Transmembrane helix interactions regulate oligomerization of the receptor tyrosine kinase EphA2.

The transmembrane helices of receptor tyrosine kinases (RTKs) have been proposed to switch between two different dimeric conformations, one associated with the inactive RTK and the other with the active RTK. Furthermore, recent work has demonstrated that some full-length RTKs are associated into oligomers that are larger than dimers, raising questions about the roles of the TM helices in the assembly and function of these oligomers. Here we probe the roles of the TM helices in the assembly of EphA2 RTK oligomers in the plasma membrane. We employ mutagenesis to evaluate the relevance of a published NMR dimeric structure of the isolated EphA2 TM helix in the context of the full-length EphA2 in the plasma membrane. We use two fluorescence methods, Förster Resonance Energy Transfer and Fluorescence Intensity Fluctuations spectrometry, which yield complementary information about the EphA2 oligomerization process. These studies reveal that the TM helix mutations affect the stability, structure, and size of EphA2 oligomers. However, the effects are multifaceted and point to a more complex role of the TM helix than the one expected from the "TM dimer switch" model.

Computational insights into inhibiting EphA2: Integrating structure-based virtual screening, docking, and molecular dynamics simulations for small molecule discovery.

Elevated expression and dysfunction of ephrin type A receptor-2 (EphA2) have been implicated in the initiation and progression of cancer, metastasis, and unfavorable clinical outcomes. A promising strategy to counteract this dysregulation involves the development of small-molecule inhibitors that target EphA2. Our study focuses on this objective. To initiate Structure-Based Virtual Screening (SBVS), we leveraged an advanced online platform, the Mcule database, which houses an extensive collection of millions of chemical compounds. Using drug similarity filters, we efficiently identified ten thousand potential hits. By further refining the selection through toxicity profiling, we prudently narrowed down the candidates to a more manageable set of 100 molecules. Using the Mcule Single Click, DockThor, and SwissDock tools, we conducted multi-scoring docking assessments of thirty-seven compounds that satisfied the ADME standards. A comprehensive evaluation of Gibbs binding free energy terms, as derived from these docking tools, facilitated the identification of top-ranking docking hits. Remarkably, among the known inhibitors, dasatinib displayed the most robust binding to EphA2 with an average ΔG of -9.0 kcal/mol. Intriguingly, alternatives have emerged in recent years. Notably, small molecules such as Mcule-1579910267 (ΔG: -9.3 kcal/mol), Mcule-1893218381 (ΔG: -9.2 kcal/mol), Mcule-3981378344 (ΔG: -9.3 kcal/mol), and Mcule-8617639093 (ΔG: -9.1 kcal/mol) exhibited a notably strong binding affinity to EphA2, rivaling dasatinib. Subsequently, the four leading ligands along with dasatinib were selected for the MD simulations. Our rigorous analyses during the MD simulation phase encompassing RMSD, RMSF, SASA, ΔGsolv, and Rg underscored the favorable stability of Mcule-8617639093. This compelling evidence ultimately signifies the potential for selective EphA2 inhibition.

Cancer-Related Mutations in the Sam Domains of EphA2 Receptor and Ship2 Lipid Phosphatase: A Computational Study.

The lipid phosphatase Ship2 interacts with the EphA2 receptor by forming a heterotypic Sam (sterile alpha motif)-Sam complex. Ship2 works as a negative regulator of receptor endocytosis and consequent degradation, and anti-oncogenic effects in cancer cells should be induced by hindering its association with EphA2. Herein, a computational approach is presented to investigate the relationship between Ship2-Sam/EphA2-Sam interaction and cancer onset and further progression. A search was first conducted through the COSMIC (Catalogue of Somatic Mutations in Cancer) database to identify cancer-related missense mutations positioned inside or close to the EphA2-Sam and Ship2-Sam reciprocal binding interfaces. Next, potential differences in the chemical-physical properties of mutant and wild-type Sam domains were evaluated by bioinformatics tools based on analyses of primary sequences. Three-dimensional (3D) structural models of mutated EphA2-Sam and Ship2-Sam domains were built as well and deeply analysed with diverse computational instruments, including molecular dynamics, to classify potentially stabilizing and destabilizing mutations. In the end, the influence of mutations on the EphA2-Sam/Ship2-Sam interaction was studied through docking techniques. This in silico approach contributes to understanding, at the molecular level, the mutation/cancer relationship by predicting if amino acid substitutions could modulate EphA2 receptor endocytosis.

