CHAPTER
Analysis of EGF Receptor Oligomerization by Homo-FRET
16
Cecilia de Heus*, Nivard Kagie{, Raimond Heukers*, Paul M.P. van Bergen en Henegouwen*, and Hans C. Gerritsen{ *
Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, Netherlands { Molecular Biophysics, Department of Soft Condensed Matter and Biophysics, Science Faculty, Utrecht University, Utrecht, Netherlands
CHAPTER OUTLINE Introduction ............................................................................................................ 306 16.1 Theory Homo-FRET Quantification ....................................................................309 16.1.1 Steady-State Fluorescence Anisotropy Imaging ............................. 309 16.1.2 Time-Resolved Fluorescence Anisotropy Imaging........................... 311 16.2 Materials........................................................................................................313 16.2.1 Plasmid Constructs..................................................................... 313 16.2.2 Cell Lines .................................................................................. 315 16.2.3 Microscope ................................................................................ 315 16.2.3.1 Confocal Time-Resolved and Steady-State Fluorescence Anisotropy Imaging Microscopy ........................................................315 16.3 Methods .........................................................................................................316 16.3.1 Slide Preparation........................................................................ 316 16.3.1.1 Reference Measurements ...................................................316 16.3.1.2 Predimerization Measurements ...........................................316 16.3.1.3 Ligand-induced Oligomerization ..........................................317 16.3.2 Analysis..................................................................................... 317 16.4 Discussion......................................................................................................317 Acknowledgments ................................................................................................... 319 References ............................................................................................................. 320
Abstract Growth factor receptors are present in the plasma membrane of resting cells as monomers or (pre)dimers. Ligand binding results in higher-order oligomerization of ligand–receptor complexes. To study the regulation of receptor clustering, several Methods in Cell Biology, Volume 117 Copyright © 2013 Elsevier Inc. All rights reserved.
ISSN 0091-679X http://dx.doi.org/10.1016/B978-0-12-408143-7.00016-5
305
306
CHAPTER 16 Analysis of EGF Receptor Oligomerization by Homo-FRET
experimental techniques have been developed in the last decades. However, many involve invasive approaches that are likely to disturb the integrity of the membrane, thereby affecting receptor interactions. In this chapter, we describe the use of a noninvasive approach to study receptor dimerization and oligomerization. This method is based upon the Fo¨rster energy transfer between identical adjacent fluorescent proteins (homo-FRET) and is determined by analyzing the change in fluorescence anisotropy. Homo-FRET takes place within a distance of 10 nm, making this an excellent approach for studying receptor–receptor interactions in intact cells. After excitation of monomeric GFP (mGFP) with polarized light, limiting anisotropy values (rinf) of the emitted light are determined, where proteins with known cluster sizes are used as references. Dimerization and oligomerization of the epidermal growth factor receptor (EGFR) in response to ligand binding is determined by using receptors that have been fused with mGFP at their C-terminus. In this chapter, we describe the involved technology and discuss the feasibility of homo-FRET experiments for the determination of cluster sizes of growth factor receptors like EGFR.
INTRODUCTION The epidermal growth factor receptor (EGFR), also called ErbB1 or Her1, is a member of the ErbB single-pass transmembrane tyrosine kinase receptor family (Ullrich & Schlessinger, 1990; Yarden & Sliwkowski, 2001). Activation of EGFR and its family members is involved in cell growth, cell proliferation, and migration. Many cancer types show overexpression or deregulation of EGFR, and it is therefore a well-studied receptor and an attractive anticancer drug target (Oliveira, van Bergen en Henegouwen, Storm, & Schiffelers, 2006; Sorkin & Goh, 2009). EGFR is composed of an extracellular domain, a transmembrane domain, and an intracellular C-terminal tail containing the tyrosine kinase and several sites involved in posttranslational modifications and signaling (Jorissen et al., 2003). The extracellular part of EGFR contains four domains of which domain I and III are involved in ligand binding and domain II in receptor dimerization. More than 20 different ligands are known for receptors from the ErbB family of which EGF is the most studied one. Ligand binding induces conformational changes of the ectodomain, which results in not only receptor dimerization but also even receptor oligomerization or clustering (Clayton et al., 2005). As a consequence of these changes, cross-phosphorylation of its C-terminal tail occurs (Jura et al., 2009). Interestingly, EGFR can form homo- or heterodimers with other ErbB family members and this already happens in resting cells. These inactive dimers on the plasma membrane are the so-called receptor predimers. Although the structure of receptor dimers is clear from crystal structures, the mechanism of receptor oligomerization remains obscure (Ferguson, 2008). Crystal structures have shown head to head interactions between receptors, suggesting the involvement of these sequences in oligomerization. Recently, also kinase activity was shown to be required for receptor clustering, which suggests the involvement of signaling in this process (Clayton et al., 2005; Hofman et al., 2010). For example,
Introduction
the production of phosphatidic acid by phospholipase D2 (PLD2) has been demonstrated to be required for EGFR clustering (Ariotti et al., 2010). Besides receptor oligomerization and downstream signaling toward cell proliferation, activation of EGF receptors results in a rapid internalization mainly via clathrin-mediated endocytosis. The intracellular transport trajectory finally ends up in lysosomes where the ligand-induced signaling is terminated by degradation of both ligand and receptors (Goh, Huang, Kim, Gygi, & Sorkin, 2010; Sorkin & Goh, 2009). For studying the clustering of EGF receptors, different experimental techniques have been developed. Dimerization of EGFR, for example, can be determined using chemical cross-linking and detection by SDS-PAGE, by co-IP with differentially tagged EGFR, or by fluorescence complementation (Moriki, Maruyama, & Maruyama, 2001; Yu, Sharma, Takahashi, Iwamoto, & Mekada, 2002; Zhu, Iaria, Orchard, Walker, & Burgess, 2003). Studying receptor clustering is more difficult and demands other experimental strategies. Receptor clustering was initially investigated using electron microscopy (EM), which can be used at high resolution, enabling