Oligomerisation of G-protein-coupled receptors

A range of approaches have recently provided evidence that G-protein-coupled receptors can exist as oligomeric complexes. Both homo-oligomers, comprising multiple copies of the same gene product, and hetero-oligomers containing more than one receptor have been detected. In several, but not all, examples, the extent of oligomerisation is regulated by the presence of agonist ligands, and emerging evidence indicates that receptor hetero-oligomers can display distinct pharmacological characteristics. A chaperonin-like role for receptor oligomerisation in effective delivery of newly synthesised receptors to the cell surface is a developing concept, and recent studies have employed a series of energy-transfer techniques to explore the presence and regulation of receptor oligomerisation in living cells. However, the majority of studies have relied largely on co-immunoprecipitation techniques, and there is still little direct information on the fraction of receptors existing as oligomers in intact cells.

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Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310461 ◽

2003 ◽

Vol 31 (3) ◽

pp. 461-471 ◽

Cited By ~ 32

Author(s):

D Devost ◽

HH Zingg

Keyword(s):

Energy Transfer ◽

Cell Surface ◽

G Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Living Cells ◽

Oxytocin Receptor ◽

G Protein Coupled Receptors ◽

Bioluminescence Resonance Energy Transfer ◽

G Protein Coupled

The nonapeptide hormone oxytocin exerts many important biological functions, including uterine contractions during parturition and milk ejection during lactation. The manifold effects of oxytocin are mediated by a single oxytocin receptor (OTR) type, a member of the super-family of G-protein-coupled receptors. There is accumulating recent evidence that certain G-protein-coupled receptors exist in the form of oligomeric complexes. Here we demonstrate, using two different co-immunoprecipitation strategies as well as bioluminescence resonance energy transfer techniques, that the OTR is capable of forming oligomeric complexes in vivo and that these complexes exist at the cell surface membrane. The human OTR was N-terminally tagged with either a Myc or Flag epitope and transiently expressed in COS-7 cells. Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by SDS-PAGE and Western blotting using an anti-Myc antibody, or vice versa. Either strategy provided evidence for the co-precipitation of Myc- or Flag-tagged OTR respectively.Biochemical characterization of OTR dimers showed that homodimer formation is not dependent on the establishment of disulfide bonds. The existence of OTR dimers and oligomers at the level of the cell surface was demonstrated by exposing intact living cells to an anti-Flag antibody and analyzing the immunoprecipitate by Western blotting with an anti-Myc antibody. This approach demonstrated furthermore that the presence of receptor oligomers at the cell surface is modulated by ligand in a time-dependent fashion. Finally, we obtained evidence that the OTR is forming oligomeric structures in intact living cells by observing the occurrence of bioluminescence resonance energy transfer in cells co-transfected with OTR constructs bearing at their C-terminus either a Renilla luciferase or the yellow fluorescent protein. Taken together, these data show that the OTR can form homodimers and oligomers in the cell model used and that these oligomers are present at the cell surface.

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Energy Transfer Technologies to Monitor the Dynamics and Signaling Properties of G-Protein-Coupled Receptors in Living Cells

Biophysical Analysis of Membrane Proteins ◽

10.1002/9783527621224.ch13 ◽

2008 ◽

pp. 309-334

Author(s):

Jean-Philippe Pin ◽

Mohammed-Akli Ayoub ◽

Damien Maurel ◽

Julie Perroy ◽

Eric Trinquet

Keyword(s):

Energy Transfer ◽

G Protein ◽

Living Cells ◽

G Protein Coupled Receptors ◽

G Protein Coupled

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Faculty Opinions recommendation of Measurement of the millisecond activation switch of G protein-coupled receptors in living cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014169.192885 ◽

2003 ◽

Author(s):

Kees Jalink

Keyword(s):

G Protein ◽

Living Cells ◽

G Protein Coupled Receptors ◽

G Protein Coupled

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Applying label-free dynamic mass redistribution technology to frame signaling of G protein–coupled receptors noninvasively in living cells

Nature Protocols ◽

10.1038/nprot.2011.386 ◽

2011 ◽

Vol 6 (11) ◽

pp. 1748-1760 ◽

Cited By ~ 111

Author(s):

Ralf Schröder ◽

Johannes Schmidt ◽

Stefanie Blättermann ◽

Lucas Peters ◽

Nicole Janssen ◽

...

