Arrestin Expression in <i>E. coli</i> and Purification

Vishnivetskiy, Sergey A.; Zhan, Xiaoli; Chen, Qiuyan; Iverson, T.M.; Gurevich, Vsevolod V.

doi:10.1002/0471141755.ph0211s67

Cited by 23 publications

(27 citation statements)

References 53 publications

(115 reference statements)

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“…Arrestin-3 (1-393) was expressed and purified as described (91)(92)(93)(94)(95) followed by an additional purification step on a Superdex S200 Increase 10/300 GL column (GE Life Sciences, Pittsburgh, PA) equilibrated in 20 mM MOPS, pH 7.5, 150 mM NaCl, and 2 mM TCEP. Protein purity was assessed by SDS-PAGE with Coomassie staining.…”

Section: Arrestin-3-(1-393) Expression and Purificationmentioning

confidence: 99%

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Kaya

Perry

Gurevich

et al. 2020

Preprint

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Agonist-activated G protein-coupled receptors (GPCRs) must correctly select from hundreds of potential downstream signaling cascades and effectors. To accomplish this, GPCRs first bind to an intermediary signaling protein, such as G protein or arrestin. These intermediaries initiate signaling cascades that promote the activity of different effectors, including several protein kinases. The relative roles of G proteins versus arrestins in initiating and directing signaling is hotly debated, and it remains unclear how the correct final signaling pathway is chosen given the ready availability of protein partners. Here, we begin to deconvolute the process of signal bias from the dopamine D1 receptor (D1R) by exploring factors that promote the activation of ERK1/2 or Src, the kinases that lead to cell growth and proliferation. We found that ERK1/2 activation involves both arrestin and Gas, while Src activation depends solely on arrestin. Interestingly, we found that the phosphorylation pattern influences both arrestin and Gas coupling, suggesting an additional way the cells regulate G protein signaling. The phosphorylation sites in the D1R intracellular loop 3 are particularly important for directing the binding of G protein versus arrestin and for selecting between the activation of ERK1/2 and Src. Collectively, these studies correlate functional outcomes with a physical basis for signaling bias and provide fundamental information on how GPCR signaling is directed. Significance Statement:The functional importance of receptor phosphorylation in GPCR regulation has been demonstrated. Over the past decade, the phospho-barcode concept was developed to explain the multidimensional nature of the arrestin-dependent signaling network downstream of GPCRs. Here, we used the dopamine-1 receptor (D1R) to explore the effect of receptor phosphorylation on G protein-dependent and arrestin-dependent ERK and Src activation. Our studies suggest that D1R intracellular loop-3 phosphorylation affects both G proteins and arrestins. Differential D1R phosphorylation can direct signaling toward ERK or Src activation. This implies that phosphorylation induces different conformations of receptor and/or bound arrestin to initiate or select different cellular signaling pathways. \body Although it is tempting to divide GPCR signaling into these two branches, G protein-dependent and arrestindependent, the nature of signaling in the cell is much more complex (reviewed in (1, 7, 8)). As a result, this classical paradigm does not fully explain the range of biological responses observed when a GPCR is activated. For example, agonist identity can affect the recruitment of different G-proteins or arrestins, which determines the selection of downstream effectors (9). In addition, some activated GPCRs may interact directly with non-canonical signaling partners, including Src-family kinases (10-21). There are also numerous studies suggesting a specific role for the nonvisual arrestins in receptor-independent activation of ERK1/2, AKT, JNK3, and NF-kB (6,(22...

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Section: Arrestin-3-(1-393) Expression and Purificationmentioning

confidence: 99%

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Kaya

Perry

Gurevich

et al. 2020

Preprint

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“…WT visual arrestin-1 from different species (bovine, mouse, and human) successfully folds even in E. coli , which has fewer chaperones than eukaryotic cells and can be purified from expressing bacteria in large quantities. 25 , 26 Therefore, next we compared the expression of human arrestin-1 C147F mutant with the WT human and bovine proteins in E. coli , as well as the soluble fraction of these proteins ( Fig. 2 C).…”

