Dimerization activation

Other parts of domain II or domain IV do not contribute significantly to the energy of dimerization. To determine whether the mutations studied here have the same effect on EGFR activation at the cell surface as they do on sEGFR dimerization in vitro, we introduced each of them into the intact receptor. Analysis of ligand-activation of intact EGFR mutants. The filled traces represent data from control parental S2 cells treated with a phycoerythrin-conjugated antibody against the EGFR extracellular region, while the open traces represent data from the transfected stable cell pools analyzed in the same fashion.

The marked right shift in each case demonstrates that each chimera is expressed appropriately at the cell surface and that our pools sample a wide-range of expression levels. A total of 10, cells were analyzed for each FACS analysis. Our mutational analysis shows that the domain II dimerization arm in disulfide-bonded module 5 [see Fig.

As shown in Fig. Ogiso et al. Through the interactions detailed in Fig. In support of this model, Ogiso et al. The contact region involving D and H in disulfide-bonded module 6 is indicated with red side chains, and the dimerization arm region in disulfide-bonded module 5 is marked with a transparent gray box.

The position of R in domain III is represented by an exaggerated protrusion from domain III that is shown projecting into domain II when swung into position and buttressing the critical dimer contacts, including those mediated by D and H This dimer contact is depicted as an exaggerated ligand-induced projection from the C-terminal part of domain II that makes contact across the dimer interface in the right-hand part of the figure.

To further test this hypothesis, we individually mutated each of the putative buttressing residues N, E, and R, and analyzed ligand binding and dimerization for each mutated sEGFR protein. Ligand-binding affinities were slightly reduced, a finding consistent with the reduced affinity of other dimerization-defective sEGFR mutants.

Effects of buttress mutations on ligand binding and receptor dimerization. A Raw analytical ultracentrifugation data are plotted as described for Fig. For comparison, solid and dashed lines from Fig. Three independent experiments were performed for each analysis. Ligand binding to the receptor does more than simply expose this region, actually altering the conformation of domain II to maximize cooperation between dimer contact sites in the fifth and sixth disulfide-bonded modules Fig.

In the model presented in Fig. This model may help to explain several unanswered questions regarding ErbB receptor homo- and heterodimerization. First, it explains why exposure of the dimerization arm is not sufficient for EGFR dimerization. An additional conformational rearrangement in the C-terminal part of domain II must also be induced in order to promote receptor dimerization Fig. Second, our findings may help to explain why ErbB3 and ErbB2 fail to homodimerize 4 , 6 , 13 , despite sharing the majority of the key dimerization arm residues with EGFR, but instead form heterodimers.

Regions outside the dimerization arm must play a key role in determining homo- versus heterodimerization specificity. In the case of EGFR, module 6 buttressed by domain III appears to provide the additional self-complementary interactions including D and H that allow efficient homodimerization. Finally, one intriguing possibility raised by these suggestions is that different ErbB ligands could induce distinct but similar domain III positions, leading to subtly different configurations of the domain II C terminus in a particular receptor.

If this is true, different ErbB ligands could potentially stabilize extended forms of their receptors with slightly altered homo- and heterodimerization specificities, and this could contribute to their distinct signaling specificities 2. We thank members of the Lemmon, Ferguson, and Schlessinger laboratories for valuable discussions and critical comments on the manuscript and Shigeyuki Yokoyama for communicating results prior to publication. National Center for Biotechnology Information , U.

Journal List Mol Cell Biol v. Mol Cell Biol. Jessica P. Ferguson 2. Mitchell B. Mark A. Kathryn M. Author information Article notes Copyright and License information Disclaimer. Phone: Fax: E-mail: ude. This article has been cited by other articles in PMC. Abstract Structural studies have shown that ligand-induced epidermal growth factor receptor EGFR dimerization involves major domain rearrangements that expose a critical dimerization arm.

Open in a separate window. TABLE 1. Analytical ultracentrifugation studies. TABLE 2. Receptor activation at the cell surface.