Keyword(s):

G Protein ◽

Living Cells ◽

G Protein Coupled Receptors ◽

Label Free ◽

Dynamic Mass ◽

Mass Redistribution ◽

G Protein Coupled ◽

Free Dynamic

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Understudied G Protein-Coupled Receptors in the Kidney

Nephron ◽

10.1159/000517355 ◽

2021 ◽

pp. 1-4

Author(s):

Nathan A. Zaidman ◽

Jennifer L. Pluznick

Keyword(s):

Renal Function ◽

Cell Surface ◽

Gene Family ◽

G Protein ◽

External Environment ◽

G Protein Coupled Receptors ◽

Surface Proteins ◽

Cell Surface Proteins ◽

Large Subset ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) are cell surface proteins which play a key role in allowing cells, tissues, and organs to respond to changes in the external environment in order to maintain homeostasis. Despite the fact that GPCRs are known to play key roles in a variety of tissues, there are a large subset of GPCRs that remain poorly studied. In this minireview, we will summarize what is known regarding the “understudied” GPCRs with respect to renal function, and in so doing will highlight the promise represented by studying this gene family.

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Imaging of persistent cAMP signaling by internalized G protein-coupled receptors

Journal of Molecular Endocrinology ◽

10.1677/jme-10-0014 ◽

2010 ◽

Vol 45 (1) ◽

pp. 1-8 ◽

Cited By ~ 47

Author(s):

Davide Calebiro ◽

Viacheslav O Nikolaev ◽

Martin J Lohse

Keyword(s):

G Protein ◽

Intracellular Signaling ◽

Current Model ◽

Living Cells ◽

G Protein Coupled Receptors ◽

Membrane Receptors ◽

Optical Methods ◽

Camp Signaling ◽

Gpcr Signaling ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. They mediate the effects of several endogenous cues and serve as important pharmacological targets. Although many biochemical events involved in GPCR signaling have been characterized in great detail, little is known about their spatiotemporal dynamics in living cells. The recent advent of optical methods based on fluorescent resonance energy transfer allows, for the first time, to directly monitor GPCR signaling in living cells. Utilizing these methods, it has been recently possible to show that the receptors for two protein/peptide hormones, the TSH and the parathyroid hormone, continue signaling to cAMP after their internalization into endosomes. This type of intracellular signaling is persistent and apparently triggers specific cellular outcomes. Here, we review these recent data and explain the optical methods used for such studies. Based on these findings, we propose a revision of the current model of the GPCR–cAMP signaling pathway to accommodate receptor signaling at endosomes.

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Inhibition of G protein-coupled receptor trafficking in neuroblastoma cells by MAP 4 decoration of microtubules

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00410.2002 ◽

2002 ◽

Vol 283 (6) ◽

pp. H2379-H2388 ◽

Cited By ~ 9

Author(s):

Guangmao Cheng ◽

Yoshihiro Iijima ◽

Yuji Ishibashi ◽

Dhandapani Kuppuswamy ◽

George Cooper

Keyword(s):

G Protein ◽

Muscarinic Receptor ◽

Receptor Binding ◽

Neuroblastoma Cells ◽

Pressure Overload ◽

G Protein Coupled Receptors ◽

Structural Protein ◽

Microtubule Network ◽

Intact Cells ◽

G Protein Coupled

One mechanism for the reappearance of G protein-coupled receptors after agonist activation is microtubule-based transport. In pressure-overload cardiac hypertrophy, there is downregulation of G protein-coupled receptors and the appearance of a densified microtubule network extensively decorated by a microtubule-associated protein, MAP 4. Our hypothesis is that overdecoration of a dense microtubule network with this structural protein, as in hypertrophied myocardium, would impede receptor recovery. We tested this hypothesis by studying muscarinic acetylcholine receptor (mAChR) internalization and recovery after agonist stimulation in neuroblastoma cells. Exposure of cells to carbachol, a muscarinic receptor agonist, decreased membrane receptor binding activity. After carbachol withdrawal, receptor binding recovered toward the initial value. When microtubules were depolymerized before carbachol withdrawal, mAChR recovery was only 44% of that in intact cells. Cells were then infected with an adenovirus containing MAP 4 cDNA. MAP 4 protein decorated the microtubules extensively, and receptor recovery upon carbachol withdrawal was reduced to 54% of control. Thus muscarinic receptor recovery after agonist exposure is microtubule dependent, and MAP 4 decoration of microtubules inhibits receptor recovery.

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The luminescent HiBiT peptide enables selective quantitation of G protein–coupled receptor ligand engagement and internalization in living cells

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011952 ◽

2020 ◽

Vol 295 (15) ◽

pp. 5124-5135 ◽

Cited By ~ 4

Author(s):

Michelle E. Boursier ◽

Sergiy Levin ◽

Kris Zimmerman ◽

Thomas Machleidt ◽

Robin Hurst ◽

...

Keyword(s):

Energy Transfer ◽

Cell Surface ◽

G Protein ◽

Dynamic Range ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Receptor Internalization ◽

Cell Surface Receptor ◽

Cellular Interactions ◽

G Protein Coupled

G protein–coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the β-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.

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