Ligand-induced Receptor Dimerization Research Articles

Antigen-induced chimeric antigen receptor multimerization amplifies on-tumor cytotoxicity

Ligand-induced receptor dimerization or oligomerization is a widespread mechanism for ensuring communication specificity, safeguarding receptor activation, and facilitating amplification of signal transduction across the cellular membrane. However, cell-surface antigen-induced multimerization (dubbed AIM herein) has not yet been consciously leveraged in chimeric antigen receptor (CAR) engineering for enriching T cell-based therapies. We co-developed ciltacabtagene autoleucel (cilta-cel), whose CAR incorporates two B-cell maturation antigen (BCMA)-targeted nanobodies in tandem, for treating multiple myeloma. Here we elucidated a structural and functional model in which BCMA-induced cilta-cel CAR multimerization amplifies myeloma-targeted T cell-mediated cytotoxicity. Crystallographic analysis of BCMA–nanobody complexes revealed atomic details of antigen–antibody hetero-multimerization whilst analytical ultracentrifugation and small-angle X-ray scattering characterized interdependent BCMA apposition and CAR juxtaposition in solution. BCMA-induced nanobody CAR multimerization enhanced cytotoxicity, alongside elevated immune synapse formation and cytotoxicity-mediating cytokine release, towards myeloma-derived cells. Our results provide a framework for contemplating the AIM approach in designing next-generation CARs.

A Multiscale Molecular Dynamic Analysis Reveals the Effect of Sialylation on EGFR Clustering in a CRISPR/Cas9-Derived Model.

Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells.

Abstract 336: Mechanism of action of MCLA-129, a bispecific antibody that targets EGFR and c-MET and impairs growth of EGFR exon 20 insertion mutant non-small cell lung cancer

Abstract Introduction: MCLA-129 is an ADCC-enhanced IgG1 bispecific antibody that targets epidermal growth factor receptor (EGFR) and c-MET. The HGF/c-MET pathway is frequently upregulated in tumors that are resistant to EGFR tyrosine kinase inhibitors (TKIs). This study investigated the MCLA-129 mechanism of action in vitro and effectiveness in vivo in an EGFR exon20 insertion (ex20ins) non-small cell lung cancer (NSCLC) model. Methods: To study the effect of MCLA-129 on c-MET and EGFR dimerization, β-galactosidase enzyme complementation assays were performed. Ligand-dependent phosphorylation of EGFR and c-MET was assessed in vitro on the NCI-H1650 NSCLC cell line by immunoblotting. Shotgun mutagenesis epitope mapping was used to identify the binding epitope of MCLA-129 to EGFR. ADCC activity of MCLA-129 was determined in NSCLC cells expressing different levels of EGFR and c-MET using an ADCC reporter assay with high affinity FcγRIII 158V-variant and low affinity 158F-variant effector cells. ADCP was measured with monocyte-derived macrophages incubated with pHrodo-labeled HCC827 NSCLC target cells with readout of tumor cell phagocytosis on the Incucyte® system. For the evaluation of efficacy in vivo in the EGFR exon20ins PDX model, MCLA-129 was administered at 2.5, 8 or 25 mg/kg i.p. weekly. Results: MCLA-129 blocks EGF and HGF binding to EGFR and c-MET, respectively, and prevents receptor dimerization. Consequently, MCLA-129 inhibits phosphorylation of EGFR and c-MET as demonstrated in NCI-H1650 cells. Mutagenesis analysis identified critical binding residues of domain III of EGFR for MCLA-129 binding that are distinct from cetuximab binding. MCLA-129 displayed potent ADCC activity on NSCLC cell lines using effector cells expressing either high or low affinity FcγRIII variant. MCLA-129 demonstrated potent dose-dependent ADCP of HCC827 NSCLC cells. Finally, MCLA-129 led to significant dose-dependent regression of a patient-derived EGFR exon20ins NSCLC tumor in a mouse PDX model. Conclusion: MCLA-129 is a Biclonics® common light chain bispecific antibody with multiple mechanisms of action including potent inhibition of c-MET and EGFR ligand binding, ligand-induced receptor dimerization and phosphorylation, ADCC and ADCP. MCLA-129 demonstrates significant growth inhibition of a patient-derived EGFR exon20ins tumor in a preclinical xenograft model. A phase 1/2 clinical trial of MCLA-129 in solid tumors is ongoing. These data support the further clinical development of MCLA-129 in patients with NSCLC, including NSCLC with EGFR exon20ins, and other solid tumors. Citation Format: David J. de Gorter, Marie O'Connor, Alexandre Deshiere, Martijn van Rosmalen, Szabolcs Fatrai, Jeroen Lammerts van Bueren, Cecile A. Geuijen. Mechanism of action of MCLA-129, a bispecific antibody that targets EGFR and c-MET and impairs growth of EGFR exon 20 insertion mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 336.