Domain II mutations. Domain IV mutations. Effects of mutations on ligand binding. Effects of mutations on sEGFR homodimer formation. Relative strengths of sEGFR dimerization. Effects of dimer interface mutations on activation of intact EGFR at the cell surface. Acknowledgments We thank members of the Lemmon, Ferguson, and Schlessinger laboratories for valuable discussions and critical comments on the manuscript and Shigeyuki Yokoyama for communicating results prior to publication.

Arteaga, C. ErbB-targeted therapeutic approaches in human cancer. Cell Res. Beerli, R. Epidermal growth factor-related peptides activate distinct subsets of ErbB receptors and differ in their biological activities. Berezov, A. Chen, Q. Liu, H. Zhang, M. Greene, and R. Disabling receptor ensembles with rationally designed interface peptidomimetics. Berger, M. Mendrola, and M. FEBS Lett. Blume-Jensen, P.

Oncogenic kinase signaling. Nature : Burgess, A. Cho, C. Eigenbrot, K. Ferguson, T. Garrett, D. Leahy, M. Lemmon, M.

Sliwkowski, C. Ward, and S. An open-and-shut case? Cell 12 : Cantor, C. Biophysical chemistry: techniques for the study of biological structure and function. Freeman, New York, N. Cho, H. Structure of the extracellular region of HER3 reveals an interdomain tether. Science : Mason, K. Ramyar, A. Stanley, S. Gabelli, D. Denney, Jr. Dancey, J. Predictive factors for epidermal growth factor receptor inhibitors: the bull's-eye hits the arrow.

Cancer Cell 5 : Elleman, T. Domagala, N. McKern, M. Nerrie, B. Lonnqvist, T. Adams, J. Lewis, G. Lovrecz, P. Disordered regions are shown as dotted lines and bound inhibitors are depicted in space-filling form. Each dimer is shown in the same reference frame as that of SLK. Conformation of activation segments. A Activation segment nomenclature. The activation segments adopt a spectrum of orientations that are dictated by the relative orientation of each monomer with each respective dimer. The disordered region of LOK is indicated by a dotted line.

Inset: overall superposition of kinases indicating area of interest shown in main panel. C Structure-based sequence alignment of activation segment regions. Known phosphorylation sites are highlighted in orange. Secondary structure elements are shown for each kinase below the alignment and coloured using the same scheme as in panel B. In all four catalytic domain structures, the exchanged activation segment forms an extended dimer interface.

In addition, the interfaces encompass a surface area that is more than fivefold larger than any interface involved in packing contacts observed in crystal contact regions of any of the kinases studied here. Nonetheless, it could not be ruled out that the observed dimeric structures of catalytic domains were related to crystal packing effects or binding of the cocrystallized inhibitor. Moreover, the conformation of the activation segment in LOK that was crystallized in a monoclinic P 2 1 form was identical to that observed in the I form presented here data not shown.

Self-association of catalytic domains was investigated by AUC and cross-linking studies. Sedimentation velocity experiments revealed the presence of catalytic domain dimers in solution at physiological pH and salt concentrations for all four kinases Figure 4. Cross-linking experiments confirmed the presence of dimers for all four kinases Figure 4B. Kinase self-association in solution. The first peak corresponds to the monomer and the second peak to the LOK dimer. B Detection of dimers by cross-linking.

Lane 1, molecular weight marker. The expected position of the T autophosphorylation peak is indicated by an arrow. Dimerization was only detected in the wild-type protein as indicated by a second peak at 3.

To address the role of dimerization in activation loop phosphorylation, we mutated residues in the dimer interface with the goal of obtaining monomeric protein. Comparison with the monomeric structure of pactivated kinase 4 PAK4, Protein Data Bank PDB code: 2BVA revealed that this residue inserts deeply into a hydrophobic pocket in the lower kinase lobe, suggesting that the generated mutants would also be destabilized in the monomeric state. Mutation of the less conserved tyrosine residue in SLK YA resulted in a stable construct that, however, still dimerized data not shown.