visualization of gold-labeled EGFR (van Belzen et al., 1988). Analysis of cluster formation can be done by determining particle distances, either by hand or automatically using Ripley’s function (Hancock & Prior, 2005). However, limitations exist with respect to determining cluster size, which is dependent on the size of the gold particle and the antibody (10 nm) (Hancock & Prior, 2005). Another approach for determining receptor clustering involves fluorescence light microscopy. However, conventional microscopy has a resolution limit of 200 nm, which makes this technique not very suitable for measuring the formation of small receptor clusters. To overcome this resolution limit problem, more advanced light microscope techniques were developed, which do provide information about small cluster sizes. One of the first examples was described by Gadella and coworkers and included time-resolved fluorescence microscopy based on Fo¨rster resonance energy transfer (FRET) analyzed by fluorescence lifetime imaging microscopy (FLIM) (Gadella & Jovin, 1995). FRET is based on the energy transfer between a donor and an acceptor fluorophore. For FRET to occur, the donor emission spectrum and acceptor excitation spectrum need sufficient spectral overlap and the two fluorophores should be within a distance of 10 nm. Because energy transfer only occurs when fluorophores are within 10 nm of each other, the detection of FRET can be used to study receptor dimerization or small cluster formation. There are different methods to detect FRET, for example, by measuring changes in the donor/acceptor emission intensity ratio, changes in fluorescence lifetime (FLIM), or differences in anisotropy. The donor/acceptor ratio is determined by measuring the emission of both fluorophores. However, this donor/ acceptor ratio is dependent on the concentrations of both fluorophores. With FLIM, the time that the donor fluorophore is in its excited state is measured. This lifetime can be determined by measuring the fluorescence decay of the donor probe after excitation by using a short laser pulse. The fluorescence lifetime changes upon its environment and also when energy transfer occurs. This method is less dependent on local concentrations of both probes and is therefore a more robust way to detect FRET (Chen, Mills, & Periasamy, 2003; Hofman et al., 2008).
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FRET-FLIM was previously used successfully to determine EGFR cluster formation by Clayton et al. (2005). EGFR-GFP in combination with anti-phosphotyrosineAlexa555 was used to label fixed and permeabilized cells. Using FRET-FLIM in combination with image correlation spectroscopy (ICS), Clayton et al. found an average cluster size of 2.2 in the absence of EGF and an average of 3.7 in the presence of EGF, suggesting the formation of higher-order clusters upon ligand binding. A limitation of this method is that it assumes the cell to be stationary and therefore only an average of the different cluster sizes in a cell can be determined instead of determining the subcellular distribution of the different cluster sizes. To solve this, Saffarian et al. studied EGFR clustering by a method based on fluorescence intensity distribution analysis (FIDA) in live cells for quantifying receptor clustering on cell membranes (Saffarian, Li, Elson, & Pike, 2007). Using this technique, the authors find an average cluster size of 1.3 in unstimulated cells, which is in contrast to results from Clayton et al. The difference between the two values might be explained by different measurement conditions, live cells versus fixed cells, and measuring the clustering distribution with FIDA instead of determining an average. A limitation of the FIDA method is that intensity fluctuations might occur during the measurements, which might disturb the clustering measurements. Furthermore, the FIDA method is relatively slow and may therefore not be suitable for measuring of EGFR clustering after EGF stimulation because of the fast internalization of EGFR. More recently, a number and brightness (N&B) analysis technique was used to study EGFR clustering, which demonstrated the formation of up to pentameric EGFR clusters (Sako, Minoghchi, & Yanagida, 2000). More recently, we introduced a method based on homo-FRET to analyze EGFR clustering. With this method, EGFR clustering can be accurately measured on intact cells using only a single label (EGFR-mGFP) (Bader, Hofman, Voortman, en Henegouwen, & Gerritsen, 2009; Hofman et al., 2010). For homo-FRET, the same fluorophore for donor and acceptor is used, which has the advantage that the detection of FRET is not dependent on concentrations of donor and acceptor. Another advantage of only one fluorophore is that sample preparation is simplified. However, homo-FRET does require a different quantification method because homo-FRET does not result in a change in the emission spectrum or in fluorescence lifetime, resulting in FRET to stay unnoticed. The decreased lifetime of the donor fluorophore in homo-FRET is compensated by the excitation by the acceptor fluorophore. The here applied analysis of energy transfer is based upon the FRETinduced loss of anisotropy. When fluorophores are excited with polarized light, only the subset that is in the right orientation will be excited. In the case of large molecules that are unable to spin on the time scale of the fluorescence lifetime, the emitted light by this subset of fluorophores is also polarized, albeit with a slightly different angular distribution. In the case of two fluorophores in close proximity, there is a high probability that they are in different orientations. Upon excitation of one fluorophore, the neighboring one could be excited through FRET, resulting in the emission of light with this different orientation. Therefore, the energy transfer between two identical fluorophores with slightly different orientation result in depolarization of the
16.1 Theory Homo-FRET Quantification
emission light; the emitted light is more isotropically distributed. Therefore, quantification of the fluorescence anisotropy can be used to determine homo-FRET. The homo-FRET method can be calibrated using reference measurements on protein clusters of known size. Subsequently, EGFR cluster sizes can be determined directly from measured fluorescence anisotropy values. As a tool for the reference measurements, we have used FKBP-mGFP, which dimerizes via its ligand AP20187. Similarly, 2xFKBP-mGFP can form oligomers by the addition of AP20187. Clusters made with the FKBP constructs can therefore be either monomeric in the absence of their ligand or dimers or oligomers in the presence of ligand. The level of anisotropy of monomers, dimers, or oligomers can be used as a reference for cluster size measurements. This homo-FRET method can be employed to study the clustering behavior of EGFR for fundamental research and anticancer drug research. For example, it has been shown that EGFR can also form inactive dimers in the absence of EGF; these predimers are formed independently of the C-terminal tail. In contrast, EGF-dependent oligomerization depends on tyrosine kinase activity and more particular on the nine tyrosine residues in the C-terminal tail of the receptor (Hofman et al., 2010).
