scholarly journals BBSome trains remove activated GPCRs from cilia by enabling passage through the transition zone

2018 ◽  
Vol 217 (5) ◽  
pp. 1847-1868 ◽  
Author(s):  
Fan Ye ◽  
Andrew R. Nager ◽  
Maxence V. Nachury
Keyword(s):  
Transition Zone ◽  
Single Molecule ◽  
Diffusion Barrier ◽  
Diffusion Barriers ◽  
G Protein Coupled Receptors ◽  
Lateral Transport ◽  
Step Process ◽  
Signaling Receptors ◽  
Guanosine Triphosphatase ◽  
G Protein Coupled

A diffusion barrier at the transition zone enables the compartmentalization of signaling molecules by cilia. The BBSome and the small guanosine triphosphatase Arl6, which triggers BBSome coat polymerization, are required for the exit of activated signaling receptors from cilia, but how diffusion barriers are crossed when membrane proteins exit cilia remains to be determined. In this study, we found that activation of the ciliary G protein–coupled receptors (GPCRs) Smoothened and SSTR3 drove the Arl6-dependent assembly of large, highly processive, and cargo-laden retrograde BBSome trains. Single-molecule imaging revealed that the assembly of BBSome trains enables the lateral transport of ciliary GPCRs across the transition zone. However, the removal of activated GPCRs from cilia was inefficient because a second periciliary diffusion barrier was infrequently crossed. We conclude that exit from cilia is a two-step process in which BBSome/Arl6 trains first move activated GPCRs through the transition zone before a periciliary barrier can be crossed.

scholarly journals BBSome trains remove activated GPCRs from cilia by enabling passage through the transition zone

2017 ◽  
Author(s):  
Fan Ye ◽  
Andrew R. Nager ◽  
Maxence V. Nachury
Keyword(s):  
Transition Zone ◽  
Single Molecule ◽  
Diffusion Barrier ◽  
Small Gtpase ◽  
Single Molecule Imaging ◽  
Lateral Transport ◽  
Step Process ◽  
On Demand ◽  
Gpcr Activation ◽  
Signaling Receptors

AbstractA diffusion barrier at the transition zone enables the compartmentalization of signaling molecules by cilia. The BBSome and the small GTPase Arl6, which triggers BBSome coat polymerization, are required for the exit of activated signaling receptors from cilia, but how diffusion barriers are crossed when membrane proteins exit cilia remains to be determined. Here we found that activation of the ciliary GPCRs Smoothened and SSTR3 drove the Arl6-dependent assembly of large, highly processive and cargo-laden retrograde BBSome trains. Single-molecule imaging revealed that the assembly of BBSome trains enables the lateral transport of ciliary GPCRs across the transition zone. Yet, the removal of activated GPCRs from cilia was inefficient because a second, periciliary diffusion barrier was infrequently crossed. We conclude that exit from cilia is a two-step process in which BBSome/Arl6 trains first moves activated GPCRs through the transition zone before a periciliary barrier can be crossed.SummaryUpon activation, GPCRs must exit cilia for appropriate signal transduction. Using bulk imaging of BBSome and single molecule imaging of GPCRs, Ye et al. demonstrate that retrograde BBSome trains assemble on-demand upon GPCR activation and ferry GPCRs across the transition zone. Yet, ciliary exit often fails because of a second diffusion barrier.

scholarly journals Near-atomic structures of the BBSome reveal the basis for BBSome activation and binding to GPCR cargoes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Shuang Yang ◽  
Kriti Bahl ◽  
Hui-Ting Chou ◽  
Jonathan Woodsmith ◽  
Ulrich Stelzl ◽  
...  
Keyword(s):  
G Protein ◽  
Transition Zone ◽  
Diffusion Barrier ◽  
Small Gtpase ◽  
G Protein Coupled Receptors ◽  
Amphipathic Helix ◽  
Membrane Recruitment ◽  
Atomic Structures ◽  
G Protein Coupled

Dynamic trafficking of G protein-coupled receptors (GPCRs) out of cilia is mediated by the BBSome. In concert with its membrane recruitment factor, the small GTPase ARL6/BBS3, the BBSome ferries GPCRs across the transition zone, a diffusion barrier at the base of cilia. Here, we present the near-atomic structures of the BBSome by itself and in complex with ARL6GTP, and we describe the changes in BBSome conformation induced by ARL6GTP binding. Modeling the interactions of the BBSome with membranes and the GPCR Smoothened (SMO) reveals that SMO, and likely also other GPCR cargoes, must release their amphipathic helix 8 from the membrane to be recognized by the BBSome.

