Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization

Calebiro, Davide; Rieken, Finn; Wagner, Julia A.; Sungkaworn, Titiwat; Zabel, Ulrike; Borzì, Alfio; Cocucci, Emanuele; Zürn, Alexander; Lohse, Martin J.

doi:10.1073/pnas.1205798110

Cited by 409 publications

(537 citation statements)

References 40 publications

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“…In the case of the neurotensin NTS 1 dimer, a half-life of 340 s has been observed (White et al, 2007) and evidence has been found that M 1 muscarinic receptor dimers have an estimated half-life of 0.5 s (Hern et al, 2010). In a study by Calebiro et al (2013), β 1 -and β 2 -adrenoceptors were monitored on the surface of living cells and the kinetics of the interactions leading to the formation of oligomers was characterized. All these receptors dynamically formed dimers and high-order oligomers, with an apparent half-life in the order of 4-6 s. Thus, a dynamic equilibrium condition was established at the cell surface, with constant formation and dissociation of new receptor complexes.…”

Section: Quaternary Structure Of Gpcr Complexesmentioning

confidence: 99%

G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication

Guidolin

Marcoli

Tortorella

et al. 2018

Reviews in the Neurosciences

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Abstract:The proposal of receptor-receptor interactions (RRIs) in the early 1980s broadened the view on the role of G protein-coupled receptors (GPCR) in the dynamics of the intercellular communication. RRIs, indeed, allow GPCR to operate not only as monomers but also as receptor complexes, in which the integration of the incoming signals depends on the number, spatial arrangement, and order of activation of the protomers forming the complex. The main biochemical mechanisms controlling the functional interplay of GPCR in the receptor complexes are direct allosteric interactions between protomer domains. The formation of these macromolecular assemblies has several physiologic implications in terms of the modulation of the signaling pathways and interaction with other membrane proteins. It also impacts on the emerging field of connectomics, as it contributes to set and tune the synaptic strength. Furthermore, recent evidence suggests that the transfer of GPCR and GPCR complexes between cells via the exosome pathway could enable the target cells to recognize/decode transmitters and/or modulators for which they did not express the pertinent receptors. Thus, this process may also open the possibility of a new type of redeployment of neural circuits. The fundamental aspects of GPCR complex formation and function are the focus of the present review article.

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Section: Quaternary Structure Of Gpcr Complexesmentioning

confidence: 99%

G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication

Guidolin

Marcoli

Tortorella

et al. 2018

Reviews in the Neurosciences

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“…It is known from studies of G-protein-coupled receptors (GPCRs) that an equilibrium may exist between monomers and oligomers (Lambert 2010, Teichmann et al 2012, Calebiro et al 2013, implying that protomer-protomer interfaces are flexible or transient. Amino acid mutations can shift this equilibrium toward a monomeric or more oligomeric state if they modify the interface(s) between the protomers.…”

Section: Transporter Oligomers and Structure-function Relationshipsmentioning

confidence: 99%

Modulation of monocarboxylate transporter 8 oligomerization by specific pathogenic mutations

Fischer¹,

Kleinau²,

Müller³

et al. 2015

Journal of Molecular Endocrinology

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The monocarboxylate transporter 8 (MCT8) is a member of the major facilitator superfamily (MFS). These membrane-spanning proteins facilitate translocation of a variety of substrates, MCT8 specifically transports iodothyronines. Mutations in MCT8 are the underlying cause of severe X-linked psychomotor retardation. At the molecular level, such mutations led to deficiencies in substrate translocation due to reduced cell-surface expression, impaired substrate binding, or decreased substrate translocation capabilities. However, the causal relationships between genotypes, molecular features of mutated MCT8, and patient characteristics have not yet been comprehensively deciphered. We investigated the relationship between pathogenic mutants of MCT8 and their capacity to form dimers (presumably oligomeric structures) as a potential regulatory parameter of the transport function of MCT8. Fourteen pathogenic variants of MCT8 were investigated in vitro with respect to their capacity to form oligomers. Particular mutations close to the substrate translocation channel (S194F, A224T, L434W, and R445C) were found to inhibit dimerization of MCT8. This finding is in contrast to those for other transporters or transmembrane proteins, in which substitutions predominantly at the outer-surface inhibit oligomerization. Moreover, specific mutations of MCT8 located in transmembrane helix 2 (del230F, V235M, and ins236V) increased the capacity of MCT8 variants to dimerize. We analyzed the localization of MCT8 dimers in a cellular context, demonstrating differences in MCT8 dimer formation and distribution. In summary, our results add a new link between the functions (substrate transport) and protein organization (dimerization) of MCT8, and might be of relevance for other members of the MFS. Finally, the findings are discussed in relationship to functional data combined with structural-mechanistical insights into MCT8.

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“…Single-molecule microscopy has the great potential of directly visualizing with high spatiotemporal resolution the dynamic behavior of individual receptors located on the surface of living cells, including their association to form dimers and higher order molecular complexes [6][7][8][9][10] . This offers several advantages compared to standard biochemical and microscopy methods, which usually report the average behavior of thousands or millions of molecules.…”

Section: Introductionmentioning

confidence: 99%

“…The reported protocol results in >90% labeling efficiency of an extracellularly SNAP-tagged cell-surface protein 10 . Further information on how to use single-molecule data to analyze the size and mobility of receptor complexes, as well as to capture transient receptor-receptor interactions, is provided.…”

mentioning

confidence: 99%

“…The protocol reported in this manuscript explains how to transfect and label SNAP-tagged 11 receptors with small organic fluorophores and use total internal reflection fluorescence (TIRF) microscopy to visualize single receptors or receptor complexes on the surface of living cells 10 . The reported protocol results in >90% labeling efficiency of an extracellularly SNAP-tagged cell-surface protein 10 .…”

mentioning

confidence: 99%

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High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Sungkaworn

Rieken

Calebiro

2014

JoVE

Self Cite

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Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., β 2 -adrenergic and γ-aminobutyric acid type B (GABA B ) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies. Video LinkThe video component of this article can be found at

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Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization

Cited by 409 publications

References 40 publications

G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication

G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication

Modulation of monocarboxylate transporter 8 oligomerization by specific pathogenic mutations

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

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