Regulation of the Oligomeric Status of CCR3 with Binding Ligands Revealed by Single-Molecule Fluorescence Imaging

Song, Yanzhuo; Ge, Baosheng; Lao, Jun; Wang, Zhencai; Yang, Bin; Wang, Xiaojuan; He, Hua; Li, Jiqiang; Huang, Fang

doi:10.1021/acs.biochem.7b00676

Cited by 13 publications

(16 citation statements)

References 58 publications

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“…The ligand-promoted formation of the CXCR4 complex is consistent with previous reports on ligand-regulated oligomerization phenomenon. 35,36 Compared with previous assessment methods such as photobleaching step counting, however, the DL-assisted tracking technique provides a realtime visualization of the complex formation and allows for direct determination of the complex lifetime.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Deep-Learning-Assisted Single-Molecule Tracking on a Live Cell Membrane

Wang

Qian

et al. 2021

Anal. Chem.

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Single-molecule fluorescence imaging is a powerful tool to study protein function by tracking molecular position and distribution, but the precise and rapid identification of dynamic molecules remains challenging due to the heterogeneous distribution and interaction of proteins on the live cell membrane. We now develop a deep-learning (DL)-assisted single-molecule imaging method that can precisely distinguish the monomer and complex for rapid and real-time tracking of protein interaction. This DL-based model, which comprises convolutional layers, max pooling layers, and fully connected layers, is trained to reach an accuracy of >98% for identifying monomer and complex. We use this method to investigate the dynamic process of chemokine receptor CXCR4 on the live cell membrane during the early signaling stage. The results show that, upon ligand activation, the CXCR4 undergoes a dynamic process of forming a receptor complex. We further demonstrate that the CXCR4 complex tends to be internalized at 2.5-fold higher rate into the cell interior than the monomer via the clathrin-dependent pathway. This study is the first example to scrutinize the early signaling process of CXCR4 at the single-molecule level on the live cell membrane. We envision that this DL-assisted imaging method would be a broadly useful technique to study more protein families for elucidating their physiological and pathological functions.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Deep-Learning-Assisted Single-Molecule Tracking on a Live Cell Membrane

Wang

Qian

et al. 2021

Anal. Chem.

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“…However, a discussion over cholesterol–receptor interaction is nonetheless incomplete without mention of what role these binding sites may play in the phenomenon of receptor multimerization. It is known that CCR3 forms functional dimers and higher-order oligomers, but the role of cholesterol in this phenomenon is unknown [ 98 ]. Therefore, the binding sites identified in this work should be considered during future evaluation of CCR3 homo- or hetero-dimerization sites.…”

Section: Discussionmentioning

confidence: 99%

“…In aggregate, these observations suggest a broadly defined swath of helical real estate involved in identified dimer interfaces in CC motif chemokine receptors, corroborated by the observed patterns in cholesterol residency predicted for CCR3 in this work. It is known that CCR3 forms dimers and higher-order oligomers in vivo [ 98 ], that dimer formation can influence receptor–ligand affinity [ 100 ], and that cholesterol typically drives dimer formation in chemokine receptors [ 10 , 13 ]. Thus, the binding sites identified herein will also serve as a starting place to explore the dimerization interfaces of CCR3.…”

Section: Discussionmentioning

confidence: 99%

In Silico Identification of Cholesterol Binding Motifs in the Chemokine Receptor CCR3

2021

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CC motif chemokine receptor 3 (CCR3) is a Class A G protein-coupled receptor (GPCR) mainly responsible for the cellular trafficking of eosinophils. As such, it plays key roles in inflammatory conditions, such as asthma and arthritis, and the metastasis of many deadly forms of cancer. However, little is known about how CCR3 functionally interacts with its bilayer environment. Here, we investigate cholesterol binding sites in silico through Coarse-Grained Molecular Dynamics (MD) and Pylipid analysis using an extensively validated homology model based on the crystal structure of CCR5. These simulations identified several cholesterol binding sites containing Cholesterol Recognition/Interaction Amino Acid Consensus motif (CRAC) and its inversion CARC motifs in CCR3. One such site, a CARC site in TM1, in conjunction with aliphatic residues in TM7, emerged as a candidate for future investigation based on the cholesterol residency time within the binding pocket. This site forms the core of a cholesterol binding site previously observed in computational studies of CCR2 and CCR5. Most importantly, these cholesterol binding sites are conserved in other chemokine receptors and may provide clues to cholesterol regulation mechanisms in this subfamily of Class A GPCRs.

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“…GPCRs constitute the largest class of membrane receptors, which regulate immune responses 26 . Among the GPCRs, the CCR3 monomer is the minimal functional unit in signal transduction though class C are known to form homodimers 27 . Additionally, in class A, the coexistence of monomers and homodimers has also been documented 28,29 .…”

Section: Discussionmentioning

confidence: 99%

The Reaction of Dimerization by Itself Reduces the Noise Intensity of the Protein Monomer

Liu

Shu

2019

Sci Rep

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Because of the small particle number of intracellular species participating in genetic circuits, stochastic fluctuations are inevitable. This intracellular noise is detrimental to precise regulation. To maintain the proper function of a cell, some natural motifs attenuate the noise at the protein level. In many biological systems, the protein monomer is used as a regulator, but the protein dimer also exists. In the present study, we demonstrated that the dimerization reaction reduces the noise intensity of the protein monomer. Compared with two common noise-buffering motifs, the incoherent feedforward loop (FFL) and negative feedback control, the coefficient of variation (COV) in the case of dimerization was 25% less. Furthermore, we examined a system with direct interaction between proteins and other ligands. Both the incoherent FFL and negative feedback control failed to buffer the noise, but the dimerization was effective. Remarkably, the formation of only one protein dimer was sufficient to cause a 7.5% reduction in the COV.

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Regulation of the Oligomeric Status of CCR3 with Binding Ligands Revealed by Single-Molecule Fluorescence Imaging

Cited by 13 publications

References 58 publications

Deep-Learning-Assisted Single-Molecule Tracking on a Live Cell Membrane

Deep-Learning-Assisted Single-Molecule Tracking on a Live Cell Membrane

In Silico Identification of Cholesterol Binding Motifs in the Chemokine Receptor CCR3

The Reaction of Dimerization by Itself Reduces the Noise Intensity of the Protein Monomer

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