publication, October 4, 1994; and in revised form, December 2, 1994)
From the
Receptor tyrosine kinases play a central role in cellular
growth, differentiation, and oncogenesis. All of these responses are
triggered by growth factors interacting with the extracellular domain
of transmembrane-spanning receptors, leading to dimerization and
activation of an intrinsic tyrosine-specific kinase activity by an
allosteric mechanism. Precise mechanisms of receptor dimerization
remain poorly understood, and current models suggest that the ligand
binding domain plays a major determining role. To examine the role of
the intracellular domain in the association of juxtaposing receptor
molecules, the full-length epidermal growth factor receptor was
transiently co-expressed in human 293 fibroblasts with a truncated
receptor that lacks the extracellular domain. After metabolic labeling
with [S]methionine, the association of these
receptor constructs was monitored by co-immunoprecipitation with an
extracellular domain-specific antibody. Specific interactions found
between these receptors were independent of ligand binding or an intact
ATP-binding site. Truncated receptors that had sequences necessary for
membrane localization, and that were capable of interacting with
full-length receptor tyrosine kinase, also displayed constitutive
kinase activity as well as the capacity to transphosphorylate
kinase-negative receptors. Receptor co-immunoprecipitation occurred
between constructs that comprise the intracellular domains of the
epidermal growth factor and
-platelet-derived growth factor
receptors, and HER-2. Subsequent deletion analysis has identified the
major region of epidermal growth factor receptor intracellular
interaction to be within the kinase domain.
The epidermal growth factor (EGF) ()receptor is a
member of a family of evolutionary conserved transmembrane molecules
that bind to polypeptide growth factors in their external domain and
transmit growth/differentiation signals via an intracellular tyrosine
kinase domain (reviewed by Ullrich and Schlessinger(1992)). The
activation of these receptor tyrosine kinases (RTKs) has been the
subject of many investigations, not least because of their implication
in the progression of certain neoplastic diseases. EGF stimulation of
its cognate receptor rapidly induces receptor autophosphorylation to
create phosphotyrosine residues, which, in the context of specific
peptide sequences, provide coded sites for receptor-specific substrate
interaction and subsequent phosphorylation (see Anderson et
al.(1990)). This ligand-induced activation and autophosphorylation
process is probably mediated by a mechanism common to all RTKs
involving allosteric receptor oligomerization (Schlessinger, 1988).
In the intermolecular model of activation, there is an assumed ligand-induced stabilization of an oligomeric, most likely dimeric, complex that possesses an elevated kinase activity (Yarden and Schlessinger, 1987a, 1987b; Sorokin et al., 1994). Further experimental support for this mechanism came from studies on transphosphorylation of structurally distinguishable EGF receptor mutants (Honegger et al., 1989). Similarly, the significance of receptor-receptor interactions has been confirmed by transphosphorylation of kinase-negative receptors, a phenomenon that occurs between receptors comprising both homologous (Tartare et al., 1991; Ballotti et al., 1989) and heterologous cytoplasmic domains (Lammers et al., 1990). Two related RTKs, the EGF receptor and HER-2, are clearly capable of transphosphorylation (King et al., 1988), as are structurally distinct fibroblast growth factor receptor family members (Bellot et al., 1991). Consistent with the dimerization and transphosphorylation mode of RTK activation, signaling defective EGF receptor mutants interfere with both normal and transforming signals generated by wild-type EGF receptors, presumably due to the formation of inactive heterodimers (Kashles et al., 1991; Redemann et al., 1992).
The role of the extracellular ligand binding domain in regulating EGF receptor function has been suggested by the inhibition of tyrosine kinase activity using a 100-kDa truncated receptor that retains an intact EGF-binding site (Basu et al., 1989). In addition, the purified EGF receptor extracellular domain alone has been found to undergo ligand-induced oligomerization confirming an active role for the ligand binding domain in receptor dimerization (Lax et al, 1991), interactions that are probably stabilized by conformational changes (Greenfield et al., 1989). However, the role of the cytoplasmic domains in receptor-receptor aggregation has not yet been fully explored. Studies with a naturally occurring altered form of the EGF receptor which lacks the majority of the ligand-binding domain, the v-ErbB oncoprotein, failed to identify the presence of oligomers using non-denaturing gel electrophoresis (Yarden and Schlessinger, 1987b). More recently, these observations have been re-examined using sedimentation analysis on sucrose density gradients and dimers of a truncated EGF receptor variant that resembles v-ErbB were apparent (Kwatra et al., 1992). Therefore, there are a number of conflicting observations which independently support the role of extracellular and cytoplasmic domain interactions in EGF receptor dimerization/activation. An interesting question that remains to be answered involves the identification of the receptor domain(s) mediating the protein-protein interactions required for oligomerization.