PIP2 promotes conformation-specific dimerization of the EphA2 membrane region.

The impact of the EphA2 receptor on cancer malignancy hinges on the two different ways it can be activated. EphA2 induces antioncogenic signaling after ligand binding, but ligand-independent activation of EphA2 is pro-oncogenic. It is believed that the transmembrane (TM) domain of EphA2 adopts two alternate conformations in the ligand-dependent and the ligand-independent states. However, it is poorly understood how the difference in TM helical crossing angles found in the two conformations impacts the activity and regulation of EphA2. We devised a method that uses hydrophobic matching to stabilize two conformations of a peptide comprising the EphA2 TM domain and a portion of the intracellular juxtamembrane (JM) segment. The two conformations exhibit different TM crossing angles, resembling the ligand-dependent and ligand-independent states. We developed a single-molecule technique using styrene maleic acid lipid particles to measure dimerization in membranes. We observed that the signaling lipid PIP2 promotes TM dimerization, but only in the small crossing angle state, which we propose corresponds to the ligand-independent conformation. In this state the two TMs are almost parallel, and the positively charged JM segments are expected to be close to each other, causing electrostatic repulsion. The mechanism PIP2 uses to promote dimerization might involve alleviating this repulsion due to its high density of negative charges. Our data reveal a conformational coupling between the TM and JM regions and suggest that PIP2 might directly exert a regulatory effect on EphA2 activation in cells that is specific to the ligand-independent conformation of the receptor.

Targeting Ship2-Sam with peptide ligands: Novel insights from a multidisciplinary approach.

The lipid phosphatase Ship2 binds the EphA2 receptor through a heterotypic Sam-Sam (Sterile alpha motif) interaction. Inhibitors of the Ship2-Sam/EphA2-Sam complex hold a certain potential as novel anticancer agents. The previously reported "KRI3" peptide binds Ship2-Sam working as a weak antagonist of the EphA2-Sam/Ship2-Sam interaction. Herein, the design and functional evaluation of KRI3 analogues, both linear and cyclic, are described. A multidisciplinary study was conducted through computational docking techniques, and conformational analyses by CD and NMR spectroscopies. The ability of new peptides to bind Ship2-Sam was analysed by NMR, MST and SPR assays. Studies on linear KRI3 analogues pointed out that aromatic interactions through tyrosines are important for the association with Ship2-Sam whereas, an increase of the net positive charge of the sequence or peptide cyclization through a disulfide bridge can favour unspecific interactions without a substantial improvement of the binding affinity to Ship2-Sam. Interestingly, preliminary cell-based assays demonstrated KRI3 cellular uptake even without the conjugation to a cell penetrating sequence with a main cytosolic localization. This work highlights important features of the KRI3 peptide that can be further exploited to design analogues able to hamper Sam-Sam interactions driven by electrostatic contacts.

Effective Tumor Targeting by EphA2-Agonist-Biotin-Streptavidin Conjugates.

We recently reported on a potent synthetic agent, 135H11, that selectively targets the receptor tyrosine kinase, EphA2. While 135H11 possesses a relatively high binding affinity for the ligand-binding domain of EphA2 (Kd~130 nM), receptor activation in the cell required the synthesis of dimeric versions of such agent (namely 135H12). This was expected given that the natural ephrin ligands also need to be dimerized or clustered to elicit agonistic activity in cell. In the present report we investigated whether the agonistic activity of 135H11 could be enhanced by biotin conjugation followed by complex formation with streptavidin. Therefore, we measured the agonistic EphA2 activity of 135H11-biotin (147B5) at various agent/streptavidin ratios, side by side with 135H12, and a scrambled version of 147B5 in pancreatic- and breast-cancer cell lines. The (147B5)n-streptavidin complexes (when n = 2, 3, 4, but not when n = 1) induced a strong receptor degradation effect in both cell lines compared to 135H12 or the (scrambled-147B5)4-streptavidin complex as a control, indicating that multimerization of the targeting agent resulted in an increased ability to cause receptor clustering and internalization. Subsequently, we prepared an Alexa-Fluor-streptavidin conjugate to demonstrate that (147B5)4-AF-streptavidin, but not the scrambled equivalent complex, concentrates in pancreatic and breast cancers in orthotopic nude-mouse models. Hence, we conclude that these novel targeting agents, with proper derivatization with imaging reagents or chemotherapy, can be used as diagnostics, and/or to deliver chemotherapy selectively to EphA2-expressing tumors.