16.1 THEORY HOMO-FRET QUANTIFICATION 16.1.1 Steady-state fluorescence anisotropy imaging In homo-FRET experiments, a linearly polarized excitation laser beam is used to excite the fluorophores. Next, the intensities of the parallel and perpendicular polarized fluorescence are detected. Here, parallel and perpendicular are defined with respect to the polarization direction of the excitation laser. From these intensities of the fluorescence, the anisotropy can be calculated using Eq. (16.1). Upon homo-FRET between fluorophores, the anisotropy decreases because the energy transfer between two fluorophores results in emission by a molecule with a different orientation than the molecule directly excited by the laser (Fig. 16.1) (Bader et al., 2009; Lakowicz, 2006; Runnels & Scarlata, 1995): r ðtÞ ¼
Ipar Iper Ipar 2Iper
(16.1)
Besides homo-FRET, other processes can affect the fluorescence anisotropy, such as rotational diffusion of fluorophores. If the excited fluorophore changes its orientation in the time period between excitation and emission, the anisotropy will be lowered. However, in case of a large, slowly rotating molecule like a green fluorescent protein (GFP), the depolarization due to rotations is negligible (Bader, Hofman, van Bergen en Henegouwen, & Gerritsen, 2007). The rotation correlation time of fluorescent proteins is typically in the order of 20 ns, while the fluorescence lifetime is usually 0.5. Four-gate anisotropy decays are created by binning the intensities Ipar and Iper per gate in regions of interest. In the anisotropy imaging experiments, a threshold of Ipar,inf þ 2Iper,inf > 300 counts was applied to all images. In theory, this number of counts corresponds to a standard deviation in the anisotropy of 0.05 (Bader et al., 2009; Hofman et al., 2010). An example of cluster size images derived from homo-FRET images is shown in Fig. 16.6A, where a resting cell before EGF stimulation is shown. Here, cluster sizes are small, but a clear population of predimers is visible; 40% of the EGFR is present as dimer. After stimulating the cell with EGF for 10 min average, cluster sizes have increased up to >2; and a significant fraction of the clusters are of size >3 (Fig. 16.6B).
16.4 DISCUSSION Studying EGFR dimerization and oligomerization is important to completely understand the signaling and trafficking mechanism of EGFR. EGFR dimerization has been studied by several biochemical studies like chemical cross-linking, co-IP with
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A
B
EGFR-mGFP
NAV=1
EGFR-mGFP +EGF
NAV=2
NAV ³ 3
FIGURE 16.6 (A) Homo-FRET-based cluster size image before stimulation with EGF and (B) after stimulation with 8 nM EGF. Results adapted from Hofman et al. (2010).
differentially labeled EGFR, and also crystallography to determine the monomer and dimer structure. Although there is already a lot known about the dimerization of EGFR, the mechanism of oligomerization remains obscure. Advanced microscopy techniques were developed to study EGFR oligomerization; initially EM with gold-labeled EGFR was used. The cluster size that can be determined by EM depends on the labeling of EGFR, in other words the size of the gold particles and antibody used (10 nm). Advanced fluorescence microscopy techniques were developed like FRET-FLIM, FIDA, and N&B analysis. These techniques are able to measure small cluster sizes and are complementary techniques. FRET-FLIM is rapid and accurate, while FIDA is able to measure cluster size in life cells. FIDA measures the clustering on the plasma membrane of cells and is a slow method; therefore, it is not suitable for measuring EGF-induced EGFR clustering in time. EGFR will internalize before the FIDA method is able to determine the cluster sizes on the plasma membrane. N&B uses the same principle as fluorescence correlation spectroscopy (FCS) measurements and measures the distribution and association states of molecules in live cells (Nagy, Claus, Jovin, & Arndt-Jovin, 2010). This technique demonstrated the concentration-dependent predimer formation. In contrast, FIDA is able to give information about the distribution of cluster sizes within a single pixel. FIDA and FCS, however, need high illumination intensities for accurate measurements, which can cause photodamage to the proteins in the cell. To study in more detail the EGFinduced EGFR clustering, FRET was used and more specifically the homo-FRET method. With this method, small EGFR clusters can be accurately determined and the cluster size distribution in one cell can be determined within single pixels. The advantage of homo-FRET instead of hetero-FRET is that the detection of FRET is not dependent on the concentrations of donor and acceptor, which simplifies the sample preparation. Homo-FRET is detected by a change in anisotropy, which is a different method than used for measuring hetero-FRET. Anisotropy is a measure for
Acknowledgments