This study has focused on intracellular domain interactions and utilizes an intact cell system involving the co-expression of full-length RTKs with genetically engineered truncated receptors that lack the extracellular ligand-binding domain. Specific protein-protein interactions were monitored by co-immunoprecipitation analysis with an antibody that exclusively recognizes the ligand binding domain. The data support the concept of cytoplasmic involvement in receptor homo-/hetero-interactions and, furthermore, highlight for the first time the importance of the tyrosine kinase domain in receptor oligomerization.
For immunoblotting, proteins were electrophoretically transferred onto nitrocellulose and probed with anti-EGF receptor (Sigma; monoclonal antibody E3138)) or anti-mouse PDGF receptor (Oncogene Science) antibodies. Immunoreactive proteins were visualized using the ECL system (Amersham).
Figure 1:
Schematic representation of receptor
constructs and co-immunoprecipitation of full-length and truncated
receptors. A, preparation of receptor mutants is described
under ``Experimental Procedures.'' The blackbox represents the tyrosine kinase domain and the shaded box the
transmembrane domain. EGF-R, wild-type human EGF receptor; EGF-R (-X+L), without extracellular domain, with
hydrophobic leader sequence; EGF-R (-X-L), without
extracellular domain and hydrophobic leader sequence; EGF-R
(-X-L-TM), without extracellular domain,
hydrophobic leader sequence, and transmembrane domain; PDGF-R
(-X-L), mouse -PDGF receptor without
extracellular domain and hydrophobic leader sequence.
-EGF-R
represents the extracellular
domain-specific antibody used for co-immunoprecipitation. B,
human kidney fibroblast 293 cells expressing truncated and/or
full-length receptors were metabolically labeled overnight with 60
µCi of [
S]methionine in methionine-free DMEM
containing 6% dialyzed fetal calf serum. Cells were then incubated in
the presence or absence of 100 ng/ml EGF for 10 min. After
solubilization, receptors were immunoprecipitated with the
extracellular domain-specific EGF receptor antibody and analyzed by
7.5% SDS-PAGE under reducing conditions. An autoradiograph of this
immunoprecipitate (upperpanel) and the total amount
of the expressed cytoplasmic construct (cyt.) in the lysate
before immunoprecipitation (lowerpanels) are
shown.
In the case of the EGF receptor, a strong association was found with EGF-R (-X-L), which migrated with the expected molecular mass of approximately 70 kDa, and also EGF-R (-X+L), which migrated with a molecular mass slightly higher than anticipated (Fig. 1B). A likely explanation for the discrepancy in molecular mass is that the close juxtaposition of the hydrophobic leader and transmembrane sequences sterically hinders cleavage of the leader peptide in the lumen of the endoplasmic reticulum during biosynthesis and that it remains attached at the N terminus of the truncated receptor. With both truncated receptors, a slightly smaller product is also apparent, which may reflect limited proteolytic cleavage; however, this band and the aberrant migration of EGF-R (-X+L) may also indicate differential phosphorylation. The truncated PDGF receptor (PDGF-R (-X-L)), migrating at the expected molecular mass of 75 kDa, also associated with the full-length EGF receptor; however, no association was apparent between the full-length EGF receptor and a truncated EGF receptor that lacks the leader and transmembrane sequences (Fig. 1B). The failure of this particular receptor mutant to associate could not be explained in terms of reduced expression since all of the truncated receptors were expressed in approximately equal amounts (Fig. 1B). Cellular fractionation also confirmed that EGF-R (-X-L-TM) was localized predominantly in the cytosolic compartment in contrast with other truncated receptors, which were found associated with a membrane fraction (data not shown).
