Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy

Işbilir, Ali; Moeller, Jan; Böck, Andreas; Zabel, Ulrike; Annibale, Paolo

doi:10.1101/240903

Cited by 5 publications

(5 citation statements)

References 37 publications

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“…For time course SpIDA imaging, as soon as ligand was added, different cells were imaged with 30-s intervals. Image analysis was performed using the one-population mode of the SpIDA function using a custom-written MATLAB routine, as described previously ( 34 ). Calculated quantal brightness values are real photon counts per fluorescent molecule per pixel dwell time.…”

Section: Methodsmentioning

confidence: 99%

“…These two values are used to quantify the average oligomeric state of the protein of interest. We used a C-terminally EYFP-tagged β 1 AR as a monomeric control, as we had confirmed its monomeric behavior even at overexpression levels (9,34). In addition, a β 1 AR with two adjacent C-terminal EYFP tags (separated by an (EAAAK) 4 linker) was employed as a "dimeric" control (34) ( Fig.…”

Section: Cxcr4 Is Largely Monomeric At Low Expression Levels and Formsmentioning

confidence: 99%

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Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

Işbilir

Møller

Arimont

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Although class A G protein−coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer−dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

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Section: Methodsmentioning

confidence: 99%

Section: Cxcr4 Is Largely Monomeric At Low Expression Levels and Formsmentioning

confidence: 99%

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

Işbilir

Møller

Arimont

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…49 The brightness values were calculated based on the average of the brightness values from each pixel within the ROI using a custom written routine as previously described. 50 Receptor Diffusion Measurements. The same microscope setup was used as for TB analysis.…”

Section: ■ Methodsmentioning

confidence: 99%

“…Data were analyzed as described previously utilizing a custom-written Igor Pro (Wavemetrics) routine . The brightness values were calculated based on the average of the brightness values from each pixel within the ROI using a custom written routine as previously described …”

Section: Methodsmentioning

confidence: 99%

Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells

et al. 2019

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High-end microscopy studies of G protein-coupled receptors (GPCRs) require installing onto the receptors bright and photostable dyes. Labeling must occur in quantitative yields, to allow stoichiometric data analysis, and in a minimally invasive fashion, to avoid perturbing GPCR function. We demonstrate here that the genetic incorporation of trans-cyclooct-2-ene lysine (TCO*) allows achieving quantitative single-residue labeling of the extracellular loops of the β2-adrenergic and the muscarinic M2 class A GPCRs, as well as of the corticotropin releasing factor class B GPCR. Labeling occurs within a few minutes by reaction with dye–tetrazine conjugates on the surface of live cells and preserves the functionality of the receptors. To precisely quantify the labeling yields, we devise a method based on fluorescence fluctuation microscopy that extracts the number of labeling sites at the single-cell level. Further, we show that single-residue labeling is better suited for studies of GPCR diffusion than fluorescent-protein tags, since the latter can affect the mobility of the receptor. Finally, by performing dual-color competitive labeling on a single TCO* site, we devise a method to estimate the oligomerization state of a GPCR without the need for a biological monomeric reference, which facilitates the application of fluorescence methods to oligomerization studies. As TCO* and the dye–tetrazines used in this study are commercially available and the described microscopy techniques can be performed on a commercial microscope, we expect our approach to be widely applicable to fluorescence microscopy studies of membrane proteins in general.

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“…Chemokine receptor oligomers cause negative cooperativity in the binding of their chemokine ligands and, in the case of some receptors, oligomerization can either enhance or inhibit receptor activation through allosteric communication (Percherancier et al, 2005;Stephens and Handel, 2013;Armando et al, 2014). The current methodologies that are able to probe dimerization are mainly based on fluorescence and bioluminescence resonance energy transfer approaches (Percherancier et al, 2005;Goddard and Watts, 2012;Fumagalli et al, 2019;Heuninck et al, 2019), fluorescence fluctuation spectroscopy (Isbilir et al, 2017;Briddon et al, 2018), and spatial intensity distribution analysis (Pediani et al, 2018). Such biophysical approaches are very sensitive to detect oligomers but are not able to identify the oligomerization interface between protomers, which is key for molecular understanding of the quaternary GPCR structures.…”

Section: Lessons From Chemokine Receptor X-ray Structuresmentioning

confidence: 99%

Chemokine Receptor Crystal Structures: What Can Be Learned from Them?

et al. 2019

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Chemokine receptors belong to the class A of G protein-coupled receptors (GPCRs) and are implicated in a wide variety of physiologic functions, mostly related to the homeostasis of the immune system. Chemokine receptors are also involved in multiple pathologic processes, including immune and autoimmune diseases, as well as cancer. Hence, several members of this GPCR subfamily are considered to be very relevant therapeutic targets. Since drug discovery efforts can be significantly reinforced by the availability of crystal structures, substantial efforts in the area of chemokine receptor structural biology could dramatically increase the outcome of drug discovery campaigns. This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications. SIGNIFICANCE STATEMENT This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications.

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Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy

Cited by 5 publications

References 37 publications

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells

Chemokine Receptor Crystal Structures: What Can Be Learned from Them?

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