Design and analysis of EphA2-SAM peptide ligands: A multi-disciplinary screening approach.

EphA2 receptor plays a critical and debatable function in cancer and is considered a target in drug discovery. Lately, there has been a growing interest in its cytosolic C-terminal SAM domain (EphA2-SAM) as it engages protein modulators of receptor endocytosis and stability. Interestingly, EphA2-SAM binds the SAM domain from the lipid phosphatase Ship2 (Ship2-SAM) mainly producing pro-oncogenic outcomes. In an attempt to discover novel inhibitors of the EphA2-SAM/Ship2-SAM complex with possible anticancer properties, we focused on the central region of Ship2-SAM (known as Mid-Loop interface) responsible for its binding to EphA2-SAM. Starting from the amino acid sequence of the Mid-Loop interface virtual peptide libraries were built through ad hoc inserted mutations with either l- or d- amino acids and screened against EphA2-SAM by docking techniques. A few virtual hits were synthesized and experimentally tested by a variety of direct and competition-type interaction assays relying on NMR (Nuclear Magnetic Resonance), SPR (Surface Plasmon Resonance), MST (Microscale Thermophoresis) techniques. These studies guided the discovery of an original EphA2-SAM ligand antagonist of its interaction with Ship2-SAM.

Impact of membrane lipid composition on the structure and stability of the transmembrane domain of amyloid precursor protein.

Cleavage of the amyloid precursor protein (APP) by γ-secretase is a crucial first step in the evolution of Alzheimer's disease. To discover the cleavage mechanism, it is urgent to predict the structures of APP monomers and dimers in varying membrane environments. We determined the structures of the C9923-55 monomer and homodimer as a function of membrane lipid composition using a multiscale simulation approach that blends atomistic and coarse-grained models. We demonstrate that the C9923-55 homodimer structures form a heterogeneous ensemble with multiple conformational states, each stabilized by characteristic interpeptide interactions. The relative probabilities of each conformational state are sensitive to the membrane environment, leading to substantial variation in homodimer peptide structure as a function of membrane lipid composition or the presence of an anionic lipid environment. In contrast, the helicity of the transmembrane domain of monomeric C991-55 is relatively insensitive to the membrane lipid composition, in agreement with experimental observations. The dimer structures of human EphA2 receptor depend on the lipid environment, which we show is linked to the location of the structural motifs in the dimer interface, thereby establishing that both sequence and membrane composition modulate the complete energy landscape of membrane-bound proteins. As a by-product of our work, we explain the discrepancy in structures predicted for C99 congener homodimers in membrane and micelle environments. Our study provides insight into the observed dependence of C99 protein cleavage by γ-secretase, critical to the formation of amyloid-ß protein, on membrane thickness and lipid composition.

Sam domain-based stapled peptides: Structural analysis and interaction studies with the Sam domains from the EphA2 receptor and the lipid phosphatase Ship2.

Sam (Sterile alpha motif) domains represent small helical protein-protein interaction modules which play versatile functions in different cellular processes. The Sam domain from the EphA2 receptor binds the Sam domain of the lipid phosphatase Ship2 and this interaction modulates receptor endocytosis and degradation primarily generating pro-oncogenic effects in cell. To identify molecule antagonists of the EphA2-Sam/Ship2-Sam complex with anti-cancer activity, we focused on hydrocarbon helical stapled peptides. EphA2-Sam and one of its interactors (i.e., the first Sam domain of the adaptor protein Odin) were used as model systems for peptide design. Increase in helicity in the stapled peptides, with respect to the corresponding linear/native-like regions, was proved by structural studies conducted through CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance). Interestingly, interaction assays by means of NMR, SPR (Surface Plasmon Resonance) and MST (MicroScale Thermophoresis) techniques led to the discovery of a novel ligand of Ship2-Sam.

Pinpointed Stimulation of EphA2 Receptors via DNA-Templated Oligovalence.

DNA nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. Here, the efficacy of SWL, an ephrin-mimicking peptide that binds specifically to EphrinA2 (EphA2) receptors, increased by presenting up to three of these peptides on small DNA nanostructures in an oligovalent manner. Ephrin signaling pathways play crucial roles in tumor development and progression. Moreover, Eph receptors are potential targets in cancer diagnosis and treatment. Here, the quantitative impact of SWL valency on binding, phosphorylation (key player for activation) and phenotype regulation in EphA2-expressing prostate cancer cells was demonstrated. EphA2 phosphorylation was significantly increased by DNA trimers carrying three SWL peptides compared to monovalent SWL. In comparison to one of EphA2's natural ligands ephrin-A1, which is known to bind promiscuously to multiple receptors, pinpointed targeting of EphA2 by oligovalent DNA-SWL constructs showed enhanced cell retraction. Overall, we show that DNA scaffolds can increase the potency of weak signaling peptides through oligovalent presentation and serve as potential tools for examination of complex signaling pathways.