Section: Resultsmentioning

confidence: 99%

“… 25 Test expression in 3 mL bacterial culture, cell lysis, and separation of soluble protein from cell debris were performed, as described. 26 In vitro transcription, translation, preparation of phosphorylated and unphosphorylated rhodopsin were performed as described recently. 27 , 28 …”

Section: Methodsmentioning

confidence: 99%

“…The protein was subjected to SDS-PAGE in 10% (GAPDH and arrestin) or 8% (GRP78/BiP) acrylamide gel and transferred to PVDF membrane. The proteins were visualized using the following primary antibodies: anti-GAPDH (loading control, mouse, 1:1000), anti-arrestin (F431 rabbit polyclonal, 26 1:5000), and anti-BIP (rabbit polyclonal, 1:1000 dilution; Cell Signaling, Danvers, MA, USA). Blots were incubated with primary antibodies overnight at 4°C, washed three times with TBS-0.1% Tween-20, then incubated with corresponding HRP-conjugated secondary antibodies (1:5000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).…”

Section: Methodsmentioning

confidence: 99%

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Molecular Defects of the Disease-Causing Human Arrestin-1 C147F Mutant

Vishnivetskiy

Sullivan

Bowne

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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PurposeThe purpose of this study was to identify the molecular defect in the disease-causing human arrestin-1 C147F mutant.MethodsThe binding of wild-type (WT) human arrestin-1 and several mutants with substitutions in position 147 (including C147F, which causes dominant retinitis pigmentosa in humans) to phosphorylated and unphosphorylated light-activated rhodopsin was determined. Thermal stability of WT and mutant human arrestin-1, as well as unfolded protein response in 661W cells, were also evaluated.ResultsWT human arrestin-1 was selective for phosphorylated light-activated rhodopsin. Substitutions of Cys-147 with smaller side chain residues, Ala or Val, did not substantially affect binding selectivity, whereas residues with bulky side chains in the position 147 (Ile, Leu, and disease-causing Phe) greatly increased the binding to unphosphorylated rhodopsin. Functional survival of mutant proteins with bulky substitutions at physiological and elevated temperature was also compromised. C147F mutant induced unfolded protein response in cultured cells.ConclusionsBulky Phe substitution of Cys-147 in human arrestin-1 likely causes rod degeneration due to reduced stability of the protein, which induces unfolded protein response in expressing cells.

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“…To assess whether the differences in downstream signaling of TRV027 and SII could result from differential conformational rearrangements of β-arrestin as previously shown for other AT 1 receptor ligands (Shukla et al, 2008 ; Zimmerman et al, 2012 ; Gurevich and Gurevich, 2014 ), we used a BRET-based biosensor that monitors the conformational rearrangement of β-arrestins by measuring intra-molecular BRET between RLucII and GFP 10 fused to the N- and C-termini of β-arrestins respectively (RlucII-βarrestin-GFP 10 ) (Charest et al, 2005 ). Using this approach, the amplitude of BRET changes provides an indirect reflection of the conformational rearrangement of β-arrestin upon its recruitment to the receptor promoted by a given ligand.…”

Section: Resultsmentioning

confidence: 99%

Comparative analyses of downstream signal transduction targets modulated after activation of the AT1 receptor by two β-arrestin-biased agonists

et al. 2015

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G protein-coupled receptors (GPCRs) are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with β-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or β-arrestin pathways. The angiotensin II (AngII) AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known β-arrestin-biased agonists, such as [Sar1, Ile4, Ile8]-AngII (SII), and [Sar1, D-Ala8]-AngII (TRV027). The aim of this study was to comparatively analyze the two above mentioned β-arrestin-biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between “G protein-dependent” and “β-arrestin-dependent” signaling. We observed clusters of activation profiles common to AngII, SII, and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of β-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization “G protein-dependent” vs. “β-arrestin-dependent” signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with distinct side effects.

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Arrestin Expression in E. coli and Purification

Cited by 23 publications

References 53 publications

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Molecular Defects of the Disease-Causing Human Arrestin-1 C147F Mutant

Comparative analyses of downstream signal transduction targets modulated after activation of the AT1 receptor by two β-arrestin-biased agonists

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