Modeling the Cluster Size Distribution of Vascular Endothelial Growth Factor (VEGF) Receptors.

We previously developed a method of defining receptor clusters in the membrane based on mutual distance and applied it to a set of transmission microscopy images of vascular endothelial growth factor receptors. An optimal length parameter was identified, resulting in cluster identification and a procedure that assigned a geometric shape to each cluster. We showed that the observed particle distribution results were consistent with the random placement of receptors within the clusters and, to a lesser extent, the random placement of the clusters on the cell membrane. Here, we develop and validate a stochastic model of clustering, based on a hypothesis of preexisting domains that have a high affinity for receptors. The proximate objective is to clarify the mechanism behind cluster formation and to estimate the effect on signaling. Receptor-enriched domains may significantly impact signaling pathways that rely on ligand-induced dimerization of receptors. We define a simple statistical model, based on the preexisting domain hypothesis, to predict the probability distribution of cluster sizes. The process yielded sets of parameter values that can readily be used in dynamical calculations as the estimates of the quantitative characteristics of the clustering domains.

The anti-Müllerian hormone prodomain is displaced from the hormone/prodomain complex upon bivalent binding to the hormone receptor

Noncovalent complexes of transforming growth factor-β family growth/differentiation factors with their prodomains are classified as latent or active, depending on whether the complexes can bind their respective receptors. For the anti-Müllerian hormone (AMH), the hormone–prodomain complex is active, and the prodomain is displaced upon binding to its type II receptor, AMH receptor type-2 (AMHR2), on the cell surface. However, the mechanism by which this displacement occurs is unclear. Here, we used ELISA assays to measure the dependence of prodomain displacement on AMH concentration and analyzed results with respect to the behavior expected for reversible binding in combination with ligand-induced receptor dimerization. We found that, in solution, the prodomain has a high affinity for the growth factor (GF) (Kd = 0.4 pM). Binding of the AMH complex to a single AMHR2 molecule does not affect this Kd and does not induce prodomain displacement, indicating that the receptor binding site in the AMH complex is fully accessible to AMHR2. However, recruitment of a second AMHR2 molecule to bind the ligand bivalently leads to a 1000-fold increase in the Kd for the AMH complex, resulting in rapid release of the prodomain. Displacement occurs only if the AMHR2 is presented on a surface, indicating that prodomain displacement is caused by a conformational change in the GF induced by bivalent binding to AMHR2. In addition, we demonstrate that the bone morphogenetic protein 7 prodomain is displaced from the complex with its GF by a similar process, suggesting that this may represent a general mechanism for receptor-mediated prodomain displacement in this ligand family.

Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases

Receptor tyrosine kinases (RTKs) orchestrate embryonic development and tissue homeostasis by controlling key cellular processes like growth and proliferation. Consequently, the vast majority of the 58 RTKs in humans are linked to cancer or metabolic disorders. For instance, RTKs represent known oncogenic drivers (such as EGFR in non-small cell lung cancer), contribute to cancer hallmarks (such as VEGFR in pro-tumor angiogenesis), and mediate resistance to therapeutics or hormones (such as the Insulin Receptor in diabetes). Classically, RTKs transduce signals through various modes of ligand-induced receptor dimerization and subsequent tyrosine kinase-mediated signaling; however, ~10% of all metazoan RTKs are pseudokinases—that is, they lack the capability to catalyze phosphotransfer. Our objective is to ascertain how RTK pseudokinases relay extracellular cues without kinase activity and whether they can be targeted therapeutically. Using structural, biophysical, and cell signaling approaches, we investigated this class of RTKs to shed light on potential non-catalytic signaling mechanisms. We identified structural elements conserved between the pseudokinases and their kinase relatives, found their pseudokinase domains to be conformationally dynamic, and uncovered a putative route to drugging these receptors via “conformational disrupting” small molecules. Our findings suggest that RTK pseudokinases could serve as viable drug targets and set the scene for future dissection of signaling pathways that have long been considered enigmatic.