the depolarization of the linear polarized excitation beam. To be able to convert the obtained anisotropy values into cluster sizes, we use reference proteins like EGFRFKBP-mGFP constructs, which form clusters of known sizes when AP20187 is added. With this method, cluster sizes of EGFR in the order of N ¼ 1, N ¼ 2, or N 3 can be determined. A conventional fluorescence microscope can be converted in a homo-FRET-detecting system when a set of polarizers is integrated and sufficient signal is generated. The limitation of this method is that it is up till now only able to distinguish monomers, dimers, and oligomers. This method is not able to measure the precise amount of molecules in higher-order clusters. Homo-FRET was used to study the predimer formation of EGFR and showed to be receptor concentration-independent by homo-FRET measurements. This is in contrast to what was found by the N&B analyses (Hofman et al., 2010; Nagy et al., 2010). This difference might be explained by the different sample preparations as homo-FRET was measured in fixed cells and N&B analysis in live cells. We note that the N&B method does not include the immobile fraction of the receptors, which is known to exist. Another explanation for the difference could be that the N&B analysis cannot distinguish the cluster size distribution within a single pixel and homo-FRET can. It might be that the amount of predimers changes upon receptor concentration but that there are still predimers formed when there are low EGFR concentrations present. This is just one example of contradicting results of EGFR clustering measurements. Combining these different methods is required to overcome these contradictions. Most probably, the most ideal way to measure clustering is by combining FRET studies with highresolution microscopy methods like FIDA, N&B, EM, or others because in that situation, you measure both the larger cluster organizations and the smaller subclusters using homo-FRET. The super resolution microscopy technique STED would be a good possibility to combine with FRET because the resolution of STED can be up to 20 nm, which is slightly higher than homo-FRET, which makes it possible when the two methods are combined to measure small clusters simultaneously with larger clusters. STED can also be applied in life cells and gives clustering information about a whole cell, which is not the case with, for example, single-molecule tracking where only a set of clusters can be analyzed (Pellett et al., 2011). In this chapter, we have described the homo-FRET method for measuring EGFR clustering and compared them with other experimental techniques. This method can equally be applied for other proteins, provided their functioning is not affected by the fusion to mGFP. Essential are the reference proteins, which are mGFP fused to FKBP for cytoplasmic proteins and FKBP fused to a receptor can be used as reference for the analysis of receptor clustering. Homo-FRET has the advantage of simple sample preparation and accurate cluster size determination. However, homo-FRET is not able to detect larger cluster organization.
Acknowledgments The authors wish to thank Dr. Erik G. Hofman and Dr. Arjan N. Bader for their fruitful discussions.
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References Ariotti, N., Liang, H., Xu, Y., Zhang, Y., Yonekubo, Y., Inder, K., et al. (2010). Epidermal growth factor receptor activation remodels the plasma membrane lipid environment to induce nanocluster formation. Molecular and Cellular Biology, 30(15), 3795–3804. Bader, A. N., Hofman, E. G., van Bergen en Henegouwen, P. M., & Gerritsen, H. C. (2007). Imaging of protein cluster sizes by means of confocal time-gated fluorescence anisotropy microscopy. Optics Express, 15(11), 6934–6945. Bader, A. N., Hofman, E. G., Voortman, J., en Henegouwen, P. M., & Gerritsen, H. C. (2009). Homo-FRET imaging enables quantification of protein cluster sizes with subcellular resolution. Biophysical Journal, 97(9), 2613–2622. Bancroft, J. D., & Stevens, A. (1982). Theory and practice of histological techniques. London, UK: Churchill Livingstone. Chen, Y., Mills, J. D., & Periasamy, A. (2003). Protein localization in living cells and tissues using FRET and FLIM. Differentiation, 71(9–10), 528–541. Clayton, A. H., Walker, F., Orchard, S. G., Henderson, C., Fuchs, D., Rothacker, J., et al. (2005). Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor—A multidimensional microscopy analysis. Journal of Biological Chemistry, 280(34), 30392–30399. De Grauw, C. J., & Gerritsen, H. C. (2001). Multiple time-gate module for fluorescence lifetime imaging. Applied Spectroscopy, 55(6), 670. Ferguson, K. M. (2008). Structure-based view of epidermal growth factor receptor regulation. Annual Review of Biophysics, 37, 353–373. Gadella, T. W., Jr., & Jovin, T. M. (1995). Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. The Journal of Cell Biology, 129(6), 1543–1558. Gautier, I., Tramier, M., Durieux, C., Coppey, J., Pansu, R. B., Nicolas, J. C., et al. (2001). Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFPtagged proteins. Biophysical Journal, 80(6), 3000–3008. Goh, L. K., Huang, F., Kim, W., Gygi, S., & Sorkin, A. (2010). Multiple mechanisms collectively regulate clathrin-mediated endocytosis of the epidermal growth factor receptor. The Journal of Cell Biology, 189(5), 871–883. Hancock, J. F., & Prior, I. A. (2005). Electron microscopic imaging of ras signaling domains. Methods, 37(2), 165–172. Hofman, E. G., Bader, A. N., Voortman, J., van den Heuvel, D. J., Sigismund, S., Verkleij, A. J., et al. (2010). Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization. Journal of Biological Chemistry, 285(50), 39481–39489. Hofman, E. G., Ruonala, M. O., Bader, A. N., van den Heuvel, D., Voortman, J., Roovers, R. C., et al. (2008). EGF induces coalescence of different lipid rafts. Journal of Cell Science, 121(Pt 15), 2519–2528. Jorissen, R. N., Walker, F., Pouliot, N., Garrett, T. P., Ward, C. W., & Burgess, A. W. (2003). Epidermal growth factor receptor: Mechanisms of activation and signalling. Experimental Cell Research, 284(1), 31–53. Jura, N., Endres, N. F., Engel, K., Deindl, S., Das, R., Lamers, M. H., et al. (2009). Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment. Cell, 137(7), 1293–1307.
