Single-molecule force spectroscopy of G-protein-coupled receptors

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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Title: Single-molecule diffusion-based estimation of ligand effects on G protein-coupled receptors

10.1101/205161 ◽

2017 ◽

Author(s):

Masataka Yanagawa ◽

Michio Hiroshima ◽

Yuichi Togashi ◽

Mitsuhiro Abe ◽

Takahiro Yamashita ◽

...

Keyword(s):

Diffusion Coefficient ◽

Cell Surface ◽

G Protein ◽

Single Molecule ◽

G Protein Coupled Receptors ◽

Single Molecule Imaging ◽

Imaging Analysis ◽

Coated Pits ◽

Ligand Effects ◽

G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) are major drug targets and have high potential for drug discovery. The development of a method for measuring the activities of GPCRs is essential for pharmacology and drug screening. However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface, and changes in the concentrations of downstream signaling molecules, which depend on signaling pathway selectivity of the receptor, are used as an index of the receptor activity. Here, we show that single-molecule imaging analysis provides an alternative method for assessing ligand effects on GPCR. We monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions by using total internal reflection fluorescence microscopy (TIRFM). The single-molecule tracking analysis demonstrates that changes in the average diffusion coefficient of mGluR3 quantitatively reflect the ligand-dependent activity. Then, we reveal that the diffusion of receptor molecules is altered by the common physiological events associated with GPCRs, including G protein binding or accumulation in clathrin-coated pits, by inhibition experiments and dual-color single-molecule imaging analysis. We also confirm the generality of agonist-induced diffusion change in class A and B GPCRs, demonstrating that the diffusion coefficient is a good index for estimating the ligand effects on many GPCRs regardless of the phylogenetic groups, chemical properties of the ligands, and G protein-coupling selectivity.One Sentence Summary: Single-molecule imaging for evaluating ligand effects on GPCRs by monitoring the diffusion dynamics on the cell surface.

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The stoichiometric ‘signature’ of Rhodopsin-family G protein-coupled receptors

10.1101/112466 ◽

2017 ◽

Author(s):

James H. Felce ◽

Sarah L. Latty ◽

Rachel G. Knox ◽

Susan R. Mattick ◽

Yuan Lui ◽

...

Keyword(s):

G Protein ◽

Single Molecule ◽

Large Scale ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Human Cell Line ◽

G Protein Coupled Receptors ◽

Bioluminescence Resonance Energy Transfer ◽

Entire Family ◽

G Protein Coupled

AbstractWhether Rhodopsin-family G protein-coupled receptors (GPCRs) form dimers is highly controversial, with much data both for and against emerging from studies of mostly individual receptors. The types of large-scale comparative studies from which a consensus could eventually emerge have not previously been attempted. Here, we sought to determine the stoichiometric “signatures” of 60 GPCRs expressed by a single human cell-line using orthogonal bioluminescence resonance energy transfer-based and single-molecule microscopy assays. We observed that a relatively small fraction of Rhodopsin-family GPCRs behaved as dimers and that these receptors otherwise appeared to be monomeric. Mapped onto the entire family the analysis predicted that fewer than 20% of the ~700 Rhodopsin- family receptors form dimers. The clustered distribution of Rhodopsin-family dimers, and a striking correlation between receptor stoichiometry and GPCR family-size that we also identified, suggested that evolution has tended to favor the lineage expansion of monomers rather than dimers.One Sentence SummaryAnalysis of 71 GPCRs from a single cell reveals the strong tendency of Rhodopsin-family receptors to exist as monomers rather than form dimers.

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Revealing the Stoichiometry of G Protein-Coupled Receptors (GPCRs) at the Cell Surface using Single Molecule Imaging

Biophysical Journal ◽

10.1016/j.bpj.2012.11.2904 ◽

2013 ◽

Vol 104 (2) ◽

pp. 525a ◽

Cited By ~ 1

Author(s):

Laura Weimann ◽

Steven F. Lee ◽

James H. Felce ◽

Simon J. Davis ◽

David Klenerman

Keyword(s):

Cell Surface ◽

G Protein ◽

Single Molecule ◽

G Protein Coupled Receptors ◽

Single Molecule Imaging ◽

G Protein Coupled

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The Ciliary Lumen Accommodates Passive Diffusion and Vesicle Trafficking in Cytoplasmic-Ciliary Transport

10.1101/704213 ◽

2019 ◽

Cited By ~ 1

Author(s):

Andrew Ruba ◽

Wangxi Luo ◽

Jingjie Yu ◽

Daisuke Takao ◽

Athanasios Evangelou ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

G Protein ◽

Single Molecule ◽

Primary Cilium ◽

Intraflagellar Transport ◽

Vesicle Transport ◽

Passive Diffusion ◽

G Protein Coupled Receptors ◽

Live Cells ◽

G Protein Coupled

AbstractTransport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Using single molecule microscopy, we mapped the movement of membrane and soluble proteins at the base of the primary cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary cilium - passive diffusional and vesicle transport routes - that are adopted by proteins for cytoplasmic-cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins.