scholarly journals Near-atomic structures of the BBSome reveal a novel mechanism for transition zone crossing

2020 ◽  
Author(s):  
Kriti Bahl ◽  
Shuang Yang ◽  
Hui-Ting Chou ◽  
Jonathan Woodsmith ◽  
Ulrich Stelzl ◽  
...  
Keyword(s):  
Transition Zone ◽  
G Protein Coupled Receptors ◽  
Amphipathic Helix ◽  
Functional Importance ◽  
Bardet Biedl Syndrome ◽  
Lipid Packing ◽  
Atomic Structures ◽  
Amphipathic Helices ◽  
Signaling Receptors ◽  
G Protein Coupled

ABSTRACTThe BBSome is a complex of eight Bardet-Biedl Syndrome (BBS) proteins that removes signaling receptors from cilia. The GTPase ARL6/BBS3 recruits the BBSome to the ciliary membrane where the BBSome–ARL6GTP complex ferries G protein-coupled receptors (GPCRs) across the transition zone, a diffusion barrier at the base of cilia. Here, we find that the BBSome undergoes a conformational change upon recruitment to membranes by ARL6GTP. Modeling the binding of the BBSome to membranes and to the GPCR Smoothened (SMO) reveals that the amphipathic helix 8 of SMO must be released from the membrane for SMO to be recognized by the BBSome. Underscoring the functional importance of amphipathic helix extraction in TZ crossing, we find that exchanging the amphipathic helix of ARL6 for one that embeds deeper into the membrane blocks BBSome-mediated exit of GPCRs from cilia. We propose that the rigid curvature and dense lipid packing of the transition zone reject asymmetric insertions in the inner leaflet and that the BBSome licenses transition zone crossing by extracting bulky amphipathic helices from the inner leaflet.

scholarly journals Single-Molecule Detection Technologies in Miniaturized High Throughput Screening: Binding Assays for G Protein-Coupled Receptors Using Fluorescence Intensity Distribution Analysis and Fluorescence Anisotropy

2001 ◽  
Vol 6 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Martin Rudiger ◽  
Ulrich Haupts ◽  
Keith J. Moore ◽  
Andrew J. Pope
Keyword(s):  
Single Molecule ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
G Protein Coupled Receptors ◽  
Single Molecule Detection ◽  
Distribution Analysis ◽  
Binding Assays ◽  
Fluorescence Intensity Distribution Analysis ◽  
Fluorescent Ligands ◽  
G Protein Coupled

G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based binding assays that are highly amenable to miniaturization. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those induced by binding of fluorescent ligands to membrane particles with multiple receptor sites. As a confocal detection technology, FIDA inherently allows reduction of the assay volume to the microliter range and below without any loss of signal. Binding and displacement experiments are demonstrated for various types of GPCRs, such as chemokine, peptide hormone, or small-molecule ligand receptors, demonstrating the broad applicability of this method. The results correlate quantitatively with radioligand binding data. We compare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. While FA requires a higher degree of binding, FIDA is sensitive down to lower levels of receptor expression. Both methods are, within these boundary conditions, applicable to uHTS.

scholarly journals Conformational pathway provides unique sensitivity to a synaptic mGluR

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Chris H. Habrian ◽  
Joshua Levitz ◽  
Vojtech Vyklicky ◽  
Zhu Fu ◽  
Adam Hoagland ◽  
...  
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptors ◽  
Dynamic Range ◽  
High Sensitivity ◽  
G Protein Coupled Receptors ◽  
Metabotropic Glutamate ◽  
Single Molecule Fret ◽  
Broad Dynamic Range ◽  
Maximal Activation ◽  
G Protein Coupled

AbstractMetabotropic glutamate receptors (mGluRs) are dimeric G-protein–coupled receptors that operate at synapses. Macroscopic and single molecule FRET to monitor structural rearrangements in the ligand binding domain (LBD) of the mGluR7/7 homodimer revealed it to have an apparent affinity ~4000-fold lower than other mGluRs and a maximal activation of only ~10%, seemingly too low for activation at synapses. However, mGluR7 heterodimerizes, and we find it to associate with mGluR2 in the hippocampus. Strikingly, the mGluR2/7 heterodimer has high affinity and efficacy. mGluR2/7 shows cooperativity in which an unliganded subunit greatly enhances activation by agonist bound to its heteromeric partner, and a unique conformational pathway to activation, in which mGluR2/7 partially activates in the Apo state, even when its LBDs are held open by antagonist. High sensitivity and an unusually broad dynamic range should enable mGluR2/7 to respond to both glutamate transients from nearby release and spillover from distant synapses.