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Lakowicz, J. R. (2006). In J. R. Lakowicz (Ed.), Principles of fluorescence spectroscopy. (3th ed.). New York: Springer. Moriki, T., Maruyama, H., & Maruyama, I. N. (2001). Activation of preformed EGF receptor dimers by ligand-induced rotation of the transmembrane domain. Journal of Molecular Biology, 311(5), 1011–1026. Nagy, P., Claus, J., Jovin, T. M., & Arndt-Jovin, D. J. (2010). Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis. Proceedings of the National Academy of Sciences of the United States of America, 107(38), 16524–16529. Oliveira, S., van Bergen en Henegouwen, P. M., Storm, G., & Schiffelers, R. M. (2006). Molecular biology of epidermal growth factor receptor inhibition for cancer therapy. Expert Opinion on Biological Therapy, 6(6), 605–617. Pellett, P. A., Sun, X., Gould, T. J., Rothman, J. E., Xu, M. Q., Correa, I. R., Jr., et al. (2011). Two-color STED microscopy in living cells. Biomedical Optics Express, 2(8), 2364–2371. Runnels, L. W., & Scarlata, S. F. (1995). Theory and application of fluorescence homotransfer to melittin oligomerization. Biophysical Journal, 69(4), 1569–1583. Saffarian, S., Li, Y., Elson, E. L., & Pike, L. J. (2007). Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis. Biophysical Journal, 93(3), 1021–1031. Sako, Y., Minoghchi, S., & Yanagida, T. (2000). Single-molecule imaging of EGFR signalling on the surface of living cells. Nature Cell Biology, 2(3), 168–172. Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., et al. (2004). Nanoscale organization of multiple GPI-anchored proteins in living cell membranes. Cell, 116(4), 577–589. Sorkin, A., & Goh, L. K. (2009). Endocytosis and intracellular trafficking of ErbBs. Experimental Cell Research, 315(4), 683–696. Tanaka, F., & Mataga, N. (1979). Theory of time-dependent photo-selection in interacting fixed systems. Photochemistry and Photobiology, 29(6), 1091–1097. Ullrich, A., & Schlessinger, J. (1990). Signal transduction by receptors with tyrosine kinase activity. Cell, 61(2), 203–212. van Belzen, N., Rijken, P. J., Hage, W. J., de Laat, S. W., Verkleij, A. J., & Boonstra, J. (1988). Direct visualization and quantitative analysis of epidermal growth factor-induced receptor clustering. Journal of Cellular Physiology, 134(3), 413–420. Yarden, Y., & Sliwkowski, M. X. (2001). Untangling the ErbB signalling network. Nature Reviews. Molecular Cell Biology, 2(2), 127–137. Yu, X., Sharma, K. D., Takahashi, T., Iwamoto, R., & Mekada, E. (2002). Ligand-independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signaling. Molecular Biology of the Cell, 13(7), 2547–2557. Zacharias, D. A., Violin, J. D., Newton, A. C., & Tsien, R. Y. (2002). Partitioning of lipidmodified monomeric GFPs into membrane microdomains of live cells. Science, 296(5569), 913–916. Zhu, H. J., Iaria, J., Orchard, S., Walker, F., & Burgess, A. W. (2003). Epidermal growth factor receptor: Association of extracellular domain negatively regulates intracellular kinase activation in the absence of ligand. Growth Factors, 21(1), 15–30.
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Analysis of EGF Receptor Oligomerization by Homo-FRET
16
Cecilia de Heus*, Nivard Kagie{, Raimond Heukers*, Paul M.P. van Bergen en Henegouwen*, and Hans C. Gerritsen{ *
Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, Netherlands { Molecular Biophysics, Department of Soft Condensed Matter and Biophysics, Science Faculty, Utrecht University, Utrecht, Netherlands
CHAPTER OUTLINE Introduction ............................................................................................................ 306 16.1 Theory Homo-FRET Quantification ....................................................................309 16.1.1 Steady-State Fluorescence Anisotropy Imaging ............................. 309 16.1.2 Time-Resolved Fluorescence Anisotropy Imaging........................... 311 16.2 Materials........................................................................................................313 16.2.1 Plasmid Constructs..................................................................... 313 16.2.2 Cell Lines .................................................................................. 315 16.2.3 Microscope ................................................................................ 315 16.2.3.1 Confocal Time-Resolved and Steady-State Fluorescence Anisotropy Imaging Microscopy ........................................................315 16.3 Methods .........................................................................................................316 16.3.1 Slide Preparation........................................................................ 316 16.3.1.1 Reference Measurements ...................................................316 16.3.1.2 Predimerization Measurements ...........................................316 16.3.1.3 Ligand-induced Oligomerization ..........................................317 16.3.2 Analysis..................................................................................... 317 16.4 Discussion......................................................................................................317 Acknowledgments ................................................................................................... 319 References ............................................................................................................. 320
Abstract Growth factor receptors are present in the plasma membrane of resting cells as monomers or (pre)dimers. Ligand binding results in higher-order oligomerization of ligand–receptor complexes. To study the regulation of receptor clustering, several Methods in Cell Biology, Volume 117 Copyright © 2013 Elsevier Inc. All rights reserved.
