Using Bioluminescence Resonance Energy Transfer (BRET) to Characterize Agonist‐Induced Arrestin Recruitment to Modified and Unmodified G Protein‐Coupled Receptors

Donthamsetti, Prashant; Quejada, Jose R.; Javitch, Jonathan A.; Gurevich, Vsevolod V.; Lambert, Nevin A.

doi:10.1002/0471141755.ph0214s70

Cited by 51 publications

(70 citation statements)

References 36 publications

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“…96 For the D2R-mediated arrestin recruitment assay, we transfected D2R (2 μ g), Rluc8-arrestin3-Sp1 (0.25 μ g), mem-linker-citrine-SH3 (5 μ g), and GRK2 (5 μ g). 97 …”

Section: Methodsmentioning

confidence: 99%

Optical Control of Dopamine Receptors Using a Photoswitchable Tethered Inverse Agonist

et al. 2017

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Family A G protein-coupled receptors (GPCRs) control diverse biological processes and are of great clinical relevance. Their archetype rhodopsin becomes naturally light sensitive by binding covalently to the photoswitchable tethered ligand (PTL) retinal. Other GPCRs, however, neither bind covalently to ligands nor are light sensitive. We sought to impart the logic of rhodopsin to light-insensitive Family A GPCRs in order to enable their remote control in a receptor-specific, cell-type-specific, and spatiotemporally precise manner. Dopamine receptors (DARs) are of particular interest for their roles in motor coordination, appetitive, and aversive behavior, as well as neuropsychiatric disorders such as Parkinson’s disease, schizophrenia, mood disorders, and addiction. Using an azobenzene derivative of the well-known DAR ligand 2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), we were able to rapidly, reversibly, and selectively block dopamine D1 and D2 receptors (D1R and D2R) when the PTL was conjugated to an engineered cysteine near the dopamine binding site. Depending on the site of tethering, the ligand behaved as either a photoswitchable tethered neutral antagonist or inverse agonist. Our results indicate that DARs can be chemically engineered for selective remote control by light and provide a template for precision control of Family A GPCRs.

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“…96 For the D2R-mediated arrestin recruitment assay, we transfected D2R (2 μ g), Rluc8-arrestin3-Sp1 (0.25 μ g), mem-linker-citrine-SH3 (5 μ g), and GRK2 (5 μ g). 97 …”

Section: Methodsmentioning

confidence: 99%

Optical Control of Dopamine Receptors Using a Photoswitchable Tethered Inverse Agonist

et al. 2017

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“…Mammalian angiotensin receptor 1 (AT1R) had a signal peptide and snap tag attached to the N-terminal. The constructs for the direct and indirect β-arrestin recruitment assays were made by exchanging the donor and acceptor with the NanoLuc splits from the BRET arrestin method previously described (12) as well as removal of the helper peptides SH3 and Sp1 to make D2R-linker-N1, mem-linker-N1 (membrane tethered N-terminal split named MeN) and N2-linker-β-arrestin2 (arrestin tethered C-terminal split named ArC). The multicistronic vector was made by inserting the 22 amino acid P2A cleavage peptide between sequence of MeN and ArC, creating ArC-P2A-MeN in pcDNA3.1+ hygromycin.…”

Section: Plasmid Constructsmentioning

confidence: 99%

“…LinkLight uses a modified luciferase attached to βarrestin that is cleaved off by a protease attached to the GPCR of interest, thereby activating the luciferase and producing light (11). BRET between a receptor fused at its C-terminus to a luciferase and arrestin tagged at its N-terminus with mVenus is also commonly used to measure arrestin recruitment (12). Notably, all of these β-arrestin assays require C-terminal fusions to the receptor of interest.…”

Section: Introductionmentioning

confidence: 99%

“…In contrast, we previously introduced a modified BRET assay in which the donor (Rluc8) is anchored to the membrane and the acceptor (Venus) is fused to β-arrestin (12). In this assay, which is based on "bystander" BRET, translocation of β-arrestin to the receptor at the plasma membrane leads to an increase in BRET from a membrane-anchored donor.…”