Most importantly, the mutant did not autophosphorylate to a significant level, demonstrating that dimerization is necessary for SLK autophosphorylation Figure 4 C and D. Comparison of active site and phosphorylated activation segment regions. Critical residues for kinase function are shown in ball and stick representation and the major secondary structure elements are labelled.

B , C Active site stabilization via activation segment phosphorylation. The colour scheme is as in B. Hydrogen bonds are indicated by dotted lines.

The catalytic base Asp is coloured with green carbons and labelled in red. We were interested to determine whether the observed conformation and dimerization can also occur between phosphorylated catalytic domains and determined the structure of both di- and unphosphorylated SLKs in the presence of the same ligand. Superimposition of the main chains of both structures revealed that the overall structures were identical r. Phosphorylation increased the number of direct hydrogen bonds formed in the dimer interface from 18 to 29, as well as the surface area buried in the interface Table II.

This is mainly a consequence of ordering of the R and R side chains flanking the phosphothreonine at position T However, AUC experiments showed that the dimer concentration for unphosphorylated SLK was twofold higher than for the phosphorylated catalytic domain data not shown. The role of phosphate moieties in activated kinases is to stabilize the activation segment in a conformation suitable for substrate binding. Typical interactions stabilizing the activation segment of the monomeric kinase PAK4 are shown in Figure 5B.

In SLK, phosphorylation of T results in formation of a hydrogen bond network involving the activation segment residues R and R Figure 5C , as well as two hydrogen bonds to main chain atoms located on both termini of the activation segment R and S In the absence of phosphorylation at T, the side chains of R and R are disordered. The second phosphorylation site, identified at S, is orientated towards the solvent and is unlikely to contribute to the stability of the activation segment in its dimeric state.

However, comparison with known activation sites in STE20 kinases e. We mutated the SLK residues T and S to alanine and determined the autophosphorylation activity of the dephosphorylated protein in vitro. The SA mutant autophosphorylated to a similar level as wild type SLK, confirming that phosphorylation at this site is not relevant for phosphorylation on T We determined the sequence specificity of the kinase catalytic domains using degenerate peptide libraries Figure 6.

Aliquots of each reaction were subsequently spotted onto a streptavidin membrane, which was washed, dried and exposed to a phosphor screen. Importantly, for none of the four studied kinases the determined consensus sequences matched the activation segment phosphorylation sites Figure 3C , but corresponded well with sequences of known substrates for the respective kinases.

The structural data revealed that the activation segment, a key regulatory element of kinase function, may exchange with the activation segment of an adjacent kinase molecule forming an active kinase in trans conformation. Structural comparison, mutagenesis and biophysical characterization suggest a model for activation segment autophosphorylation, which may be common to kinases from diverse families.

The high degree of flexibility in a region that constitutes the substrate-binding site in inactive kinases raises the question of how activation segments are specifically recognized.

The segment exchange observed in the four catalytic domain structures leads to ordering of the activation segment in the nonphosphorylated state. This conformation is stabilized by a large number of interactions at the dimer interface. However, no activation segment exchange has been reported for the published structures Ohren et al , However, DAPK3 has been reported not to require activation segment phosphorylation for catalytic activity.

This is supported by our observation that DAPK3 is autophosphorylated at two sites S50 and T in the absence of a phosphorylation within the activation segment. Autophosphorylation at the activation segment residue T has been suggested to be of functional importance for DAPK3 activity, because a TA mutant is resistant to activation and suppresses DAPK3 function in cells Graves et al , The proposed mechanism for autophosphorylation requires that the activation segment-exchanged kinase dimers retain an active conformation.

In the structure of diphosphorylated dimeric SLK, T forms a hydrogen bond network with the activation segment residues R and R, whereas phosphorylation of S did not result in formation of new polar interactions in dimeric SLK. This suggests that the role of T phosphorylation is to promote activation segment phosphorylation at S positioned in close proximity to the catalytic aspartate D of the interacting protomer Figure 5C.