ISSN 0091-679X http://dx.doi.org/10.1016/B978-0-12-408143-7.00016-5
305
306
CHAPTER 16 Analysis of EGF Receptor Oligomerization by Homo-FRET
experimental techniques have been developed in the last decades. However, many involve invasive approaches that are likely to disturb the integrity of the membrane, thereby affecting receptor interactions. In this chapter, we describe the use of a noninvasive approach to study receptor dimerization and oligomerization. This method is based upon the Fo¨rster energy transfer between identical adjacent fluorescent proteins (homo-FRET) and is determined by analyzing the change in fluorescence anisotropy. Homo-FRET takes place within a distance of 10 nm, making this an excellent approach for studying receptor–receptor interactions in intact cells. After excitation of monomeric GFP (mGFP) with polarized light, limiting anisotropy values (rinf) of the emitted light are determined, where proteins with known cluster sizes are used as references. Dimerization and oligomerization of the epidermal growth factor receptor (EGFR) in response to ligand binding is determined by using receptors that have been fused with mGFP at their C-terminus. In this chapter, we describe the involved technology and discuss the feasibility of homo-FRET experiments for the determination of cluster sizes of growth factor receptors like EGFR.
INTRODUCTION The epidermal growth factor receptor (EGFR), also called ErbB1 or Her1, is a member of the ErbB single-pass transmembrane tyrosine kinase receptor family (Ullrich & Schlessinger, 1990; Yarden & Sliwkowski, 2001). Activation of EGFR and its family members is involved in cell growth, cell proliferation, and migration. Many cancer types show overexpression or deregulation of EGFR, and it is therefore a well-studied receptor and an attractive anticancer drug target (Oliveira, van Bergen en Henegouwen, Storm, & Schiffelers, 2006; Sorkin & Goh, 2009). EGFR is composed of an extracellular domain, a transmembrane domain, and an intracellular C-terminal tail containing the tyrosine kinase and several sites involved in posttranslational modifications and signaling (Jorissen et al., 2003). The extracellular part of EGFR contains four domains of which domain I and III are involved in ligand binding and domain II in receptor dimerization. More than 20 different ligands are known for receptors from the ErbB family of which EGF is the most studied one. Ligand binding induces conformational changes of the ectodomain, which results in not only receptor dimerization but also even receptor oligomerization or clustering (Clayton et al., 2005). As a consequence of these changes, cross-phosphorylation of its C-terminal tail occurs (Jura et al., 2009). Interestingly, EGFR can form homo- or heterodimers with other ErbB family members and this already happens in resting cells. These inactive dimers on the plasma membrane are the so-called receptor predimers. Although the structure of receptor dimers is clear from crystal structures, the mechanism of receptor oligomerization remains obscure (Ferguson, 2008). Crystal structures have shown head to head interactions between receptors, suggesting the involvement of these sequences in oligomerization. Recently, also kinase activity was shown to be required for receptor clustering, which suggests the involvement of signaling in this process (Clayton et al., 2005; Hofman et al., 2010). For example,
Introduction
the production of phosphatidic acid by phospholipase D2 (PLD2) has been demonstrated to be required for EGFR clustering (Ariotti et al., 2010). Besides receptor oligomerization and downstream signaling toward cell proliferation, activation of EGF receptors results in a rapid internalization mainly via clathrin-mediated endocytosis. The intracellular transport trajectory finally ends up in lysosomes where the ligand-induced signaling is terminated by degradation of both ligand and receptors (Goh, Huang, Kim, Gygi, & Sorkin, 2010; Sorkin & Goh, 2009). For studying the clustering of EGF receptors, different experimental techniques have been developed. Dimerization of EGFR, for example, can be determined using chemical cross-linking and detection by SDS-PAGE, by co-IP with differentially tagged EGFR, or by fluorescence complementation (Moriki, Maruyama, & Maruyama, 2001; Yu, Sharma, Takahashi, Iwamoto, & Mekada, 2002; Zhu, Iaria, Orchard, Walker, & Burgess, 2003). Studying receptor clustering is more difficult and demands other experimental strategies. Receptor clustering was initially investigated using electron microscopy (EM), which can be used at high resolution, enabling visualization of gold-labeled EGFR (van Belzen et al., 1988). Analysis of cluster formation can be done by determining particle distances, either by hand or automatically using Ripley’s function (Hancock & Prior, 2005). However, limitations exist with respect to determining cluster size, which is dependent on the size of the gold particle and the antibody (10 nm) (Hancock & Prior, 2005). Another approach for determining receptor clustering involves fluorescence light microscopy. However, conventional microscopy has a resolution limit of 200 nm, which makes this technique not very suitable for measuring the formation of small receptor clusters. To overcome this resolution limit problem, more advanced light microscope techniques were developed, which do provide information about small cluster sizes. One of the first examples was described by Gadella and coworkers and included time-resolved fluorescence microscopy based on Fo¨rster resonance energy transfer (FRET) analyzed by fluorescence lifetime imaging microscopy (FLIM) (Gadella & Jovin, 1995). FRET is based on the energy transfer between a donor and an acceptor fluorophore. For FRET to occur, the donor emission spectrum and acceptor excitation spectrum need sufficient spectral overlap and the two fluorophores should be within a distance of 10 nm. Because energy transfer only occurs when fluorophores are within 10 nm of each other, the detection of FRET can be used to study receptor dimerization or small cluster formation. There are different methods to detect FRET, for example, by measuring changes in the donor/acceptor emission intensity ratio, changes in fluorescence lifetime (FLIM), or differences in anisotropy. The donor/acceptor ratio is determined by measuring the emission of both fluorophores. However, this donor/ acceptor ratio is dependent on the concentrations of both fluorophores. With FLIM, the time that the donor fluorophore is in its excited state is measured. This lifetime can be determined by measuring the fluorescence decay of the donor probe after excitation by using a short laser pulse. The fluorescence lifetime changes upon its environment and also when energy transfer occurs. This method is less dependent on local concentrations of both probes and is therefore a more robust way to detect FRET (Chen, Mills, & Periasamy, 2003; Hofman et al., 2008).