Theoretical Model of EphA2-Ephrin A1 Inhibition.

This work aims at the theoretical description of EphA2-ephrin A1 inhibition by small molecules. Recently proposed ab initio-based scoring models, comprising long-range components of interaction energy, is tested on lithocholic acid class inhibitors of this protein⁻protein interaction (PPI) against common empirical descriptors. We show that, although limited to compounds with similar solvation energy, the ab initio model is able to rank the set of selected inhibitors more effectively than empirical scoring functions, aiding the design of novel compounds.

Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain?

Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an α-helix and two short ß-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains.

Emerging and Diverse Functions of the EphA2 Noncanonical Pathway in Cancer Progression.

Erythropoietin-producing hepatocellular receptor A2 (EphA2) receptor tyrosine kinase controls multiple physiological processes to maintain homeostasis in normal cells. In many types of solid tumors, it has been reported that EphA2 is overexpressed and plays a critical role in oncogenic signaling. However, in recent years, the opposing functions of EphA2 have been explained by the canonical and noncanonical signaling pathways. Ligand- and tyrosine kinase-dependent EphA2 activation (the canonical pathway) inhibits cancer cell proliferation and motility. In contrast, ligand- and tyrosine kinase-independent EphA2 signaling (the noncanonical pathway) promotes tumor survival and metastasis and controls acquired drug resistance and maintenance of cancer stem cell-like properties. Evidence has accumulated showing that the EphA2 noncanonical pathway is mainly regulated by inflammatory cytokines and growth factors via phosphorylation at Ser-897 in the intracellular C-tail region via some serine/threonine kinases, including p90 ribosomal S6 kinase. In this review, we focus on the regulation of Ser-897 phosphorylation and its functional importance in tumor malignancy and discuss future therapeutic targeting.

Dissociation of a Dynamic Protein Complex Studied by All-Atom Molecular Simulations.

The process of protein complex dissociation remains to be understood at the atomic level of detail. Computers now allow microsecond timescale molecular-dynamics simulations, which make the visualization of such processes possible. Here, we investigated the dissociation process of the EphA2-SHIP2 SAM-SAM domain heterodimer complex using unrestrained all-atom molecular-dynamics simulations. Previous studies on this system have shown that alternate configurations are sampled, that their interconversion can be fast, and that the complex is dynamic by nature. Starting from different NMR-derived structures, mutants were designed to stabilize a subset of configurations by swapping ion pairs across the protein-protein interface. We focused on two mutants, K956D/D1235K and R957D/D1223R, with attenuated binding affinity compared with the wild-type proteins. In contrast to calculations on the wild-type complexes, the majority of simulations of these mutants showed protein dissociation within 2.4 µs. During the separation process, we observed domain rotation and pivoting as well as a translation and simultaneous rolling, typically to alternate and weaker binding interfaces. Several unsuccessful recapturing attempts occurred once the domains were moderately separated. An analysis of protein solvation suggests that the dissociation process correlates with a progressive loss of protein-protein contacts. Furthermore, an evaluation of internal protein dynamics using quasi-harmonic and order parameter analyses indicates that changes in protein internal motions are expected to contribute significantly to the thermodynamics of protein dissociation. Considering protein association as the reverse of the separation process, the initial role of charged/polar interactions is emphasized, followed by changes in protein and solvent dynamics. The trajectories show that protein separation does not follow a single distinct pathway, but suggest that the mechanism of dissociation is common in that it initially involves transitions to surfaces with fewer, less favorable contacts compared with those seen in the fully formed complex.

EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling.

The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression.

Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies.