This interpretation is supported strongly by the impairment of autophosphorylation observed for the TA mutant.

Multiple activation segment phosphorylation sites have been reported for a number of kinases. From our results, it could be deduced that these sites play functional roles in activation segment phosphorylation and kinase activation, but may be less relevant for the catalytic activity once the kinase is activated. A common feature of the discussed kinases is that all activation segment autophosphorylation sites identified in this study or reported in the literature do not match the sequence requirements for consensus substrate binding or phosphorylation.

Furthermore, it was shown recently that full-length, activated CHK2 does not recognize an isolated CHK2 kinase domain as a bona fide substrate despite rapid autophosphorylation Oliver et al , Thus, the mechanism for the recognition of autophosphorylation residues differs from substrate recognition and likely requires dimerization of the catalytic domain as observed in the crystal structure. In addition, improper activation across kinase cascades would also be prevented. Yet, in the absence of dimerization and activation segment exchange, CHK1 cannot phosphorylate activation segment residues and activate CHK2, as they do not resemble CHK1 consensus substrates.

AUC demonstrated that dimerization is also observed in solution. Intrinsic FRET is a measure of the separation between the fluorescent proteins in the dimer. Two different intrinsic FRET values were measured for fgf1 and fgf2. Therefore, the binding of these two ligands to the extracellular domains leads to different separation of the fluorescent proteins on the cytoplasmic side of the membrane Table 2.

Expression of the receptors was probed with antibodies to the extracellular domains of the three receptors. Two bands are observed for all receptors. Only the top bands, corresponding to the fully glycosylated mature receptors that reside primarily in the plasma membrane, were considered in our analysis. Phosphorylation was calculated by dividing the intensities of the anti-phospho-Y bands to the intensities of the anti-receptor bands, and scaled to the fgf2 case.

Left: graphic not to scale representation of the finding that the average distance between the fluorescent proteins is larger when fgf1 is bound, as compared with the fgf2-bound case.

Right: graphic representation of the finding that phosphorylation is higher when fgf2 is bound. The representation of the kinase domains is a cartoon, not based on structural data. Typical western blot results are shown in Fig. Only the top bands, corresponding to the mature fully glycosylated receptors, were quantified. The western blot results therefore support the finding of two distinct ligand-bound active FGFR1 and FGFR3 states, and demonstrate that these different structural states correlate with biological activity.

We used the same method to measure phosphorylation in the absence of ligand. Supplementary Figure 6 shows typical western blot results in the absence of ligand, along with the fgf1 and fgf2 results. Thus, the unliganded FGFR1 and FGFR3 dimers exhibit significant phosphorylation even in the absence of ligand, consistent with previous reports 48 , 49 , This finding is consistent with the literature 45 , and may be due to the fact that FGFR2 interacts with soluble adaptor proteins, such as Grb2, which can regulate its dimerization and activity 44 , The TM helices are almost parallel, and wrap around each other in a tight, closed-packed configuration Supplementary Fig.

To investigate if either the observed NMR interface or the putative GxxxG-like interfaces are related to the structures that we observed in our experiments, we created two sets of amino-acid mutations, each designed to destabilize one of the two TM dimer interfaces.

First, we mutated residues L, G and A to Ile. The width of the bar represents the standard error from the fit. The intrinsic FRET decreases due to both mutations, suggesting that the fluorescent proteins in the mutant dimers move away from each other due to the mutations.

Cartoons are not drawn to scale. The histograms of measured intrinsic FRET values in single vesicles for the wild type are shown in grey for the fgf1 case and in green for the fgf2 case.

The histograms for the mutants are shown in black in the presence of fgf1 and in olive in the presence of fgf2. Both sets of mutations decreased the intrinsic FRET, indicating that the fluorescent proteins were further away from each other in the mutants, as compared with the wild type Fig. Thus, the TM dimer interface in the fgf1-bound dimer does not involve the amino acids in the NMR interface, but instead likely involves the alternative GxxxG-like motifs.