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FRET-FLIM was previously used successfully to determine EGFR cluster formation by Clayton et al. (2005). EGFR-GFP in combination with anti-phosphotyrosineAlexa555 was used to label fixed and permeabilized cells. Using FRET-FLIM in combination with image correlation spectroscopy (ICS), Clayton et al. found an average cluster size of 2.2 in the absence of EGF and an average of 3.7 in the presence of EGF, suggesting the formation of higher-order clusters upon ligand binding. A limitation of this method is that it assumes the cell to be stationary and therefore only an average of the different cluster sizes in a cell can be determined instead of determining the subcellular distribution of the different cluster sizes. To solve this, Saffarian et al. studied EGFR clustering by a method based on fluorescence intensity distribution analysis (FIDA) in live cells for quantifying receptor clustering on cell membranes (Saffarian, Li, Elson, & Pike, 2007). Using this technique, the authors find an average cluster size of 1.3 in unstimulated cells, which is in contrast to results from Clayton et al. The difference between the two values might be explained by different measurement conditions, live cells versus fixed cells, and measuring the clustering distribution with FIDA instead of determining an average. A limitation of the FIDA method is that intensity fluctuations might occur during the measurements, which might disturb the clustering measurements. Furthermore, the FIDA method is relatively slow and may therefore not be suitable for measuring of EGFR clustering after EGF stimulation because of the fast internalization of EGFR. More recently, a number and brightness (N&B) analysis technique was used to study EGFR clustering, which demonstrated the formation of up to pentameric EGFR clusters (Sako, Minoghchi, & Yanagida, 2000). More recently, we introduced a method based on homo-FRET to analyze EGFR clustering. With this method, EGFR clustering can be accurately measured on intact cells using only a single label (EGFR-mGFP) (Bader, Hofman, Voortman, en Henegouwen, & Gerritsen, 2009; Hofman et al., 2010). For homo-FRET, the same fluorophore for donor and acceptor is used, which has the advantage that the detection of FRET is not dependent on concentrations of donor and acceptor. Another advantage of only one fluorophore is that sample preparation is simplified. However, homo-FRET does require a different quantification method because homo-FRET does not result in a change in the emission spectrum or in fluorescence lifetime, resulting in FRET to stay unnoticed. The decreased lifetime of the donor fluorophore in homo-FRET is compensated by the excitation by the acceptor fluorophore. The here applied analysis of energy transfer is based upon the FRETinduced loss of anisotropy. When fluorophores are excited with polarized light, only the subset that is in the right orientation will be excited. In the case of large molecules that are unable to spin on the time scale of the fluorescence lifetime, the emitted light by this subset of fluorophores is also polarized, albeit with a slightly different angular distribution. In the case of two fluorophores in close proximity, there is a high probability that they are in different orientations. Upon excitation of one fluorophore, the neighboring one could be excited through FRET, resulting in the emission of light with this different orientation. Therefore, the energy transfer between two identical fluorophores with slightly different orientation result in depolarization of the
16.1 Theory Homo-FRET Quantification
emission light; the emitted light is more isotropically distributed. Therefore, quantification of the fluorescence anisotropy can be used to determine homo-FRET. The homo-FRET method can be calibrated using reference measurements on protein clusters of known size. Subsequently, EGFR cluster sizes can be determined directly from measured fluorescence anisotropy values. As a tool for the reference measurements, we have used FKBP-mGFP, which dimerizes via its ligand AP20187. Similarly, 2xFKBP-mGFP can form oligomers by the addition of AP20187. Clusters made with the FKBP constructs can therefore be either monomeric in the absence of their ligand or dimers or oligomers in the presence of ligand. The level of anisotropy of monomers, dimers, or oligomers can be used as a reference for cluster size measurements. This homo-FRET method can be employed to study the clustering behavior of EGFR for fundamental research and anticancer drug research. For example, it has been shown that EGFR can also form inactive dimers in the absence of EGF; these predimers are formed independently of the C-terminal tail. In contrast, EGF-dependent oligomerization depends on tyrosine kinase activity and more particular on the nine tyrosine residues in the C-terminal tail of the receptor (Hofman et al., 2010).
16.1 THEORY HOMO-FRET QUANTIFICATION 16.1.1 Steady-state fluorescence anisotropy imaging In homo-FRET experiments, a linearly polarized excitation laser beam is used to excite the fluorophores. Next, the intensities of the parallel and perpendicular polarized fluorescence are detected. Here, parallel and perpendicular are defined with respect to the polarization direction of the excitation laser. From these intensities of the fluorescence, the anisotropy can be calculated using Eq. (16.1). Upon homo-FRET between fluorophores, the anisotropy decreases because the energy transfer between two fluorophores results in emission by a molecule with a different orientation than the molecule directly excited by the laser (Fig. 16.1) (Bader et al., 2009; Lakowicz, 2006; Runnels & Scarlata, 1995): r ðtÞ ¼
Ipar Iper Ipar 2Iper
(16.1)
Besides homo-FRET, other processes can affect the fluorescence anisotropy, such as rotational diffusion of fluorophores. If the excited fluorophore changes its orientation in the time period between excitation and emission, the anisotropy will be lowered. However, in case of a large, slowly rotating molecule like a green fluorescent protein (GFP), the depolarization due to rotations is negligible (Bader, Hofman, van Bergen en Henegouwen, & Gerritsen, 2007). The rotation correlation time of fluorescent proteins is typically in the order of 20 ns, while the fluorescence lifetime is usually 0.5. Four-gate anisotropy decays are created by binning the intensities Ipar and Iper per gate in regions of interest. In the anisotropy imaging experiments, a threshold of Ipar,inf þ 2Iper,inf > 300 counts was applied to all images. In theory, this number of counts corresponds to a standard deviation in the anisotropy of 0.05 (Bader et al., 2009; Hofman et al., 2010). An example of cluster size images derived from homo-FRET images is shown in Fig. 16.6A, where a resting cell before EGF stimulation is shown. Here, cluster sizes are small, but a clear population of predimers is visible; 40% of the EGFR is present as dimer. After stimulating the cell with EGF for 10 min average, cluster sizes have increased up to >2; and a significant fraction of the clusters are of size >3 (Fig. 16.6B).
