Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors

Lohse, Martin J.; Nikolaev, Viacheslav O.; Hein, Peter; Hoffmann, Carsten; Vilardaga, Jean-Pierre; Bünemann, Moritz

doi:10.1016/j.tips.2007.12.002

Cited by 114 publications

(91 citation statements)

References 74 publications

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“…Therefore, we analyzed the kinetics of PTH-induced interaction among PTHR, NHERF1, and ␤-arrestin2 using FRET in living cells. Here, we used a PTHR with a C-terminal CFP fusion because several previous studies evidenced that measuring protein-protein interactions with intracellular parts of a GPCR by FRET required an intracellular fusion of a fluorophore to receptor (33). To test whether this receptor was still able to bind to NHERF1, coimmunoprecipitation experiments were performed.…”

Section: Figure 2 Nherf1 Forms a Ternary Complex With Pthr And ␤-Arrmentioning

confidence: 99%

Formation of a Ternary Complex among NHERF1, β-Arrestin, and Parathyroid Hormone Receptor

Klenk

Vetter

Zürn

et al. 2010

Journal of Biological Chemistry

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␤-Arrestins are crucial regulators of G-protein coupled receptor (GPCR) signaling, desensitization, and internalization. Despite the long-standing paradigm that agonist-promoted receptor phosphorylation is required for ␤-arrestin2 recruitment, emerging evidence suggests that phosphorylation-independent mechanisms play a role in ␤-arrestin2 recruitment by GPCRs. Several PDZ proteins are known to interact with GPCRs and serve as cytosolic adaptors to modulate receptor signaling and trafficking. Na ؉ /H ؉ exchange regulatory factors (NHERFs) exert a major role in GPCR signaling. By combining imaging and biochemical and biophysical methods we investigated the interplay among NHERF1, ␤-arrestin2, and the parathyroid hormone receptor type 1 (PTHR). We show that NHERF1 and ␤-arrestin2 can independently bind to the PTHR and form a ternary complex in cultured human embryonic kidney cells and Chinese hamster ovary cells. Although NHERF1 interacts constitutively with the PTHR, ␤-arrestin2 binding is promoted by receptor activation. NHERF1 interacts directly with ␤-arrestin2 without using the PTHR as an interface. Fluorescence resonance energy transfer studies revealed that the kinetics of PTHR and ␤-arrestin2 interactions were modulated by NHERF1. These findings suggest a model in which NHERF1 may serve as an adaptor, bringing ␤-arrestin2 into close proximity to the PTHR, thereby facilitating ␤-arrestin2 recruitment after receptor activation.Scaffold proteins play an increasingly recognized role in the regulation and the signal transduction of G protein coupled receptors (GPCRs).2 The interaction of GPCRs with ␤-arrestins results in several distinct effects: (i) signal termination by homologous receptor desensitization, (ii) recruitment of elements of the internalization machinery thereby promoting GPCR endocytosis, and (iii) switching GPCR signaling to nonclassical pathways such as the mitogen-activated protein kinase pathway (1-3). Despite their limited sequence homology, nearly all GPCRs bind to ␤-arrestins, and ␤-arrestin recruitment is considered to be a universal mechanism for GPCR regulation. Rather than binding to a specific motif, ␤-arrestins appear to interact with several parts of the receptor involving the second and third intracellular loops and the C terminus (4 -6). It is generally thought that ␤-arrestin-receptor interaction involves an initial weak interaction of cytosolic ␤-arrestins with an active and phosphorylated receptor conformation, followed by conformational rearrangements of ␤-arrestins that further stabilize the receptor⅐arrestin complex (7,8).The receptor for parathyroid hormone (PTH) and PTH-related protein is involved in regulating calcium homeostasis and bone remodeling (9). Agonist occupancy of the PTH/PTH-related protein receptor (PTHR) leads to G s -mediated stimulation of adenylyl cyclase, resulting in cAMP production and PKA activation, and G q/11 -mediated phosphatidylinositol-specific phospholipase C␤ stimulation, leading to inositol 1,4,5-trisphosphate formation, calcium mobilization...

