살아있는 세포의 세포막에서 단일 분자의 확산을 통한 리간드와 수용체의 결합 분석 : Analyzing Interactions between Ligand and Receptor Using Single-Molecule Diffusivity in a Living Cell Membrane

다국어 초록 (Multilingual Abstract)

Ligand-receptor interactions are the fundamental biochemical reactions that generate cellular functions. The investigation of membrane-protein interactions with ligands has been challenging, despite of their biological and therapeutic importance, because the integrity of a membrane protein is highly dependent on a lipid membrane; the biochemical properties of their interactions identified using purified membrane proteins have not often appeared under real physiological conditions. Although recent attempts to analyze these interactions utilizing membrane proteins embedded in lipid vesicles have expanded my understanding of the membrane-protein interactions, these methods still cannot reflect the genuine properties produced only in an intact plasma membrane of a live cell.

I develop a novel method that analyze interactions between membrane and soluble proteins in an intact living cell membrane utilizing single particle tracking with super-resolution microscopy. My method (termed single-molecule diffusional mobility shift assay, smDIMSA) overcomes the fundamental limitations of current methods for the analysis of ligand-receptor interactions by providing unprecedented biochemical information on the ligand-receptor interactions occurred in the interface between the solution and crowded cell membrane at the single-molecule population level.

smDIMSA is developed based on the novel finding on membrane protein’s diffusivity in a living cell membrane. It has been believed, over 40 years, that the effect of the water-soluble regions of a membrane protein on its diffusivity is negligible due to the orders of magnitude higher viscosity of a plasma membrane than aqueous solution surrounding a cell. This inherent property of membrane protein’s diffusivity fundamentally restrains the diffusion-based imaging assays from measuring the interactions involving water-soluble regions of membrane proteins. However, I unexpectedly observed that the diffusion-coefficient of EGFR decreased up to 40% by the direct binding of its antibody. Because the detection limit of the diffusion-coefficient changes in a living cell membrane using the current methods including FRAP and FCS is typically larger than 400%, this phenomenon can be detected only by single-molecule population analysis that is capable to probe less than 5% of diffusion-coefficient changes. This remarkable finding was extensively validated with various membrane proteins including ErbB receptor family (receptor tyrosine kinases) and adrenergic receptor family (G-protein coupled receptors) with different soluble ligands.

Analysis of the ligand-receptor interactions using smDIMSA is extraordinary because it provides novel features that traditional methods, such as using radioactive-isotope labeled ligand, surface plasmon resonance, and co-immunoprecipitation, cannot provide. First, smDIMSA is ligand-label free, eliminating the difficulties associated with ligand labeling and enabling its application to a broad range of ligand concentrations or types. Second, smDIMSA is sensitive to the size of binding partners, generating an unprecedented level of information that can be used to reduce the complexity of a pool of interacting proteins; it can unravel the multiple steps of complex formation on a plasma membrane. Third, it provides quantitative biochemical information to assess binding constants, interaction modes, and kinetics, which is critical to understand the detailed mechanisms of biomolecular reactions. Fmyth, compared with popular label-free assays utilizing SPR, which requires labor-intensive membrane protein purification and expensive gold surfaces, smDISMA directly utilize a single cell seeded on an inexpensive glass surface, greatly reducing both the experimental labor and cost. Last but not least, smDIMSA provides genuine interaction information on a living cell membrane by significantly reducing the false-positives and -negatives derived from chemical purification of membrane proteins for in vitro assays.

An immediate biochemical application of smDIMSA was further demonstrated. I revealed, for the first time, the positive cooperativity of cetuximab binding to EGFR L858R on a living cell membrane. Furthermore, based on information about the binding cooperativity of various antibodies targeting different epitopes of the extracellular domain of EGFR L858R, I unraveled the conformation of the extracellular domain of dimeric EGFR L858R, which was controversial in previous studies. By measuring interaction cooperativity and deducing reaction mechanism, my results provide a novel biochemical insight on how dimeric EGFR L858R can be constitutively active without natural ligands on a cell membrane.

In conclusion, I developed a novel method that analyze interactions between membrane and soluble proteins in an intact living cell membrane utilizing single particle tracking with super-resolution microscopy. My method overcomes the fundamental limitations of current methods for the analysis of ligand-receptor interactions by providing unprecedented biochemical information on the ligand-receptor interactions occurred in the interface between the solution and crowded cell membrane at the single-molecule population level, which allows detecting a new biochemical aspect of ligand-receptor interactions that appeared only in a crowded living cell membrane, not a solution or a vesicle, as shown in this study.

