The efficacy of receptor tyrosine kinase EphA2 autophosphorylation increases with EphA2 oligomer size

The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell–cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms high-order oligomers upon ligand binding. Here, we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used fluorescence intensity fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Our comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, an autophosphorylation response contributing to EphA2 activation, correlates with EphA2 mean oligomer size. However, we found that other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. Taken together, this work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features. The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell–cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms high-order oligomers upon ligand binding. Here, we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used fluorescence intensity fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Our comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, an autophosphorylation response contributing to EphA2 activation, correlates with EphA2 mean oligomer size. However, we found that other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. Taken together, this work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features. Assessment of oligomer sizes of membrane protein complexes in live cells poses unique challenges, as most methods used for soluble proteins are not applicable in the context of the native plasma membrane. Fluorescence-based methods are often the only option available to probe the oligomerization of membrane proteins suitably labeled with fluorophores. Widely used fluorescent-based techniques are FRET, fluorescence lifetime imaging, and fluorescence fluctuation spectroscopy (1Kozer N. Henderson C. Jackson J.T. Nice E.C. Burgess A.W. Clayton A.H.A. Evidence for extended YFP-EGFR dimers in the absence of ligand on the surface of living cells.Phys. Biol. 2011; 8: 066002Crossref PubMed Scopus (21) Google Scholar, 2Wallrabe H. Periasamy A. 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Some of these fluorescence fluctuation methods are based on fluorescence cross-correlation spectroscopy (FCCS), a technique in which the dynamics, concentration, and interactions of diffusing proteins labeled with a fluorophore are determined by spatial autocorrelation analysis (10Digman M.A. Sengupta P. Wiseman P.W. Brown C.M. Horwitz A.R. Gratton E. Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure.Biophys. J. 2005; 88: L33-36Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). FCCS often utilizes pulsed interleaved excitation via two synchronized lasers with a time delay between them, such that the energy transfer to the acceptor and the direct excitation of the acceptor are separated in time (11Hendrix J. Lamb D.C. Implementation and application of pulsed interleaved excitation for dual-color FCS and RICS.Met. Mol. Biol. 2014; 1076: 653-682Crossref PubMed Scopus (10) Google Scholar). 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Biol. 2014; 1076: 653-682Crossref PubMed Scopus (10) Google Scholar). An example of a technique that measures fluorescence fluctuation on a standard confocal microscope is number and brightness (N&B) (15Digman M.A. Dalal R. Horwitz A.F. Gratton E. Mapping the number of molecules and brightness in the laser scanning microscope.Biophys. J. 2008; 94: 2320-2332Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 16Nagy P. Claus J. Jovin T.M. Arndt-Jovin D.J. Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 16524-16529Crossref PubMed Scopus (129) Google Scholar, 17Paul M.D. Rainwater R. Zuo Y. Gu L. Hristova K. Probing membrane protein association using concentration-dependent number and brightness.Angew. Chem. Int. Ed. Engl. 2021; 60: 6503-6508Crossref PubMed Scopus (5) Google Scholar). N&B works by rapidly acquiring a stack of images of the same region of a cell and then computing the mean fluorescence intensity and the variance of the intensity across the stack for each pixel. The molecular brightness, defined as the ratio of the variance of the fluorescence intensity over time to the mean fluorescence intensity, is known to scale with the oligomer size. The average oligomer size is then easily calculated by normalizing the molecular brightness measured for a protein of interest to the molecular brightness of a monomer control. However, a caveat is that a large immobile oligomer would be invisible in N&B analysis, as no fluctuations would arise over time. While N&B monitors fluorescence fluctuations over time, other techniques such as spatial intensity distribution analysis (SPIDA) quantify fluctuations over space (18Godin A.G. Costantino S. Lorenzo L.