16.4 DISCUSSION Studying EGFR dimerization and oligomerization is important to completely understand the signaling and trafficking mechanism of EGFR. EGFR dimerization has been studied by several biochemical studies like chemical cross-linking, co-IP with
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A
B
EGFR-mGFP
NAV=1
EGFR-mGFP +EGF
NAV=2
NAV ³ 3
FIGURE 16.6 (A) Homo-FRET-based cluster size image before stimulation with EGF and (B) after stimulation with 8 nM EGF. Results adapted from Hofman et al. (2010).
differentially labeled EGFR, and also crystallography to determine the monomer and dimer structure. Although there is already a lot known about the dimerization of EGFR, the mechanism of oligomerization remains obscure. Advanced microscopy techniques were developed to study EGFR oligomerization; initially EM with gold-labeled EGFR was used. The cluster size that can be determined by EM depends on the labeling of EGFR, in other words the size of the gold particles and antibody used (10 nm). Advanced fluorescence microscopy techniques were developed like FRET-FLIM, FIDA, and N&B analysis. These techniques are able to measure small cluster sizes and are complementary techniques. FRET-FLIM is rapid and accurate, while FIDA is able to measure cluster size in life cells. FIDA measures the clustering on the plasma membrane of cells and is a slow method; therefore, it is not suitable for measuring EGF-induced EGFR clustering in time. EGFR will internalize before the FIDA method is able to determine the cluster sizes on the plasma membrane. N&B uses the same principle as fluorescence correlation spectroscopy (FCS) measurements and measures the distribution and association states of molecules in live cells (Nagy, Claus, Jovin, & Arndt-Jovin, 2010). This technique demonstrated the concentration-dependent predimer formation. In contrast, FIDA is able to give information about the distribution of cluster sizes within a single pixel. FIDA and FCS, however, need high illumination intensities for accurate measurements, which can cause photodamage to the proteins in the cell. To study in more detail the EGFinduced EGFR clustering, FRET was used and more specifically the homo-FRET method. With this method, small EGFR clusters can be accurately determined and the cluster size distribution in one cell can be determined within single pixels. The advantage of homo-FRET instead of hetero-FRET is that the detection of FRET is not dependent on the concentrations of donor and acceptor, which simplifies the sample preparation. Homo-FRET is detected by a change in anisotropy, which is a different method than used for measuring hetero-FRET. Anisotropy is a measure for
Acknowledgments
the depolarization of the linear polarized excitation beam. To be able to convert the obtained anisotropy values into cluster sizes, we use reference proteins like EGFRFKBP-mGFP constructs, which form clusters of known sizes when AP20187 is added. With this method, cluster sizes of EGFR in the order of N ¼ 1, N ¼ 2, or N 3 can be determined. A conventional fluorescence microscope can be converted in a homo-FRET-detecting system when a set of polarizers is integrated and sufficient signal is generated. The limitation of this method is that it is up till now only able to distinguish monomers, dimers, and oligomers. This method is not able to measure the precise amount of molecules in higher-order clusters. Homo-FRET was used to study the predimer formation of EGFR and showed to be receptor concentration-independent by homo-FRET measurements. This is in contrast to what was found by the N&B analyses (Hofman et al., 2010; Nagy et al., 2010). This difference might be explained by the different sample preparations as homo-FRET was measured in fixed cells and N&B analysis in live cells. We note that the N&B method does not include the immobile fraction of the receptors, which is known to exist. Another explanation for the difference could be that the N&B analysis cannot distinguish the cluster size distribution within a single pixel and homo-FRET can. It might be that the amount of predimers changes upon receptor concentration but that there are still predimers formed when there are low EGFR concentrations present. This is just one example of contradicting results of EGFR clustering measurements. Combining these different methods is required to overcome these contradictions. Most probably, the most ideal way to measure clustering is by combining FRET studies with highresolution microscopy methods like FIDA, N&B, EM, or others because in that situation, you measure both the larger cluster organizations and the smaller subclusters using homo-FRET. The super resolution microscopy technique STED would be a good possibility to combine with FRET because the resolution of STED can be up to 20 nm, which is slightly higher than homo-FRET, which makes it possible when the two methods are combined to measure small clusters simultaneously with larger clusters. STED can also be applied in life cells and gives clustering information about a whole cell, which is not the case with, for example, single-molecule tracking where only a set of clusters can be analyzed (Pellett et al., 2011). In this chapter, we have described the homo-FRET method for measuring EGFR clustering and compared them with other experimental techniques. This method can equally be applied for other proteins, provided their functioning is not affected by the fusion to mGFP. Essential are the reference proteins, which are mGFP fused to FKBP for cytoplasmic proteins and FKBP fused to a receptor can be used as reference for the analysis of receptor clustering. Homo-FRET has the advantage of simple sample preparation and accurate cluster size determination. However, homo-FRET is not able to detect larger cluster organization.
Acknowledgments The authors wish to thank Dr. Erik G. Hofman and Dr. Arjan N. Bader for their fruitful discussions.
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