©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Kinase Domain and Membrane Localization Determine Intracellular Interactions between Epidermal Growth Factor Receptors (*)

(Received for publication, October 4, 1994; and in revised form, December 2, 1994)

Andrew Chantry

From the Departments of Biochemistry and Medical Oncology, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RP, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 beta-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.


INTRODUCTION

The epidermal growth factor (EGF) (^1)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.


EXPERIMENTAL PROCEDURES

Materials

Constructs encoding the human EGF receptor (HER), chimeric receptors comprising the extracellular domain of the human EGF receptor connected to either the cytoplasmic domain of the mouse beta-PDGF receptor (EP-R; Seedorf et al., 1991) or HER-2 (HER-1/2; Lee et al., 1989), and 85-kDa non-catalytic subunit of phosphatidylinositol 3-kinase (p85), and antibodies specific to the extracellular domain of the EGF receptor (monoclonal antibody 108.1, Honegger et al., 1989) were kindly provided by Dr. Axel Ullrich. The PDGF-R (-X-L) construct was prepared by Dr. Klaus Seedorf. Human embryonic kidney fibroblast 293 cells (ATCC CRL 1573) were maintained in DMEM with 4500 mg/liter (Life Technologies, Inc.) containing 10% fetal calf serum (Life Technologies, Inc.) and 1 mML-glutamine at 37 °C in a 5% CO(2) atmosphere.

Construction of Receptor Mutants

The human EGF receptor cDNA was subcloned from the plasmid pCVN-HERc (Riedel et al., 1989) into the cytomegalovirus promoter-based pCMV-1 expression vector (Gorman et al., 1989). Single-stranded uracil-containing phage DNA was rescued from strain CJ236 Escherichia coli that had been transformed with pCMV-HERc and then infected with 2 times 10^7 plaque-forming units/ml M13K07 helper phage. This DNA template was used for site-directed mutagenesis, which was performed essentially according to the method outlined in the Bio-Rad Mutagene kit. The following oligonucleotides were used as primers; EGF-R (-X+L; without extracellular domain, with hydrophobic leader sequence), ACCATCCCAGTGGCGATAGCCCGACTCGCCGGGC; EGF-R (-X-L; without extracellular domain and hydrophobic leader sequence), ACCATCCCAGTGGCGATCATCGCTGCTCCCCGAA; EGF-R (-X-L-TM; without extracellular domain, hydrophobic leader sequence and transmembrane domain), ACGATGTGGCGCCTTCGCATCGCTGCTCCCCGAA; EGF-R kinase-negative (lysine 721 to alanine), GCTTCTCTTAATTCCGCGATAGCGACGGGAAT; EGF-R Deltacytoplasmic-tail (stop at residue 973), TCCATCAGGGCACGGTAGAAGTTGGAGTCT; EGF-R Deltakinase/cytoplasmic-tail (stop at residue 682), AATTCAGTTTCCTTTCAGATCCTCAAGAGA; HER Deltakinase (residues 683-972 deleted), TCCATCAGGGCACGGTAGATCCTCAAGAGAGCTT. The numbering for the human EGF receptor sequence is from Ullrich et al.(1984). Mutations were confirmed by dideoxy sequencing and restriction enzyme analysis. The kinase-negative truncated EGF receptor (EGF-R (-X+L)kinase -ve) and truncated receptor without the cytoplasmic tail (EGF-R (-X+L) DeltaC-terminal) were prepared by subcloning appropriate restriction enzyme fragments from the above mutants into the truncated receptor background.

Transient Expression, Metabolic Labeling, and Cell Lysis

Human 293 fibroblasts were seeded at 5 times 10^5 cells/well into six-well plates (Costar) approximately 8 h prior to transfection. Cells were then transfected with 3 µg of plasmid DNA using the calcium phosphate procedure of Chen and Okayama(1987) in a 3% CO(2) atmosphere. For co-transfections, a total of 6 µg of plasmid DNA was used and compensation to a total of 6 µg of plasmid DNA was made with empty vector. 18 h after the addition of the precipitate, the cells were washed once and supplied with fresh medium. After another 8 h in culture, cells were washed once with methionine-free DMEM (Life Technologies, Inc.) containing 6% dialyzed fetal calf serum and metabolically radiolabeled for 18h in the same medium with 60 µCi of [S]methionine (Amersham Corp.). After washing with phosphate-buffered saline, cells were lysed at 4 °C with 0.5 ml of ``lysis buffer'' (50 mM HEPES, pH 7.5, containing 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM sodium orthovanadate) and solubilized proteins clarified by centrifugation (17,000 times g for 15 min at 4 °C).

Immunoprecipitation, Gel Electrophoresis, and Immunoblotting

About 0.5 µg of anti-EGF receptor monoclonal antibody (108.1; Honegger et al., 1989) and 20 µl of prewashed protein A-Sepharose were added to the lysates, and protein-antibody complexes were allowed to form during rotation for 2 h at 4 °C. Immunoprecipitates were washed four times with 1 ml of ``washing buffer'' (50 mM HEPES, pH 7.5, containing 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM sodium orthovanadate). SDS sample buffer containing beta-mercaptoethanol (60 µl) was added, samples were heated (100 °C; 5 min), and proteins were separated by SDS-polyacrylamide gel electrophoresis. Gels were stained for 10 min in 0.05% Coomassie Blue in 10% acetic acid/40% methanol and destained for 30 min in the same buffer without the dye. After incubation with Amplify (Amersham) for 20 min, gels were dried and exposed for 1-2 days to film (Kodak X-Omat).