국문 초록 (Abstract)

리간드-수용체간 상호작용은 세포 안과 밖의 신호전달을 매개함으로써 생명유지에 필수적인 역할을 하는 것뿐만이 아니라, 수많은 질병의 원인 인자로서 현재 개발된 50% 이상의 신약들이 리간드-수용체간 상호작용을 대상으로 하고 있다. 이러한 중요성에도 불구하고, 세포막 위에서 일어나고 있는 리간드-수용체간 상호작용에 대한 이해가 많이 부족한 상황이다. 이는 전체 유전자의 15% 이상 되는 다양한 단백질들이 세포막 위에서 매우 높은 농도로 존재함으로써 나타나는 복잡성과 친수성 및 소수성을 모두 가지는 세포막의 생화학적 특이성이 만들어 내는 특수한 환경 때문인데, 현재까지는 기술적인 제약으로 인해 리간드-수용체 간의 상호작용의 연구가 대부분 시험관 수준에서 이루어졌지만 실제 세포막 위에서 나타나는 이들 상호작용의 본질적인 성질을 밝혀내는데 큰 한계가 있었다. 하지만 본 연구에서 최초로 살아있는 세포의 세포막 위에서 리간드-수용체간 상호작용을 연구할 수 있는 기술을 개발함으로써 그 한계를 근본적으로 극복하였다.

이 기술의 핵심은 세포막 위에서 끊임없이 움직이는 수용체가 리간드와 결합하였을 때 단일 분자 수준에서 수용체의 움직임이 둔화된다는 원리에 있다. 40여 년간 세포막 단백질 유동의 표준모델로서 받아들여져 왔던 샤프만-델브룩 (Saffman-Delbrück) 모델에 따르면 세포막의 높은 점성도를 가지는 지질 특성으로 인해 수용성인 리간드가 수용체에 결합하더라도 수용체의 움직임이 변화하지 않는다고 믿어져 왔다. 그러나 본인은 최근에 급속도로 발전하고 있는 초해상도 현미경과 단일 분자 영상 기술들은 응용하여 기존에 측정이 불가능했던 단일 분자 수준에서의 세포막 수용체의 움직임을 직접 관찰하였고, 우연히 리간드가 수용체에 결합하였을 때 그 움직임이 변화한다는 결과를 얻어내었다.

세포막 단백질 유동에 대한 새로운 원리를 토대로 개발된 기술인 smDIMSA (single-molecule DIffusional Mobility Shift Assay, 본 논문에서 명명함)는 리간드-비표지적 방법으로 수용체에 결합하는 리간드의 크기를 정량적으로 인식하기 때문에 대상 수용체에 대한 신약 리간드의 후보 선별에 매우 적합하며, 기존의 방법들과 비교하였을 때 혁명적인 장점들을 가지고 있다. 현재 상용화되어 주로 사용되고 있는 표면 플라즈몬 공명 (Surface Plasmon Resonance)을 이용한 방법은 복잡하고 힘들게 정제한 대상 단백질을 값비싼 금표면에 부착시켜 리간드와의 결합을 측정하게 된다. 특히, 수용체의 경우 높은 세포막 의존도로 인해 정제가 어려워 그 측정이 더욱 힘들었다. 그러나 smDIMSA 기술은 살아있는 세포에서 리간드-수용체간 상호작용을 직접 측정함으로써 시험관 실험에서 오는 양성 및 음성 오류들을 현저히 줄일 수 있고, 까다로운 세포막 단백질 정제 과정을 완전히 없애고, 비용적인 측면에서 기존대비 수천 배 이상 절약할 수 있다.

목차 (Table of Contents)

• I. Introduction 1
• 1.1 Receptors 1
• 1.2 Epidermal Growth Factor Receptors 2
• 1.3 Diffusion-based Imaging Assays 3
• 1.4 Single Particle Tracking 4
• 1.5 Super-Resolution Microscopy 5
• 1.6 Saffman-Delbruck Model 6
• 1.7 Introduction to the Research 6
• II. Materials and Methods 8
• 2.1 Reagents 8
• 2.2 Plasmid Vectors and DNA Cloning 9
• 2.3 Cell Lines and Culture 12
• 2.4 Total Internal Reflection Fluorescence Microscopy 13
• 2.5 Sample Preparation 13
• 2.6 Image Acquisition 14
• 2.7 Multiple Particle Tracking 15
• 2.8 Diffusion Coefficient Determination 17
• 2.9 Single-Molecule Diffusional Mobility Shift Assays 19
• 2.10 Flow Cytometry 23
• 2.11 Western Blot 24
• 2.12 Confocal Microscopy 24
• III. Results 26
• 3.1 Development of smDIMSA 26
• 3.2 Validation of smDIMSA 44
• 3.3 Size-sensitive measurement of EGFR interactions with water-soluble binders using smDIMSA 52
• 3.4 Quantitative measurement of the EGFR interaction with cetuximab by smDIMSA 58
• 3.5 Molecule-specific measurement of ligand-receptor interactions on the cell membrane by smDIMSA 61
• 3.6 smDIMSA reveals the positive cooperativity of the interaction between EGFR L858R and cetuximab 66
• IV. Discussion 72
• V. Conclusion 78
• VI. References 79
• VII. Summary in Korean (요약문) 86
• VIII. Acknowledgements 88
• IX. Curriculum Vitae 90