Figure 2: Tyrosine autophosphorylation and transphosphorylation of full-length and truncated receptors. A, cells overexpressing the full-length or truncated receptors were lysed, and total extracts separated on 10% SDS-PAGE before immunoblotting against antibodies specific for the intracellular domain of the receptor (left panel) or anti-phosphotyrosine antibody (right panel). Before lysis, cells overexpressing the EGF receptor were stimulated with 100 µg of EGF for 10 min. Cells transfected with an empty p-CMV vector were used as a control. B, the wild-type EGF-R or EGF-R kinase -ve were co-expressed in 293 cells with empty vector (pCMV) or the various truncated receptors indicated. Receptors were immunoprecipitated, separated on 7.5% SDS-PAGE and probed with the antibodies indicated.
To assess whether truncated receptors that associate with full-length receptors are capable of transphosphorylation, a kinase-negative EGF receptor was co-transfected with each truncated receptor. Following immunoprecipitation with an extracellular domain-specific antibody, blotting against anti-phosphotyrosine antibody indicated that those truncated receptors that associate, i.e. EGF-R (-X+L), EGF-R (-X-L), and PDGF-R (-X-L), have undergone autophosphorylation and are able to significantly transphosphorylate a kinase-negative receptor (Fig. 2B).
Both EGF-R (-X-L) and PDGF-R (-X-L) show a strong association with full-length RTKs comprising the intracellular domains of the EGF, PDGF receptors, and HER-2 (Fig. 3). As an added positive control, the non-catalytic subunit of phosphatidylinositol 3-kinase (p85) was co-expressed and then co-immunoprecipitated with the full-length RTKs. The p85 subunit is known to form a high affinity complex with some RTKs, in particular the PDGF receptor, which contains the most appropriate motif for interaction with the p85 SH2 domains (Escobedo et al., 1991; Otsu et al., 1991; Skolnik et al., 1991). Although not quantitated, the association of the truncated receptors appears as great as the RTK-p85 interaction and in some instances greater, notably with the EGF receptor and HER-2 (Fig. 3). The specificity of these interactions was confirmed by the inability of the truncated receptors when expressed alone to immunoprecipitate with the extracellular domain-specific antibody (Fig. 3, rightmost two lanes).
Figure 3:
Homo- and
hetero-association of receptors comprising the cytoplasmic domains of
EGF and PDGF receptors, and HER-2. Cells co-expressing full-length and
truncated receptors or p85, the non-catalytic subunit of
phosphatidylinositol 3-kinase, were labeled overnight with
[S]methionine and receptors immunoprecipitated
from lysed cells with an antibody against the extracellular domain of
the EGF receptor. Samples were separated by 7.5% SDS-PAGE and receptors
visualized by autoradiography. The full-length PDGF receptor and HER-2
represent chimeric molecules that have the extracellular domain of the
EGF receptor connected to the respective cytoplasmic domains. As
immunoprecipitation controls, the EGF and PDGF receptor cytoplasmic
constructs were transfected alone.
Figure 4:
Identification of the domain responsible
for EGF receptor intracellular interactions by site-directed deletion
and co-immunoprecipitation analysis. A, receptor deletion and
point mutants were generated as described under ``Experimental
Procedures,'' and their topology relative to the wild-type EGF
receptor is depicted schematically. The white area in the
kinase domain of EGF-R kinase -ve indicates the position of the
lysine-721 to alanine point mutation. The receptors were co-transfected
with the cytoplasmic domain construct, EGF-R (-X+L), cells
were metabolically labeled and receptors immunoprecipitated with the
extracellular domain-specific antibody as described under
``Experimental Procedures'' before separation on 7.5%
SDS-PAGE. An autoradiogram of the immunoprecipitate (upperpanel) and the total amount of the expressed EGF-R
(-X-L) in the lysate before immunoprecipitation (lowerpanel) are shown. B, the truncated receptors
EGF-R (-X+L) kinase -ve or EGF-R (-X+L)
C-terminal were co-expressed with the indicated receptors and
analyzed as described in a on 10%
SDS-PAGE.
The present study provides the first demonstration that an intact kinase domain is a prerequisite for stable intracellular domain association within the EGF receptor and other related RTKs, in a manner that is independent of an intact ATP-binding site but requires sequences that are presumably necessary for membrane localization. These receptor-receptor interactions are not due to enzyme-substrate interactions between an active kinase domain and the autophosphorylation sites in the C-terminal tail, since two receptors that both lack all of the known EGF receptor phosphorylation sites in the C-terminal tail also underwent co-immunoprecipitation. Since this association is independent of ligand binding and occurs with kinase-negative receptors, it seems that the ability of RTK cytoplasmic domains to oligomerize is an intrinsic property that reflects the likely existence of a ``dimerization motif.''
