Receptor Quaternary Organization Explains G Protein-Coupled Receptor Family Structure

Felce, James H.; Latty, Sarah L.; Knox, Rachel; Mattick, Susan R.; Lui, Yuan; Lee, Steven F.; Klenerman, David; Davis, Simon J.

doi:10.1016/j.celrep.2017.08.072

Cited by 41 publications

(60 citation statements)

References 57 publications

(94 reference statements)

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“…The use of BRET‐ and single‐molecule microscopy‐based assays on several members of the rhodopsin family of GPCR recently showed that a relatively small fraction of these receptors behave as dimers, while the major proportion appears to be monomeric . In this analysis, CXCR4 was included in a subgroup of dimer‐forming GPCR whereas closely related CKR such as CCR6 or CCR1 were not.…”

Section: Chemokine Receptors: Additional Levels Of Complexitymentioning

confidence: 90%

“…57 The use of BRET-and single-molecule microscopy-based assays on several members of the rhodopsin family of GPCR recently showed that a relatively small fraction of these receptors behave as dimers, while the major proportion appears to be monomeric. 58 In this analysis, CXCR4 was included in a subgroup of dimer-forming GPCR whereas closely related CKR such as CCR6 or CCR1 were not. This study was performed in the absence of ligands, that is, under steady-state conditions, therefore, possible ligand-mediated clustering effects were not considered.…”

Section: Chemokine Receptors: Additional Levels Of Complexitymentioning

confidence: 99%

“…68 Although experimental evidence indicates that the formation of homo-and heterodimers is a general process in the chemokine receptor field, a recent report on class A GPCR members, predicts that this is not always the case, as some receptors exist as monomeric entities. 58 In this regard, it should be noted that FRET and BRET experiments only allow evaluation of complexes but cannot quantify monomers. Therefore, the coexistence of dimers and oligomers with monomeric entities must not be discarded and its relevance analyzed.…”

Section: Membrane Lipids Membrane Proteins F I G U R E 1 Chemokine Rementioning

confidence: 99%

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Remodeling our concept of chemokine receptor function: From monomers to oligomers

Martínez-Muñoz

Villares

Rodrı́guez-Fernández

et al. 2018

Journal of Leukocyte Biology

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The chemokines direct leukocyte recruitment in both homeostatic and inflammatory conditions, and are therefore critical for immune reactions. By binding to members of the class A G protein-coupled receptors, the chemokines play an essential role in numerous physiological and pathological processes. In the last quarter century, the field has accumulated much information regarding the implications of these molecules in different immune processes, as well as mechanistic insight into the signaling events activated through their binding to their receptors. Here, we will focus on chemokine receptors and how new methodological approaches have underscored the role of their conformations in chemokine functions. Advances in biophysical-based techniques show that chemokines and their receptors act in very complex networks and therefore should not be considered isolated entities. In this regard, the chemokine receptors can form homo- and heterodimers as well as oligomers at the cell surface. These findings are changing our view as to how chemokines influence cell biology, identify partners that regulate chemokine function, and open new avenues for therapeutic intervention.

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Section: Chemokine Receptors: Additional Levels Of Complexitymentioning

confidence: 90%

Section: Chemokine Receptors: Additional Levels Of Complexitymentioning

confidence: 99%

Section: Membrane Lipids Membrane Proteins F I G U R E 1 Chemokine Rementioning

confidence: 99%

See 1 more Smart Citation

Remodeling our concept of chemokine receptor function: From monomers to oligomers

Martínez-Muñoz

Villares

Rodrı́guez-Fernández

et al. 2018

Journal of Leukocyte Biology

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“…If the RLuc8 donor fusion molecule catalyzed the coelenterazine 400a and the GFP 2 acceptor fusion molecule is located less than 10 nm from the donor, a transfer of energy will occur from the donor to the acceptor. This method has been widely used to study PPI (Bery et al., ; Felce et al., ; Mercier et al., ) and also PPI inhibition by small molecules (Beautrait et al., ; Corbel et al., ; Corbel et al., ; Lavoie et al., ; Mazars & Fahraeus, ; Quevedo et al., ) or macromolecules (Bery et al., ; Guillard et al., ; Spencer‐Smith et al., ). It offers several advantages: it is a very sensitive technique as it is based on luminescence, there is no background signal from cellular autofluorescence.…”