-E. Swift J.L. Sergeev M. 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FIF spectrometry calculates the molecular brightness of fluorescent protein–tagged receptors in small segments of the plasma membrane and creates a histogram of these molecular brightness values derived from thousands of such segments. Here, we explore the utility of FIF in studies of EphA2 association in the plasma membrane. The EphA2 receptor is highly expressed in epithelial and endothelial cells, where it triggers diverse downstream signaling pathways that control the assembly of cell–cell junctions. This receptor has been implicated in many physiological and disease processes, such as cancer (19Pasquale E.B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond.Nat. Rev. Cancer. 2010; 10: 165-180Crossref PubMed Scopus (897) Google Scholar, 20Barquilla A. Pasquale E.B. Eph receptors and ephrins: therapeutic opportunities.Annu. Rev. Pharmacol. Toxicol. 2015; 55: 465-487Crossref PubMed Scopus (184) Google Scholar, 21Wilson K. Shiuan E. Brantley-Sieders D.M. 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In many cases, ligand-induced EphA2 signaling has been recognized as antioncogenic, and thus agents that activate EphA2 could be useful as cancer therapeutics (36Noberini R. Lamberto I. Pasquale E.B. Targeting Eph receptors with peptides and small molecules: progress and challenges.Semin. Cell Dev. Biol. 2012; 23: 51-57Crossref PubMed Scopus (82) Google Scholar). EphA2 belongs to the receptor tyrosine kinase (RTK) family. It is a single-pass transmembrane receptor with an extracellular region that binds the activating ligands (ephrins) and an intracellular region that contains the tyrosine kinase domain. The kinase domain is activated by autophosphorylation of tyrosine residues occurring upon close contact of neighboring EphA2 molecules. Therefore, lateral interactions of EphA2 molecules are the first required step in EphA2 signal transduction in the plasma membrane. While most of the 58 RTKs signal mainly as dimers, EphA2, in addition, can form high-order oligomers (37Singh D.R. 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However, the exact functional dependence of EphA2 signaling on the oligomerization state of the receptor is unknown. Challenges that have plagued such investigations have been (1) limited ability to control the oligomer size of EphA2 assemblies in cells and (2) limited methods to quantify heterogeneous distributions of oligomer sizes for membrane receptors. In this study, we overcome these limitations to investigate if a correlation exists between EphA2 oligomer size and signaling properties. This work was empowered by the recent discovery of a series of small engineered peptides that bind specifically to EphA2 and activate it (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). 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Pasquale E.B. Targeting the Eph system with peptides and peptide conjugates.Curr. Drug Targets. 2015; 16: 1031-1047Crossref PubMed Scopus (39) Google Scholar). The engineered peptides have been designed to modulate the association of EphA2 molecules (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar) and thus potentially modulate EphA2 oligomer size. The different dimeric peptide ligands used in this study have different potencies and different efficacies, depending on their sequence and configuration (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). These dimeric peptides are highly selective, and some of them have been shown to stimulate EphA2 signaling responses with unprecedented subnanomolar potency (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). Furthermore, the peptides and the monomeric soluble form of the ephrinA1 ligand (m-ephrinA1) have been shown to induce biased signaling compared with the widely used ligand ephrinA1-Fc, which consists of the ephrinA1 extracellular region dimerized by fusion to the Fc portion of an antibody (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). In particular, these ligands can differentially modulate two EphA2 signaling responses: EphA2 autophosphorylation on tyrosine 588 (Y588, a site in the juxtamembrane segment whose phosphorylation promotes EphA2 kinase activity and activation of downstream signaling) and inhibition of AKT phosphorylation on serine 473 (a site critical for AKT activation) (51Miao H. Li D.Q. Mukherjee A. Guo H. Petty A. Cutter J. et al.EphA2 mediates ligand-dependent inhibition and ligand-independent promotion of cell migration and invasion via a reciprocal regulatory loop with akt.Cancer Cell. 2009; 16: 9-20Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 52Yang N.Y. Fernandez C. Richter M. Xiao Z. Valencia F. Tice D.A. et al.Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells.Cell Signal. 2011; 23: 201-212Crossref PubMed Scopus (85) Google Scholar). The dimeric peptide ligands have been engineered from monomeric precursors through N-terminal, C-terminal, or N–C-terminal linkages (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). Based on molecular modeling, we previously hypothesized that these dimeric peptides stabilize different types of EphA2 dimers, engaging different interfaces and perhaps exhibiting different signaling properties (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). However, we found that all the dimeric peptides induce the formation of EphA2 oligomers. Using mutagenesis of two crystallographic extracellular interfaces, the “dimerization” interface and the “clustering” interface (38Himanen J.P. Yermekbayeva L. Janes P.W. Walker J.R. Xu K. Atapattu L. et al.Architecture of Eph receptor clusters.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 10860-10865Crossref PubMed Scopus (187) Google Scholar), we showed that a C-terminally linked dimeric peptide induces EphA2 oligomers that utilize both interfaces (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). In contrast, an N-terminally linked dimeric peptide induces EphA2 oligomers that utilize the dimerization but not the clustering interface (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). Here, we investigate differences in the size of EphA2 oligomers that form in response to ephrinA1-Fc, m-ephrinA1, three monomeric peptide ligands, and three dimeric peptide ligands with different configurations (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). We sought to assess the oligomerization state of EphA2, labeled with enhanced YFP (eYFP), using FIF spectrometry (8Stoneman M.R. Biener G. Ward R.J. Pediani J.D. Badu D. Eis A. et al.A general method to quantify ligand-driven oligomerization from fluorescence-based images.Nat. Met. 2019; 16: 493-496Crossref PubMed Scopus (33) Google Scholar). Attachment of eYFP to the C terminus of EphA2 via a 15 amino acid (GGS)5 flexible linker has been previously shown to not affect EphA2 autophosphorylation (53Singh D.R. Ahmed F. Paul M.D. Gedam M. Pasquale E.B. Hristova K. The SAM domain inhibits EphA2 interactions in the plasma membrane.Biochim. Biophys. Acta. 2016; 1864: 31-38Crossref Scopus (33) Google Scholar). Following EphA2-eYFP expression in transiently transfected human embryonic kidney 293T (HEK293T) cells without ligand treatment (Fig. 1A) or treated with different ligands (Fig. 1B), the plasma membrane in contact with the substrate was imaged by confocal microscopy as previously described (54Ahmed F. Zapata-Mercado E. Rahman S. Hristova K. The biased ligands NGF and NT-3 differentially stabilize Trk-A dimers.Biophys. J. 2021; 120: 55-63Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). We observed that the plasma membrane exhibits homogeneous EphA2-eYFP fluorescence in the absence of ligands (Fig. 1A). However, upon ligand addition, heterogeneities appear within a minute or two (Fig. 1B). The appearance of such “puncta” of EphA2 fluorescence in response to ligand binding has been reported in the literature (40Gomez-Soler M. Gehring M.P. Lechtenberg B.C. Zapata-Mercado E. Ruelos A. Matsumoto M.W. et al.Ligands with different dimeric configurations potently activate the EphA2 receptor and reveal its potential for biased signaling.iScience. 2022; 25103870Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar, 55Seiradake E. Schaupp A. Ruiz D.D.T. Kaufmann R. Mitakidis N. Harlos K. et al.Structurally encoded intraclass differences in EphA clusters drive distinct cell responses.Nat. Struct. Mol. Biol. 2013; 20: 958-964Crossref PubMed Scopus (70) Google Scholar) and used to determine whether EphA2 mutations affect receptor functionality (39Seiradake E. Harlos K. Sutton G. Aricescu A.R. Jones E.Y. An extracellular steric seeding mechanism for Eph-ephrin signaling platform assembly.Nat. Struct. Mol. Biol. 2010; 17: 398-402Crossref PubMed Scopus (161) Google Scholar). Interestingly, the appearance of the puncta is characteristically distinct for the different ligands (Fig. 1B). Fluorescence micrographs including ∼200 to 300 cells were analyzed with the FIF spectrometry software (8Stoneman M.R. Biener G. Ward R.J. Pediani J.D. Badu D. Eis A. et al.A general method to quantify ligand-driven oligomerization from fluorescence-based images.Nat. Met. 2019; 16: 493-496Crossref PubMed Scopus (33) Google Scholar). In the first step of the analysis, a selected area of the plasma membrane (Fig. 1A, P1) is divided into smaller