Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9

Luttrell, Louis M.; Wang, Jialu; Plouffe, Bianca; Smith, Jeffrey S.; Yamani, Lama; Kaur, Suneet; Jean-Charles, Pierre-Yves; Gauthier, Christophe; Lee, Mi-Hye; Pani, Biswaranjan; Kim, Jihee; Ahn, Seungkirl; Rajagopal, Sudarshan; Reiter, Eric; Bouvier, Michel; Shenoy, Sudha K.; Laporte, Stéphane A.; Rockman, Howard A.; Lefkowitz, Robert J.

doi:10.1126/scisignal.aat7650

Cited by 192 publications

(243 citation statements)

References 111 publications

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“…However, insight in the dependence of this recruitment on phosphorylation requires additional experiments. ß-arrestins constitute important regulators of GPCR function, whose central role is to desensitize G protein-mediated signaling (Alvarez-Curto et al 2016, Grundmann et al 2018, Luttrell et al 2018, reviewed in Gurevich & Gurevich 2019. At the same time, ß-arrestins can scaffold various downstream effectors and were shown to have a stimulatory effect on the selected aspects of GPCR signaling (Jean-Charles et al 2017, Grundmann et al 2018.…”

Section: Discussionmentioning

confidence: 99%

Seeing β-arrestin in action: The role of β-arrestins in Histamine 1 Receptor signaling

Pietraszewska-Bogiel

Goedhart

2019

Preprint

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ß-arrestins regulate G protein-coupled receptor functions by influencing their signaling activity and intracellular location. Histamine is a major chemical mediator of allergic reactions, and its action is mainly mediated by the G q/11 -and G i -coupled H1R.Contrary to accumulating insights into G protein-mediated signaling downstream of H1R, very little is known about the function of ß-arrestins in H1R signaling. Here, we describe dynamic, live cell measurements of ß-arrestin recruitment upon H1R activation in HEK293TN cells. Our observations classify H1R as a class A receptor, undergoing transient interactions with ß-arrestin. To investigate the relative contributions of G proteins and ß-arrestins to H1R signaling, we use specific G protein inhibitors, as well as ß-arrestin overexpression and depletion, and quantify various signaling outcomes in a panel of dynamic, live cell biosensor assays. Overall, we link ß-arrestins to desensitization of H1R-mediated signaling and show that ERK activation downstream of endogenous (HeLa, HUVEC) or transiently expressed (HEK293TN) H1R is largely G q -mediated.

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Section: Discussionmentioning

confidence: 99%

Seeing β-arrestin in action: The role of β-arrestins in Histamine 1 Receptor signaling

Pietraszewska-Bogiel

Goedhart

2019

Preprint

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“…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)(23)(24)(25)(26). Complexity of the signaling process is further increased by cross-talk between the signaling pathways initiated by G proteins and arrestins (27).…”

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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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“…In this case, the increase in ERK activation is seen in the mutants which also showed reduction in b-arrestin recruitment when activated with SLIGRL-NH2 further supporting the ideas the ERK activation here is predominantly occurring through a G-proteinmediated pathway. Overall, mechanisms regulating ERK activation downstream of PAR2 are likely agonist and context specific as reported for other GPCRs (53,54) and warrant further study.…”

Section: Discussionmentioning

confidence: 86%

Role of the C-terminal tail in regulating Proteinase Activated Receptor 2 (PAR2) signalling

Thibeault

Ramachandran

2020

Preprint

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The C-terminal tail of G-proteincoupled receptors contain important regulatory sites that enable interaction with intracellular signalling effectors. Here we examine the relative contribution of the C-tail serine/threonine phosphorylation sites (Ser 383-385 , Ser 387 -Thr 392 ) and the helix-8 palmitoylation site (Cys 361 ) in signalling regulation downstream of the proteolytically-activated GPCR, PAR2. We examined Gaq/11-coupled calcium signalling, b-arrestin-1/-2 recruitment, and MAPK activation (p44/42 phosphorylation) by wild-type and mutant receptors expressed in a CRISPR/Cas9 PAR2knockout HEK-293 cell background. We find that alanine substitution of the membrane proximal serine residues (Ser 383-385 Ala) had no effect on SLIGRL-NH2-or trypsin-stimulated b-arrestin recruitment. Alanine substitutions in the Ser 387 -Thr 392 cluster resulted in a large (~50%) decrease in b-arrestin-1/2 recruitment triggered by the activating peptide, SLIGRL-NH2, but was without effect on trypsinactivated b-arrestin-1/-2 recruitment. Additionally, we find that alanine substitution of the helix-8 cysteine residue (Cys 361 Ala) led to a (~50%) decrease in b-arrestin-1/-2 recruitment in response to both trypsin and SLIGRL-NH2. We further show that Gaq/11 inhibition with YM254890, inhibited ERK phosphorylation by PAR2 agonists, while genetic deletion of b-arrestin-1/-2 by CRISPR/Cas9 enhanced MAPK activation. Knockout of b-arrestins also enhanced Gaq/11mediated calcium signalling. In line with these findings, C-tail serine/threonine and cysteine

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Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9

Cited by 192 publications

References 111 publications

Seeing β-arrestin in action: The role of β-arrestins in Histamine 1 Receptor signaling

Seeing β-arrestin in action: The role of β-arrestins in Histamine 1 Receptor signaling

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Role of the C-terminal tail in regulating Proteinase Activated Receptor 2 (PAR2) signalling

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