A method for functional testing constitutive and ligand-induced interactions of lysin motif receptor proteins

BackgroundPlant receptors with lysin motifs (LsyM) recognize microbial signals such as fungal chitin and lipo-chitooligosaccharidic Nod factors of nitrogen-fixing rhizobia. It is generally assumed that ligand-induced dimerization of LysM receptors is an essential step in activation of intracellular kinase domains and downstream signaling. Consequently, genes required for plant defense and establishment of symbiosis are expressed. We recently found that three LysM receptor proteins (namely LYK1, LYK4 and LYK5) of Arabidopsis thaliana form a tripartite receptor complex to perceive chitin. However, constitutive and ligand-induced interactions of LysM receptors generally remain difficult to be characterized.ResultsInteractions between ectodomains of LYK1, LYK4 and LYK5 were investigated by a chimeric receptor approach using hairy roots of the legume Lotus japonicus. Synthetic receptor pairs consisting of a LYK ectodomain and the intracellular domain of a L. japonicus Nod factor receptor (NFR1 and NFR5, respectively) were tested for their capacity to activate expression of the symbiotic NIN (nodule inception) gene. The results indicated constitutive (LYK4ED–LYK4ED, LYK4ED–LYK5ED) and chitin-induced interactions (LYK1ED–LYK1ED, LYK1ED–LYK5ED) of the examined ectodomains.ConclusionWe present a method to functionally analyze constitutive and ligand-induced interactions of LysM-type proteins.

Cooperativity transitions driven by higher-order oligomer formations in ligand-induced receptor dimerization.

While cooperativity in ligand-induced receptor dimerization has been linked with receptor-receptor couplings via minimal representations of physical observables, effects arising from higher-order oligomer, e.g., trimer and tetramer, formations of unobserved receptors have received less attention. Here we propose a dimerization model of ligand-induced receptors in multivalent form representing physical observables under basis vectors of various aggregated receptor states. Our simulations of multivalent models not only reject Wofsy-Goldstein parameter conditions for cooperativity, but show that higher-order oligomer formations can shift cooperativity from positive to negative.

Two-detector number and brightness analysis reveals spatio-temporal oligomerization of proteins in living cells.

Number and brightness analysis (N&B) is a useful tool for the simultaneous visualization of protein oligomers and their localization, with single-molecule sensitivity. N&B determines particle brightness (fluorescence intensity per particle) and maps the spatial distribution of fluorescently labeled proteins by performing statistical analyses of the image series obtained using laser scanning microscopy. The brightness map reveals presence of the oligomers of the targeted protein and their distribution in living cells. However, even when corrections are applied, conventional N&B is affected by afterpulsing, shot noise, thermal noise, dead time, and overestimation of particle brightness when the concentration of the fluorescent particles changes during measurement. The drawbacks of conventional N&B can be circumvented by using two detectors, a novel approach that we henceforth call two-detector number and brightness analysis (TD-N&B), and introducing a linear regression of fluorescence intensity. This statistically eliminates the effect of noise from the detectors, and ensures that the correct particle brightness is obtained. Our method was theoretically assessed by numerical simulations and experimentally validated using a dilution series of purified enhanced green fluorescent protein (EGFP), EGFP tandem oligomers in cell lysate, and EGFP tandem oligomers in living cells. Furthermore, this method was used to characterize the complex process of ligand-induced glucocorticoid receptor dimerization and their translocation to the cell nucleus in live cells. Our method can be applied to other oligomer-forming proteins in cell signaling, or to aggregations of proteins such as those that cause neurodegenerative diseases.