This fact prompted us to further investigate the behaviour of the mutant. We also characterized the phosphorylation of the full-length mutant receptor in the presence of saturating fgf1 and fgf2 concentrations. The comprehensive characterization of the dimerization of the AE pathogenic mutant, in the absence and presence of fgf1 and fgf2, is shown in Supplementary Fig. The intrinsic FRET values as well as the western blots that report the phosphorylation of the mutants in the presence of fgf1 or fgf2 are shown in Fig.

The phosphorylation of the full-length AE mutant in the presence of saturating fgf1 or fgf2 concentrations was also the same, and was the same as in the wild-type fgf2-bound state Fig. The AE mutation is the genetic cause for Crouzon syndrome with acanthosis nigricans, a cranial abnormality 57 , and has been linked to bladder cancer 8.

The histograms for the wild type are shown in grey in the presence of fgf1 and in green in the presence of fgf2. The histograms for the AE mutant are shown in black in the presence of fgf1 and in olive in the presence of fgf2. The intrinsic FRET values measured for the AE mutant in the presence of fgf1 shift up, such that they overlap with the fgf2 wild-type values.

Thus, the AE mutation abolished the fgf1-bound state. Data are from three independent experiments. Thus, the AE mutation increases the phosphorylation in the fgf1 state to fgf2-state levels. Left: graphic representation of the finding that the average distance between the fluorescent proteins is the same in the presence of both fgf1 and fgf2. Distances are not drawn to scale. Right: graphic representation of the finding that phosphorylation is also the same in the presence of fgf1 and fgf2.

The representation of the kinase domains is not based on structural data. Taken together, published data 36 and the results reported here show that the AE mutation abolishes the fgf1 state and traps the FGFR3 dimer in the fgf2 state even in the absence of ligand. This finding can be explained by the formation of a stabilizing hydrogen bond between the mutant Glu and the neighbouring helix, an idea that is supported by molecular modelling Once formed, this structure does not change significantly on binding fgf1 or fgf2.

The AE mutation therefore mimics the action of fgf2 in enforcing a close-packed TM dimer structure that leads to increased phosphorylation and thus disregulated signalling and disease. Phosphorylation, measured using western blotting, was normalized to the phosphorylation of fgf2-bound wild type, which was assigned a value of 1. An example of a western blot, used to arrive at the relationship, is shown in Supplementary Fig.

The final results, shown in Fig. In Fig. At least three independent experiments were performed for each mutant. The phosphorylation of the wild type in the fgf2-bound state is assigned a value of 1, and all other measured phosphorylation levels are scaled accordingly.

Since the discovery of receptor tyrosine kinases RTKs in the s, researchers have been searching for a model that captures the essence of RTK signal transduction across the plasma membrane. These unliganded dimers are stabilized through contacts between the TM domains and the IC domains Fig. We also show that the unliganded FGFR dimers are phosphorylated, providing an explanation of the fact that FGFR overexpession leads to cancer 10 , 11 , 12 , 13 , 14 , The dimers undergo structural changes in response to ligand binding, and these structural changes increase phosphorylation.

Thus, all RTKs may follow a universal model of activation, which includes unliganded dimers of various stabilities as intermediates. We provide a direct experimental demonstration that ligand-induced structural changes occur in FGFR dimers within the plasma membrane. Ultimately, the ligand controls the structure of the TM domain by triggering a switch to a specific configuration, and the resulting structure of the TM dimer controls the activity of the receptor.

The structural changes in response to fgf1 and fgf2 binding are very different, resulting in different distances between the intracellular domains, and different phosphorylation levels for the fgf1- and fgf2-bound dimers. Thus, there exist multiple active ligand-bound states for the FGF receptors. On fgf1 binding, FGFR3 TM domains change conformation and engage in interactions that likely involve small amino-acid residues in the N-terminal portion of the receptors Fig.

