Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

Kasai, Rinshi S.; Suzuki, Kenichi; Prossnitz, Eric R.; Koyama-Honda, Ikuko; Nakada, Chieko; Fujiwara, Takeshi; Kusumi, Akihiro

doi:10.1083/jcb.201009128

Cited by 311 publications

(344 citation statements)

References 67 publications

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“…3F). Knowing τ * 1 and τ * 2 , the true lifetime of receptor-receptor interactions can be roughly estimated as τ int = τ * 2 − τ * 1 ≈ 4 s at 20°C (27).…”

Section: Resultsmentioning

confidence: 99%

“…Another characteristic of our approach is the use of the SNAP-tag technology (28) to directly label cell-surface GPCRs, which has several advantages compared with the use of fluorescent ligands (26,27). First, whereas the latter methods are associated with partial labeling and/or possible influences of negative cooperativity in ligand binding (38,39), virtually complete receptor labeling can be achieved.…”

Section: Discussionmentioning

confidence: 99%

“…Of these, single-molecule microscopy has the great potential of directly observing the state and behavior of individual proteins. Interestingly, two recent single-molecule microscopy studies using fluorescent ligands showed dynamic dimerization of M1 muscarinic and N-formyl peptide receptors (26,27).…”

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confidence: 99%

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

Calebiro

Rieken

Wagner

et al. 2012

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

395

455

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G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the β 1 -adrenergic receptor (β 1 AR), the β 2 -adrenergic receptor (β 2 AR), and the γ-aminobutyric acid (GABA B ) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for β 1 ARs (monomers/dimers) and highest for GABA B receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas β 1 -/ β 2 ARs were apparently freely diffusing on the cell surface, GABA B receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA B receptor complexes. These data provide a spatiotemporal characterization of β 1 -/β 2 ARs and GABA B receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.live cell imaging | protein-protein interactions

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“…3F). Knowing τ * 1 and τ * 2 , the true lifetime of receptor-receptor interactions can be roughly estimated as τ int = τ * 2 − τ * 1 ≈ 4 s at 20°C (27).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Calebiro

Rieken

Wagner

et al. 2012

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

395

455

View full text Add to dashboard Cite

show abstract

“…Although there is an increasing body of evidence supporting dimerization of GPCRs as a widespread feature of GPCR biology, including numerous studies on family A GPCRs, whether these are stable, transient, constitutive, or ligand dependent, and how they impact on receptor function and drug discovery are less clear, and general rules for oligomeric behavior are not evident (9)(10)(11)(12)(13)(14)(15)(16). Even where effects on signaling are studied, these are generally linked to a single pathway and the role of dimerization in the control of receptor engagement and preference for distinct intracellular signaling intermediates (i.e., signal bias) is virtually unstudied.…”

mentioning

confidence: 99%

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

Harikumar

Wootten

Pinon

et al. 2012

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

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The glucagon-like peptide-1 receptor (GLP-1R) is a family B G proteincoupled receptor and an important drug target for the treatment of type II diabetes, with activation of pancreatic GLP-1Rs eliciting glucose-dependent insulin secretion. Currently, approved therapeutics acting at this receptor are peptide based, and there is substantial interest in small molecule modulators for the GLP-1R. Using a variety of resonance energy transfer techniques, we demonstrate that the GLP-1R forms homodimers and that transmembrane helix 4 (TM4) provides the primary dimerization interface. We show that disruption of dimerization using a TM4 peptide, a minigene construct encoding TM4, or by mutation of TM4, eliminates G protein-dependent highaffinity binding to GLP-1(7-36)NH 2 but has selective effects on receptor signaling. There was <10-fold decrease in potency in cAMP accumulation or ERK1/2 phosphorylation assays but marked loss of intracellular calcium mobilization by peptide agonists. In contrast, there was near-complete abrogation of the cAMP response to an allosteric agonist, compound 2, but preservation of ERK phosphorylation. Collectively, this indicates that GLP-1R dimerization is important for control of signal bias. Furthermore, we reveal that two small molecule ligands are unaltered in their ability to allosterically modulate signaling from peptide ligands, demonstrating that these modulators act in cis within a single receptor protomer, and this has important implications for small molecule drug design.allosteric modulation | biased signaling | G protein-coupled receptors | family B GPCRs G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface proteins and play crucial roles in virtually every physiological process. Their widespread abundance, yet selective distribution, and ability to couple to a variety of signaling and effector systems make them extremely attractive targets for drug development (1). Recently, there has been increasing interest in the stoichiometry of receptors involved in GPCR signaling complexes and how this may impact on receptor function and drug discovery (2-4).With the exception of family C GPCRs, where obligate dimerization can occur (4), the role of oligomerization in GPCR function has remained controversial (2-8), and this has been the subject of a number of recent reviews (9-11). Although there is an increasing body of evidence supporting dimerization of GPCRs as a widespread feature of GPCR biology, including numerous studies on family A GPCRs, whether these are stable, transient, constitutive, or ligand dependent, and how they impact on receptor function and drug discovery are less clear, and general rules for oligomeric behavior are not evident (9-16). Even where effects on signaling are studied, these are generally linked to a single pathway and the role of dimerization in the control of receptor engagement and preference for distinct intracellular signaling intermediates (i.e., signal bias) is virtually unstudied.For family B GPCRs, which encompass many ther...

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“…0, assuming a constant concentration of molecules N/V [9]. The number of reacting molecules in the plasma membrane is of order N ¼ 10 3 to 10 5 [10,11], implying an infinitesimal rate of switching between macroscopic states of activity and inactivity in the well-mixed approximation. In spatially extended reactors, the characteristic size of the well-mixed subcompartment is effectively controlled by diffusion.…”

Section: State-to-state Transitions In Homogeneous and Heterogeneous mentioning

confidence: 99%

Stochastic transitions in a bistable reaction system on the membrane

Kochańczyk

Jaruszewicz

Lipniacki

2013

J. R. Soc. Interface.

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Transitions between steady states of a multi-stable stochastic system in the perfectly mixed chemical reactor are possible only because of stochastic switching. In realistic cellular conditions, where diffusion is limited, transitions between steady states can also follow from the propagation of travelling waves. Here, we study the interplay between the two modes of transition for a prototype bistable system of kinase-phosphatase interactions on the plasma membrane. Within microscopic kinetic Monte Carlo simulations on the hexagonal lattice, we observed that for finite diffusion the behaviour of the spatially extended system differs qualitatively from the behaviour of the same system in the well-mixed regime. Even when a small isolated subcompartment remains mostly inactive, the chemical travelling wave may propagate, leading to the activation of a larger compartment. The activating wave can be induced after a small subdomain is activated as a result of a stochastic fluctuation. Such a spontaneous onset of activity is radically more probable in subdomains characterized by slower diffusion. Our results show that a local immobilization of substrates can lead to the global activation of membrane proteins by the mechanism that involves stochastic fluctuations followed by the propagation of a semi-deterministic travelling wave.

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Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

Cited by 311 publications

References 67 publications

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

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

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

Stochastic transitions in a bistable reaction system on the membrane

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