Section: Introductionmentioning

confidence: 99%

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A novel luminescence-based β-arrestin membrane recruitment assay for unmodified GPCRs

Pedersen

Pham²,

Mancebo³

et al. 2020

Preprint

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G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, G protein-independent signaling via the arrestin pathway has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either the G protein or arrestin-signaling pathways. Currently available screening techniques that measure arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure arrestin recruitment to any unmodified receptor.NanoLuc, an engineered luciferase from ophlorus gracilirostris (deep sea shrimp), is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. Here, we have identified a novel split site and have fused the N-terminal fragment to a membrane tether and the C-terminal fragment to the N-terminus of either β-arrestin 1 or 2. Upon receptor activation, arrestin is recruited to the plasma membrane in an agonist concentrationdependent manner and the two NanoLuc fragments complement to reconstitute functional luciferase, which allows quantification of recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification. The split NanoLuc arrestin recruitment assay has promise as a new generic assay for measuring arrestin recruitment to diverse GPCR types in heterologous or native cells.

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“…Ligand induced recruitment of arrestin by GPCRs, has been dynamically monitored using BRET (Donthamsetti et al, 2015;Pfleger et al, 2006;Stoddart et al, 2015). To study arrestin recruitment by GPCRs, proteins are modified by means of fusion to accessory protein fragments, that act as donor and acceptor.…”

Section: Bret Assay To Study Arrestin Recruitmentmentioning

confidence: 99%

BRET Assays to Determine Altered Function of a D2DR Variant in G protein-Independent and -Dependent Pathways

Kunz¹

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G protein-dependent and independent signaling pathways perform crucial roles in dopaminerelated physiological and pathological processes. A pivotal role of dopamine in the CNS is the control of locomotion; disruption to dopaminergic systems, such as reduced striatal D2 receptor availability, contributes to the development of movement disorders. Affected members of a family with dystonia, a movement disorder, are heterozygous for a SNP in exon 4 of D2DR (c.634A>T p.I212F), a missense variant located within the third intracellular loop; specifically, the variation is in the middle of a short stretch of residues that have been found to be critical for binding of arrestin and several other dopamine receptor-interacting proteins. The comparative actions between D2-WT and D2-I212F were analyzed in their effects on both the G proteinmediated pathway and the arrestin3 pathway. It was shown that the D2-I212F variant was only 34% as effective as D2-WT at recruiting arrestin3, a deficiency that is likely to have important behavioral consequences. Additionally, quinpirole stimulation of D2-I212F produced dosedependent inhibition of cAMP accumulation; it was observed that lower concentrations of quinpirole were able to fully activate the rare variant. To further investigate whether this missense variant causes movement disorders, a condition that mimics the heterozygosity (D2-WT/D2-I212F), for the SNP affected patients have, was utilized and tested. Co-transfection of D2-WT and D2-I212F trended toward a shift in the EC50, where D2-WT/D2-I212F were different from D2-WT expressed alone, but the difference was not statistically different. This variant is likely to have decreased arrestin-mediated signaling and enhanced G protein-mediated signaling, so behaviors and cellular functions mediated by those two pathways will be altered according to patients with this variant. 3

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Using Bioluminescence Resonance Energy Transfer (BRET) to Characterize Agonist‐Induced Arrestin Recruitment to Modified and Unmodified G Protein‐Coupled Receptors

Cited by 51 publications

References 36 publications

Optical Control of Dopamine Receptors Using a Photoswitchable Tethered Inverse Agonist

Optical Control of Dopamine Receptors Using a Photoswitchable Tethered Inverse Agonist

A novel luminescence-based β-arrestin membrane recruitment assay for unmodified GPCRs

BRET Assays to Determine Altered Function of a D2DR Variant in G protein-Independent and -Dependent Pathways

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