Section: Commentarymentioning

confidence: 99%

Bioluminescence Resonance Energy Transfer 2 (BRET2)‐Based RAS Biosensors to Characterize RAS Inhibitors

Béry

Rabbitts

2019

CP Cell Biology

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Protein-protein interactions (PPIs) are principle biological processes that control normal cell growth, differentiation, and homeostasis but are also crucial in diseases such as malignancy, neuropathy, and infection. Despite the importance of PPIs in biology, this target class has been very challenging to convert to therapeutics. In the last decade, much progress has been made in the inhibition of PPIs involved in diseases, but many remain difficult such as RAS-effector interactions in cancers. We describe here a protocol for using Bioluminescence Resonance Energy Transfer 2 (BRET2)-based RAS biosensors to detect and characterize RAS PPI inhibition by macromolecules and small molecules. This method could be extended to any other small GTPases or any other PPIs of interest. C 2019 by John Wiley & Sons, Inc.Keywords: biosensors r BRET r protein-protein interaction inhibition r RAS family GTPases of 22RAS associating (RA) domain of RALGDS. We also used as acceptor the full-length CRAF and PI3Kα effectors to study their respective downstream signaling pathways. Therefore, using this BRET-based RAS biosensors toolbox, we characterized the cellular properties of various RAS inhibitors comprising anti-RAS Design Ankyrin Repeat Proteins (DARPins) (Guillard et al., 2017) and anti-RAS intracellular domain antibody (iDAb RAS) (Bery et al., 2018) macromolecules and antibody derived anti-RAS compounds Quevedo et al., 2018).Here we describe a set of protocols that include step-by-step instructions to monitor the interaction between RAS and a partner (Basic Protocol 1), to identify inhibitors of RAS PPIs including macromolecules (Basic Protocol 2) and small molecules (Basic Protocol 3). We also describe a protocol to detect the effect of RAS inhibitors on the RAS downstream pathways by immunoblot (Basic Protocol 4) and finally a protocol explaining how to calculate the BRET ratio obtained in the previous protocols (Basic Protocol 5). Figure 1Use of the BRET-based RAS biosensors to assess a PPI. (A) A schematic representation of the BRET-based biosensors. A protein (P1) is fused to the donor moiety RLuc8 and a protein (P2 or P3) is tagged with the acceptor moiety GFP 2 . If P1 does not bind to P2, it only produces a background BRET signal. However, when P1 interacts with P2, it induces a BRET signal, if the RLuc8 and GFP 2 domains are within 10 nm. This BRET signal can be decreased by addition of a competitor (either by a macromolecule or a small molecule inhibitor). (B) A representative donor saturation assay between KRAS G12D , KRAS WT and KRAS S17N (donors) and CRAF RBD (acceptor) with total GFP 2 and RLuc8 controls. Where error bars are presented, these correspond to mean values ± SD of quadruplicates technical repeats. Day 2: Cell transfection2. Mix together a fixed amount of RLuc8 construct (typically 50 ng) and a varying amount of GFP 2 construct (see quantity in Table 2).The pEF-myc-cyto empty plasmid is used to equalize total amount of transfected DNA between wells. The total DNA amount transfected per well is 1.6 μg. ...

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“…The bivalent ligand AP20187 is a crosslinker that enables ligand-dependent ''dimerization'' of two membrane proteins fused to mutant FKBP domains. FKBP-mediated crosslinks have also been used as positive controls for ''dimerization'' in type 1 and 3 BRET experiments with one protein fused to FKBP and GFP 2 and the other protein fused to FKBP and Rluc (6). However, in the clever type 4 experiment for heterodimers, the GFP 2 -and Rluc-fusion constructs are generated for receptor A, and the FKBP-or (FKBP) 3 -fusion constructs are generated for receptor B.…”

mentioning

confidence: 99%

Third-Party Capture of Elusive GPCR Dimers

Huber

Sakmar

2019

Biophysical Journal

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Receptor Quaternary Organization Explains G Protein-Coupled Receptor Family Structure

Cited by 41 publications

References 57 publications

Remodeling our concept of chemokine receptor function: From monomers to oligomers

Remodeling our concept of chemokine receptor function: From monomers to oligomers

Bioluminescence Resonance Energy Transfer 2 (BRET2)‐Based RAS Biosensors to Characterize RAS Inhibitors

Third-Party Capture of Elusive GPCR Dimers

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