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).


RESULTS

Co-immunoprecipitation of Full-length and Truncated Receptors

In order to address the role of the intracellular domain in RTK oligomerization, several truncated constructs were prepared by site-directed mutagenesis. All mutants lacked the extracellular domain but differed in membrane-localization motifs. EGF-R (-X+L) retained the hydrophobic leader sequence for proper insertion into the plasma membrane during biosynthesis, EGF-R (-X-L) and PDGF-R (-X-L) lacked this leader sequence but retained the hydrophobic transmembrane domain, and EGF-R (-X-L-TM) lacked both the leader and the transmembrane domain (Fig. 1A). These truncated receptors, contained within a cytomegalovirus promoter-based expression vector, were then transiently co-expressed with the full-length EGF receptor in human embryonic kidney fibroblast 293 cells. After metabolic labeling with [S]methionine, immunoprecipitation with an extracellular domain-specific anti-EGF receptor antibody and SDS-PAGE analysis, a clear association was apparent between some of these cytoplasmic domains and the full-length receptor, in a manner that was independent of ligand binding to the EGF receptor extracellular domain (Fig. 1B).


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 beta-PDGF receptor without extracellular domain and hydrophobic leader sequence. alpha-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).

Tyrosine Phosphorylation of Truncated Receptors and Transphorylation of Kinase-negative EGF Receptors

The full-length EGF receptor or truncated EGF and PDGF receptors lacking the extracellular domain were transfected into 293 fibroblasts and their potential to transmit phosphorylation-dependent signals examined by immunoblotting total lysates with an anti-phosphotyrosine antibody. All receptors were expressed in approximately equal amounts as determined by immunoblotting against RTK-specific antibodies (Fig. 2A, leftpanel). The intrinsic kinase activity of the full-length EGF receptor was rapidly engaged by stimulation with EGF, and, even in the absence of ligand, the truncated receptors EGF-R (-X+L), EGF-R (-X-L), and PDGF-R (-X-L) all displayed constitutive tyrosine autophosphorylation as well the capacity to phosphorylate substrates (Fig. 2A, rightpanel). Interestingly, EGF-R (-X-L-TM), which also lacks the transmembrane domain and failed to interact with the full-length EGF receptor (see Fig. 1B), has a markedly reduced tyrosine autophosphorylation capability with only limited substrate phosphorylation (Fig. 2A, rightpanel).


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).

Hetero-association between RTK Intracellular Domains

To further examine the role of the cytoplasmic domains in the process of receptor-receptor hetero-interactions, full-length RTKs comprising the intracellular domains of the EGF, PDGF receptors, and HER-2 were co-expressed with truncated EGF and PDGF receptors devoid of the extracellular domain. In this instance chimeric receptors comprising the extracellular domain of the EGF receptor connected to the respective EGF, PDGF, or HER-2 intracellular domains were used as full-length RTK counterparts. These chimeras have a functional EGF ligand binding domain and transmit a cell signal that corresponds to the nature of the intracellular domain (Lee et al., 1989; Seedorf et al., 1991).

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.



Domains Responsible for RTK Intracellular Interactions

The intracellular domain of the EGF receptor can be subdivided into three major parts; a short juxtamembrane region that contains major protein kinase C phosphorylation sites at threonine 654 and 669, a catalytic tyrosine kinase domain, and a C-terminal tail that contains most of the major autophosphorylation and substrate binding sites. In an attempt to identify which of these regions is responsible for intracellular receptor-receptor association events, a number of deletion mutants were prepared, co-expressed with the truncated EGF receptor (-X+L), and analyzed by the same co-immunoprecipitation as before. Association was apparent with the wild-type EGF receptor, a catalytically inactive point mutation (lysine 721 to alanine; EGF-R kinase -ve) and also the C-terminal tail-deleted receptor; however, no interaction occurred with mutants lacking either the kinase domain alone or the kinase and C-terminal domains together (Fig. 4A). This failure to associate could not be explained in terms of aberrant expression of either EGF-R (-X+L) or any of the other receptor mutants. In addition, a catalytically inactive EGF-R (-X+L) construct with an equivalent lysine to alanine point mutation (EGF-R (-X+L) kinase -ve) also associated with EGF-R kinase -ve and wild-type EGF-R, as well as the cytoplasmic tail-deleted receptor (Fig. 4B). A truncated EGF receptor that lacks the cytoplasmic tail (EGF-R(-X+L) DeltaC-terminal) migrating with an expected apparent molecular mass of 50 kDa also associated with the full-length EGF receptor (wild-type and kinase-negative) as well as EGF-R DeltaC-terminal (Fig. 4B).


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) DeltaC-terminal were co-expressed with the indicated receptors and analyzed as described in a on 10% SDS-PAGE.




DISCUSSION

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.


FOOTNOTES

*
Funding was provided by the Trustees Research Committee of Charing Cross Hospital and the Nuffield Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: EGF, epidermal growth factor; RTK, receptor tyrosine kinase; PDGF, platelet-derived growth factor; PDGF-R, PDGF receptor; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

I thank Axel Ullrich for allowing me to acquire techniques of receptor expression within his laboratory on a fellowship provided by the Alexander von Humboldt Stiftung. I am also grateful to Rachel Cohen for technical assistance.


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