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Section: Figure 2 Nherf1 Forms a Ternary Complex With Pthr And ␤-Arrmentioning

confidence: 99%

Formation of a Ternary Complex among NHERF1, β-Arrestin, and Parathyroid Hormone Receptor

Klenk

Vetter

Zürn

et al. 2010

Journal of Biological Chemistry

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“…Whereas research performed over the past 30 y has revealed in great detail the basic mechanisms and kinetics of GPCR signaling (1,2), fundamental aspects, such as receptor di-/oligomerization or G-protein coupling and dissociation, remain controversial, mostly due to technical limitations to directly observe these phenomena (3)(4)(5).…”

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confidence: 99%

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.

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406

463

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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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“…2B). First, the kinetics of the change is very rapid (t 1/2 less than 1 s), a characteristic that suggests a conformational change rather than the recruitment or the dissociation of a partner subunit or even the internalization of the receptor (19). Second, with the rare earth cryptate Lumi4-Tb, FRET efficiency depends almost exclusively on the distance between the two fluorophores and not on the orientation (20).…”

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confidence: 99%

Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors

Doumazane

Scholler

Fabre

et al. 2013

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

109

147

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In multimeric cell-surface receptors, the conformational changes of the extracellular ligand-binding domains (ECDs) associated with receptor activation remain largely unknown. This is the case for the dimeric metabotropic glutamate receptors even though a number of ECD structures have been solved. Here, using an innovative approach based on cell-surface labeling and FRET, we demonstrate that a reorientation of the ECDs is associated with receptor and G-protein activation. Our approach helps identify partial agonists and highlights allosteric interactions between the effector and binding domains. Any approach expected to stabilize the active conformation of the effector domain increased the agonist potency in stabilizing the active ECDs conformation. These data provide key information on the structural dynamics and drug action at metabotropic glutamate receptors and validate an approach for tackling such analysis on other receptors.M any cell-surface receptors are multimers of proteins composed of several domains (1-3), including extracellular domains (ECDs) involved in endogenous ligand recognition and transmembrane domains (TMDs) responsible for intracellular signal transduction. Analysis of the conformational changes of the ECDs associated with receptor activation is crucial to understand the detailed mechanism involved in receptor activation and for the development of new innovative drugs. However, limited information is available on how the conformational changes in these proteins lead to receptor activation, especially in living cells.The eight glutamate-activated G-protein-coupled receptors (GPCRs), called "metabotropic glutamate receptors" (mGluRs), are key examples of multidomain and multimeric receptors (Fig. 1A). These receptors are strict dimers (4-6), and each subunit is made of a large ECD associated with a seven-helix TMD responsible for G-protein activation and downstream signaling (7). The mGluRs are key elements involved in the regulation of synaptic activity (8), and therefore they represent promising targets in drug development for the treatment of multiple neurologic and psychiatric diseases (9). More generally, the mGluRs are part of the class C GPCR family that contains structurally related receptors such as the receptors for sweet and umami taste, calcium, basic amino acids, and the inhibitory neurotransmitter GABA (10, 11).Crystallographic studies of the isolated dimeric ECDs and mutagenesis analyses have provided a clear view of the structure of the dimeric ECD and of the initial steps of mGluR activation. The ECD is composed of a Venus flytrap (VFT) bilobate domain containing the agonist binding site (12-15) and a cysteine-rich domain (CRD) that connects the VFT to the TMD (16). The VFTs exist in two major states: an open state (o) in absence of ligand and stabilized by antagonists, and a closed state (c) stabilized by agonists and required for receptor activation (12-15). The VFT dimer is in equilibrium between various conformations, depending on whether one or two VFTs are open or cl...

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Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors

Cited by 114 publications

References 74 publications

Formation of a Ternary Complex among NHERF1, β-Arrestin, and Parathyroid Hormone Receptor

Formation of a Ternary Complex among NHERF1, β-Arrestin, and Parathyroid Hormone Receptor

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

Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors

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