FGFR3 phosphorylation increased by a factor of 1. We note that this cannot be explained by an increase in dimerization because FGFR3 dimerization is already very high under these conditions.

Binding of fgf2 triggers a structural change towards a different TM dimer structure in which the interface likely involves contacts between L, G and A Fig. Bottom: binding of fgf2 to D2, D3, and the linker, on the other hand, triggers a switch towards a closely packed TM dimer structure.

The difference between the fgf1- and fgf2-bound states observed here provides an explanation of the different biological roles of these two ligands. Hidai et al. Our study suggests that these profoundly different biological effects may originate in structural differences of the receptor—ligand complexes on the cell surface.

An important result of this study is the strong correlation between intrinsic FRET and kinase phosphorylation Fig. Since in the full-length receptors the kinase domains are attached to the TM domains via the juxtamembrane domains, these results suggest that a correlation exists between the separation of the kinase domains in the dimer and their phosphorylation: the smaller the distance between the kinases, the higher their phosphorylation.

However, the distance between the TM domain C termini is not the only parameter that affects phosphorylation levels. There appears to be a conformational change in the TM domain on fgf1 binding that likely affects TM helix rotation, not separation, and thus cannot be captured in the FRET experiments.

These findings, and published work 62 , suggest that the relative orientation of the kinases with respect to each other is another important parameter that determines phosphorylation efficiencies.

We show that the AE mutation in FGFR3, linked to Crouzon syndrome with acanthosis nigricans and to bladder cancer 8 , 57 , mimics the structural and functional effects of fgf2 binding.

In particular, the mutation prevents the FGFR3 dimer from exploring the unliganded and fgf1-bound conformations, and traps it in its most active state, the fgf2 state.

This is a fundamentally novel mechanism through which FGFR-linked pathologies can arise. Tsien University of California, San Diego. Mohammadi, NYU. Donoghue, UCSD. All primers were purchased from Invitrogen. These constructs are shown in Supplementary Fig. Images of vesicles with FGF receptors tagged with fluorescent proteins are shown in Supplementary Fig. Gains of 8. The bleaching of the fluorescent proteins was minimized through the use of ND8 filters when exciting with the nm laser, and low pixel dwell time 1.

The acceptor concentration in each vesicle, C A , was calculated according to ref. The sensitized emission of the acceptor in each vesicle was determined as 37 :. The donor intensity in the absence of the acceptor I D,corr , and the donor concentrations C D were calculated as:. From equations 1 and 4 , the total concentration, T , and the acceptor fraction, x A , are calculated according to:. The dimeric fraction is determined from the corrected FRET efficiency according to:. This is a structural parameter, a constant for each receptor dimer, which depends only on the separation and the orientation of the two fluorescent proteins in the dimer, not on the dimerization propensity.

Based on the law of mass action, the dimeric fraction can be written as a function of the total receptor concentration, T , and the dimerization constant K according to equation 9 :. Equations 8 and 9 are used to fit the dimerization model to the data while optimizing for two adjustable parameters: the dimerization constant K , and the intrinsic FRET, Supplementary Fig.

The value of K diss can be then directly compared with expressions levels in order to evaluate the biological significance of dimerization. The standard state is defined as nm 2 per receptor 33 , and therefore:.

Thus, measurements of E and x A for each vesicle in this case allows us to directly determine the value of the intrinsic FRET, , in each vesicle. Histograms of the measured are shown throughout the manuscript, such as in Fig. Finally, the dependence of the intrinsic FRET, , on the distance between the fluorescent proteins in the dimer is given by equation The phosphorylation of the tyrosines in the activation loop of the FGFR kinases was assessed first following protein transfer to the nitrocellulose membrane.

Dilutions were , for all primary antibodies and , for the secondary antibody. Uncropped versions of gels in Fig. How to cite this article: Sarabipour, S and Hristova, K. Mechanism of FGF receptor dimerization and activation.

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Bad bones, absent smell, selfish testes: The pleiotropic consequences of human FGF receptor mutations.