Using Surface Enhanced Raman Spectroscopy to Probe Single-Cell NF-kB Dynamics during TNF-α Stimulation

Surface-enhanced Raman spectroscopy (SERS) is an optical device that is complicated but fast, that deals with high precision cells, noninvasive and label-free. Tumor necrosis factor-alpha (TNF-α) is primarily produced by macrophages an inflammatory cytokine. When TNF-α binds to TNFR, it activates nuclear factor kappa B (NF-kB) transcript factors. This will lead to oscillation in nuclear NF-kB and steering gene expression by negative feedback loops. We applied biotin anti-human TNF-α antibody to bind with TNF-α proceeded by chemical bonding for streptavidin- Phycoerythrin (PE) to detect the space structure between TNF-α and TNFR. The intracellular p65 was labeled with GFP for NF-kB tracking. We indicated the probing of spatiotemporal interaction between binding of TNF-α to TNF-receptor. PE interacted with TNF-α on a ratio of 1:1 and 1:4. Quantum dots (QDs) with TNF-α at 1:1. The signal strength had decreased in the ratio 1:4. There was low bright field in QD interaction to TNF-α. We used real-time imaging for single TNF-α molecule on the living NIH-3T3 cell surface. The result showed the ligand-induced receptor dimerization and trimerization. The bioconjugation and diameter of fluorescent probes both affected the spatial binding of TNF-α to its receptor. Using real-time imaging of GFP-P65, we successfully visualized the intracellular NF-kB nuclear translocation. Our goal is to compare the results from fluorescence imaging to SERS experiment. SERS has the potential to analyze NF-kB. With this image, label-free SERS space detection and with potentially patentable novel gold nano-particles (Au NPs) it can be seen NF-kB activation in two hours. In addition, we can conduct a 96 or 384 test in parallel and increase the throughput by two or three orders by 100-1000 faults. This will be the ultimate goal to move this device into clinical application.

Nectin-like molecule-4/cell adhesion molecule 4 inhibits the ligand-induced dimerization of ErbB3 with ErbB2

The ligand-induced dimerization of cell surface single-transmembrane receptors is essential for their activation. However, physiological molecules that inhibit their dimerization and activation have not been identified. ErbB3 dimerizes with ErbB2 upon binding of heregulin (HRG) to ErbB3, causing the ErbB2-catalyzed tyrosine phosphorylation of ErbB3, which leads to the activation of the signalling pathways for cell movement and survival. Genetic disorders of this receptor cause tumorigenesis and metastasis of cancers. We show here that nectin-like molecule-4/cell adhesion molecule 4, known to serve as a tumour suppressor, interacts with ErbB3 in the absence of HRG and inhibits the HRG-induced dimerization of ErbB3 with ErbB2 and its activation. The third immunoglobulin-like domain of nectin-like molecule-4 cis-interacts with the extracellular domain 3 of ErbB3. We describe here a novel regulatory mechanism for the activation and signalling of cell surface single-transmembrane receptors.

Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand–receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

Dimerization of Tie2 mediated by its membrane-proximal FNIII domains

Tie1 and Tie2, members of the tyrosine kinase family with immunoglobulin and EGF homology domains, are receptor tyrosine kinases found primarily in endothelial cells with key roles in development and maintenance of the vasculature and in angiogenesis. They are attractive targets for therapeutic intervention in tumor angiogenesis, inflammation, and sepsis. Tie2 is regulated directly by the multimeric angiopoietin (Ang) ligands, with Ang1 being its primary activator. Structural studies have shown how Angs bind to the Tie2 ligand-binding region, but do not explain Tie2 activation and suggest a passive role for the Tie2 extracellular region (ECR) in ligand-induced receptor dimerization. Here we show that the Tie2 ECR forms strong dimers even in the absence of bound ligand. Dimerization is mediated by membrane-proximal fibronectin type III (FNIII) domains that were omitted in previous structural studies. We describe a 2.5-Å resolution X-ray crystal structure of the membrane-proximal three Tie2 FNIII domains, Tie2(FNIIIa-c), revealing two possible dimerization modes that primarily involve the third FNIII domain, FNIIIc. Mutating these dimer interfaces implicates one of them (dimer 1) in soluble Tie2 (sTie2) dimerization in solution but suggests that both could play a role in Ang1-induced Tie2 activation, possibly modulated by Tie1. Through small-angle X-ray scattering studies of sTie2 dimers in solution and modeling based on crystal structures, we suggest that Ang1 binding may cross-link Tie2 dimers into higher-order oligomers, potentially explaining how Tie2 is differentially clustered following ligand engagement in different cellular contexts. Our results also firmly implicate FNIII domain-mediated interactions in Tie2 activation, identifying a potential Achilles' heel for therapeutic inhibition.