A much weaker physical association between full-length RTKs has been noted previously (Lammers et al., 1990). Therefore, one possible explanation for the high degree of association with truncated receptors found in this study is that, under normal circumstances, newly synthesized RTKs preparing to migrate from the endoplasmic reticulum to the cell surface are prevented from forming intracellular domain interactions by an extracellular domain-mediated structural constraint. Upon ligand binding, a number of conformational changes have been observed (Greenfield et al., 1989); therefore, the observation of spontaneous oligomerization of receptors lacking the extracellular domain reported in this study support the idea that RTK activation involves subtle structural alterations in the extracellular domain sufficient to remove any constraints, allowing intracellular domain interactions and transactivation events to proceed. The ligand-independent activation of v-ErbB oncoproteins, naturally occurring EGF receptor variants that lack the majority of the ligand binding domain (Downward et al., 1984), is also compatible with such a model.
Since a truncated receptor that lacks all membrane localization signals including the transmembrane domain (EGF-R(-X-L-TM)) failed to interact with the full-length EGF receptor, it would appear that either an intact transmembrane domain or a membrane environment is important for receptor-receptor interactions. The transmembrane domain of the EGF receptor has been extensively mutated and this region found to be non-essential for receptor dimerization and autophosphorylation (Carpenter et al., 1991). The failure of the truncated EGF receptor (EGF-R (-X+L)) to associate with an EGF receptor mutant that lacks the majority of the intracellular domain, apart from the juxtamembrane region, would also suggest a minimal involvement of the transmembrane domain in receptor interactions. However, the importance of the transmembrane domain in RTK activation in general cannot be excluded since an activating mutation has been found in this region of HER-2 resulting in constitutive kinase activity and transformation (Bargmann et al., 1986). In the EGF receptor, the transmembrane domain may play a more passive role during activation, serving more as a membrane anchor for the intracellular kinase domain. The inability of a truncated receptor that lacks the transmembrane domain (EGF-R(-X-L-TM)) to undergo both oligomerization and auto- or transphosphorylation suggests that both membrane localization and receptor-receptor intracellular domain interactions may be essential features of tyrosine kinase activation.
The domains within RTKs that are responsible for receptor-receptor interactions have not been described. The findings reported here have identified the tyrosine kinase domain as a region necessary for oligomerization and therefore likely to harbor a specific ``dimerization motif.'' Different RTKs are clearly capable of heterodimerization (Fig. 3) (this study; Lammers et al., 1990; King et al., 1988; Tartare et al., 1991; Ballotti et al., 1989), and it is now apparent that the most conserved RTK structural domain plays an integral role in this process. Transphosphorylation events between the sub-class I receptors such as the EGF receptor and HER-2 have been reported previously, and it is also apparent from this study that the sub-class III PDGF receptor also has the capacity for trans-activation (see Fig. 2B). At present, three-dimensional structural information for the tyrosine kinase domain has not been described, which precludes any refined predictional analysis of an interactive motif. Based on the capacity for heterodimerization, it seems likely that the motif responsible is itself conserved, at least between EGF and PDGF receptors, and HER-2.
A common feature of other classes of protein that undergo dimerization are small repeats of hydrophobic and acidic/basic residues, typified by the leucine zipper structural motif, which forms a repeating helical dimerization interface (Krylov et al., 1994). In the EGF and PDGF receptors and HER-2, the longest conserved stretch of amino acids in the kinase domain corresponds with residues Val-810 to Leu-820 in the human EGF receptor, a region that is particularly rich in hydrophobic and charged residues. Another highly conserved region sharing these primary sequence characteristics, albeit shorter, can be found at residues Asp-831 to Leu-837. Alignment of 65 different protein tyrosine and serine/threonine kinases has subdivided the catalytic domain into 11 major conserved subdomains (Hanks et al., 1988). Using these definitions as a structural framework, it will be possible to predict which deletions within the kinase domain could be made that do not overtly affect the overall integrity of the domain structure. Studies are now in progress, using a site-directed deletion analysis combined with co-immunoprecipitation of truncated and full-length receptor mutants, to test predictions for potential interaction motifs and to define precisely which subdomain of the tyrosine kinase domain is responsible for intracellular RTK oligomerization.