The receptor tyrosine kinase EPHA2 is overexpressed in several cancers (breast, head and neck, non-small-cell lung cancer). Small-molecule-based inhibition of the EPHA2 kinase domain (KD) is seen as an important strategy for therapeutic intervention. However, obtaining structural information by crystallography or NMR spectroscopy for drug discovery is severely hampered by the lack of pure, homogeneous protein. Here, different fragments of the EPHA2 KD were expressed and purified from both bacterial (Escherichia coli, BL21(DE3) cells) and insect cells (Spodoptera frugiperda, Sf9 cells).1 H,15 N HSQC was used to determine the proper folding and homogeneity of all the constructs. Protein from E. coli was well-folded but unstable, and it did not crystallize. However, a construct (D596-G900) produced in Sf9 cells yielded homogenous, well-folded protein that crystallized readily, thereby resulting in eleven new EPHA2-ligand crystal structures. We have also established a strategy for selective and uniform 15 N-amino acid labeling of EPHA2 KD in Sf9 cells for investigating dynamics and EPHA2-drug interactions by NMR.

Targeting EphA2-Sam and Its Interactome: Design and Evaluation of Helical Peptides Enriched in Charged Residues.

The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam-Sam interactions. Because EphA2-Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues. Several peptide sequences with high predicted helical propensities were designed, and detailed conformational analyses were conducted by diverse techniques including NMR, CD, and molecular dynamics (MD) simulations. Interaction studies were also performed by NMR, surface plasmon resonance (SPR), and microscale thermophoresis (MST) and led to the identification of two peptides capable of binding to the first Sam domain of Odin. These molecules represent early candidates for the generation of efficient Sam domain binders and antagonists of Sam-Sam interactions involving EphA2.

Antigen-specific immunoreactivity and clinical outcome following vaccination with glioma-associated antigen peptides in children with recurrent high-grade gliomas: results of a pilot study.

Recurrent high-grade gliomas (HGGs) of childhood have an exceedingly poor prognosis with current therapies. Accordingly, new treatment approaches are needed. We initiated a pilot trial of vaccinations with peptide epitopes derived from glioma-associated antigens (GAAs) overexpressed in these tumors in HLA-A2+ children with recurrent HGG that had progressed after prior treatments. Peptide epitopes for three GAAs (EphA2, IL13Rα2, survivin), emulsified in Montanide-ISA-51, were administered subcutaneously adjacent to intramuscular injections of poly-ICLC every 3 weeks for 8 courses, followed by booster vaccines every 6 weeks. Primary endpoints were safety and T-cell responses against the GAA epitopes, assessed by enzyme-linked immunosorbent spot (ELISPOT) analysis. Treatment response was evaluated clinically and by magnetic resonance imaging. Twelve children were enrolled, 6 with glioblastoma, 5 with anaplastic astrocytoma, and one with malignant gliomatosis cerebri. No dose-limiting non-CNS toxicity was encountered. ELISPOT analysis, in ten children, showed GAA responses in 9: to IL13Rα2 in 4, EphA2 in 9, and survivin in 3. One child had presumed symptomatic pseudoprogression, discontinued vaccine therapy, and responded to subsequent treatment. One other child had a partial response that persisted throughout 2 years of vaccine therapy, and continues at >39 months. Median progression-free survival (PFS) from the start of vaccination was 4.1 months and median overall survival (OS) was 12.9 months. 6-month PFS and OS were 33 and 73 %, respectively. GAA peptide vaccination in children with recurrent malignant gliomas is generally well tolerated, and has preliminary evidence of immunological and modest clinical activity.

Binding and function of phosphotyrosines of the Ephrin A2 (EphA2) receptor using synthetic sterile α motif (SAM) domains.

The sterile α motif (SAM) domain of the ephrin receptor tyrosine kinase, EphA2, undergoes tyrosine phosphorylation, but the effect of phosphorylation on the structure and interactions of the receptor is unknown. Studies to address these questions have been hindered by the difficulty of obtaining site-specifically phosphorylated proteins in adequate amounts. Here, we describe the use of chemically synthesized and specifically modified domain-length peptides to study the behavior of phosphorylated EphA2 SAM domains. We show that tyrosine phosphorylation of any of the three tyrosines, Tyr(921), Tyr(930), and Tyr(960), has a surprisingly small effect on the EphA2 SAM structure and stability. However, phosphorylation at Tyr(921) and Tyr(930) enables differential binding to the Src homology 2 domain of the adaptor protein Grb7, which we propose will lead to distinct functional outcomes. Setting up different signaling platforms defined by selective interactions with adaptor proteins thus adds another level of regulation to EphA2 signaling.

Point mutations in dimerization motifs of the transmembrane domain stabilize active or inactive state of the EphA2 receptor tyrosine kinase.

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L(535)X3G(539)X2A(542)X3V(546)X2L(549) rather than through the alternative glycine zipper motif A(536)X3G(540)X3G(544) (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr(588) and/or Tyr(594)) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.