Structure of the Full-length VEGFR-1 Extracellular Domain in Complex with VEGF-A

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and-3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4Å resolution. We combined X-ray crystallography, single-particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in immunoglobulin homology domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity.

VEGF Receptor Tyrosine Kinases: Key Regulators of Vascular Function.

Vascular endothelial growth factor receptor (VEGFR) tyrosine kinases are key regulators of vascular development in vertebrates. Their activation is regulated through a family of secreted glycoproteins, the vascular endothelial growth factors (VEGFs). Expression, proteolytic processing, and diffusion range of VEGF proteins need to be tightly regulated, due to their crucial roles in development. While some VEGFs form concentration gradients across developing tissues and act as morphogenes, others function as inhibitors of receptor activation and downstream signaling. Ligand-induced receptor dimerization leads to activation of the intrinsic tyrosine kinase activity, which results in autophosphorylation of the receptors and in turn triggers the recruitment of interacting proteins as well as the initiation of downstream signaling. Although many biochemical details of VEGFR signaling have been revealed, the in vivo relevance of certain signaling aspects still remains to be demonstrated. Here, we highlight basic principles of VEGFR signaling and discuss its crucial role during development of the vascular system in mammals.

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms.

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling.

Absolute Ligand Discrimination by Dimeric Signaling Receptors

Many signaling pathways act through shared components, where different ligand molecules bind the same receptors or activate overlapping sets of response regulators downstream. Nevertheless, different ligands acting through cross-wired pathways often lead to different outcomes in terms of the target cell behavior and function. Although a number of mechanisms have been proposed, it still largely remains unclear how cells can reliably discriminate different molecular ligands under such circumstances. Here we show that signaling via ligand-induced receptor dimerization—a very common motif in cellular signaling—naturally incorporates a mechanism for the discrimination of ligands acting through the same receptor.

Structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4 A resolution. We combined X-ray crystallography, single-particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in immunoglobulin homology domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity.

A Photogate Microscopy to Track Single Molecules in Crowded Environments

Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signaling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density in the cellular environment. Here, we developed PhotoGate, which controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary. The method enables tracking of single particles at high spatial and temporal resolution until they photobleach or dissociate from their stationary target. This technical ability opens new possibilities for tracking single molecules in live cells at concentrations two orders of magnitude higher than the diffraction limit without photoactivation. Using this method, we tracked single particles at surface densities two orders of magnitude higher than the single-molecule detection limit. We observed ligand-induced dimerization of epidermal growth factor receptors (EGFR) on a live cell membrane. Furthermore, we directly measured binding and the dissociation rate of single APPL1 molecules from early endosomes in the crowded environment of the cytoplasm. We have also applied PhotoGate to intraflagellar transport and observed the dynamics of cargo disaasembly and reassembly at the ciliary tip. Thus, PhotoGate reveals spatial and temporal features of signal transduction events at the cell surface and on intracellular compartments with single molecule resolution.

Exploring the stochastic dynamics of correlated movement of receptor proteins in plasma membranes in vivo.

Ligand-induced receptor dimerization plays a crucial role in the signaling process of living cells. In this study, we developed a theoretical model and performed single-molecule tracking to explore the correlated diffusion processes of liganded epidermal growth factor receptors prior to dimer formation. We disclosed that both an attractive potential between liganded receptor proteins in proximity and correlated fluctuations in the local environments of the proteins play an important role to produce the observed correlated movement of the receptors. This result can serve as the foundation to shed light on the way in which receptor functions are regulated in plasma membranes in vivo.