scholarly journals Imaging of G protein-coupled receptors in solid-supported planar membranes at the single molecule level

2008 ◽  
Author(s):  
Iwan Märki ◽  
Marcel Leutenegger ◽  
Matthias Geissbuehler ◽  
Rudolf Robelek ◽  
Eva-Kathrin Sinner ◽  
...  
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
G Protein Coupled Receptors ◽  
Single Molecule Level ◽  
G Protein Coupled ◽  
Planar Membranes

scholarly journals The Class-A GPCR Dopamine D2 Receptor Forms Transient Dimers Stabilized by Agonists: Detection by Single-Molecule Tracking

2017 ◽  
Vol 76 (1-2) ◽  
pp. 29-37 ◽  
Author(s):  
Rinshi S. Kasai ◽  
Shuichi V. Ito ◽  
Ryo M. Awane ◽  
Takahiro K. Fujiwara ◽  
Akihiro Kusumi
Keyword(s):  
Single Molecule ◽  
Resting State ◽  
Dopamine D2 Receptor ◽  
Critical Role ◽  
D2 Receptor ◽  
G Protein Coupled Receptors ◽  
Single Molecule Tracking ◽  
Class A ◽  
Dopamine D2 ◽  
G Protein Coupled

Abstract Whether class-A G-protein coupled receptors (GPCRs) exist and work as monomers or dimers has drawn extensive attention. A class-A GPCR dopamine D2 receptor (D2R) is involved in many physiological and pathological processes and diseases, indicating its critical role in proper functioning of neuronal circuits. In particular, D2R homodimers might play key roles in schizophrenia development and amphetamine-induced psychosis. Here, using single-molecule imaging, we directly tracked single D2R molecules in the plasma membrane at a physiological temperature of 37 °C, and unequivocally determined that D2R forms transient dimers with a lifetime of 68 ms in its resting state. Agonist addition prolonged the dimer lifetime by a factor of ~1.5, suggesting the possibility that transient dimers might be involved in signaling.

scholarly journals Simple methods for quantifying super-resolved cortical actin

2021 ◽  
Author(s):  
Evelyn Garlick ◽  
Emma L Faulkner ◽  
Stephen J Briddon ◽  
Steven G Thomas
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Movement ◽  
G Protein Coupled Receptors ◽  
Cortical Actin ◽  
Surface Receptors ◽  
Actin Networks ◽  
Development And Validation ◽  
G Protein Coupled

Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or corrals, in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by both Super Resolved Radial Fluctuations (SRRF) and expansion microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31 μm2 ± 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.

scholarly journals Metastable GPCR dimers trigger the basal signal by recruiting G-proteins

2020 ◽  
Author(s):  
Rinshi S. Kasai ◽  
Takahiro K. Fujiwara ◽  
Akihiro Kusumi
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Single Molecule ◽  
Resting State ◽  
G Protein Coupled Receptors ◽  
Β2 Adrenergic Receptor ◽  
Low Levels ◽  
Protein Recruitment ◽  
Novel Drug ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) constitute the largest family of integral membrane proteins in the human genome and are responsible for various important signaling pathways for vision, olfaction, gustation, emotion, cell migration, etc. A distinct feature of the GPCR-family proteins is that many GPCRs, including the prototypical GPCR, β2-adrenergic receptor (β2AR), elicit low levels of basal constitutive signals without agonist stimulation, which function in normal development and various diseases1–3. However, how the basal signals are induced is hardly known. Another general distinctive feature of GPCRs is to form metastable homo-dimers, with lifetimes on the order of 0.1 s, even in the resting state. Here, our single-molecule-based quantification4 determined the dissociation constant of β2AR homo-dimers in the PM (1.6 ± 0.29 copies/μm2) and their lifetimes (83.2 ± 6.4 ms), and furthermore found that, in the resting state, trimeric G-proteins were recruited to both β2AR monomers and homo-dimers. Importantly, inverse agonists, which suppress the GPCR’s basal constitutive activity, specifically blocked the G-protein recruitment to GPCR homo-dimers, without affecting that to monomers. These results indicate that the G-proteins recruited to transient GPCR homo-dimers are responsible for inducing their basic constitutive signals. These results suggest novel drug development strategies to enhance or suppress GPCR homo-dimer formation.

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