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EJE AWARD 2020: Signalling by G protein-coupled receptors: why space and time matter

Acta Endocrinologica ◽

10.1530/eje-20-0890 ◽

2021 ◽

Vol 184 (2) ◽

pp. R41-R49

Author(s):

Davide Calebiro

Keyword(s):

G Protein ◽

Single Molecule ◽

Drug Targets ◽

Endocrine System ◽

G Protein Coupled Receptors ◽

Membrane Receptors ◽

Optical Methods ◽

Endocrine Disorders ◽

Spatiotemporal Resolution ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and major drug targets. They play a fundamental role in the endocrine system, where they mediate the effects of several hormones and neurotransmitters. As a result, alterations of GPCR signalling are a major cause of endocrine disorders such as congenital hypothyroidism or Cushing’s syndrome. My group develops innovative optical methods such as fluorescence resonance energy transfer (FRET) and single-molecule microscopy, which allow us to investigate GPCR signalling in living cells with unprecedented spatiotemporal resolution. Using this innovative approach, we have contributed to elucidate some long-debated questions about the mechanisms of GPCR signalling and their involvement in human disease. Among other findings, these studies have led to the unexpected discovery that GPCRs are not only signalling at the cell surface, as previously assumed, but also at various intracellular sites. This has important implications to understand how hormones and neurotransmitters produce specific responses in our cells and might pave the way to innovative treatments for common diseases like diabetes or heart failure.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1205798110 ◽

2012 ◽

Vol 110 (2) ◽

pp. 743-748 ◽

Cited By ~ 272

Author(s):

D. Calebiro ◽

F. Rieken ◽

J. Wagner ◽

T. Sungkaworn ◽

U. Zabel ◽

...

Keyword(s):

G Protein ◽

Single Molecule ◽

G Protein Coupled Receptors ◽

Single Molecule Analysis ◽

G Protein Coupled

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Single-molecule diffusion-based estimation of ligand effects on G protein–coupled receptors

Science Signaling ◽

10.1126/scisignal.aao1917 ◽

2018 ◽

Vol 11 (548) ◽

pp. eaao1917 ◽

Cited By ~ 27

Author(s):

Masataka Yanagawa ◽

Michio Hiroshima ◽

Yuichi Togashi ◽

Mitsuhiro Abe ◽

Takahiro Yamashita ◽

...

Keyword(s):

Diffusion Coefficient ◽

G Protein ◽

Single Molecule ◽

G Protein Coupled Receptors ◽

Single Molecule Imaging ◽

Imaging Analysis ◽

Ligand Effects ◽

Average Diffusion Coefficient ◽

Average Diffusion ◽

G Protein Coupled

G protein–coupled receptors (GPCRs) are major drug targets. Developing a method to measure the activities of GPCRs is essential for pharmacology and drug screening. However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface; thus, changes in the concentrations of downstream signaling molecules, which depend on the signaling pathway selectivity of the receptor, are often used as an index of receptor activity. We show that single-molecule imaging analysis provides an alternative method for assessing the effects of ligands on GPCRs. Using total internal reflection fluorescence microscopy (TIRFM), we monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions. Our single-molecule tracking analysis demonstrated that increases and decreases in the average diffusion coefficient of mGluR3 quantitatively reflected the ligand-dependent inactivation and activation of receptors, respectively. Through experiments with inhibitors and dual-color single-molecule imaging analysis, we found that the diffusion of receptor molecules was altered by common physiological events associated with GPCRs, including G protein binding, and receptor accumulation in clathrin-coated pits. We also confirmed that agonist also decreased the average diffusion coefficient for class A and B GPCRs, demonstrating that this parameter is a good index for estimating ligand effects on many GPCRs regardless of their phylogenetic groups, the chemical properties of the ligands, or G protein–coupling selectivity.

Download Full-text