Structure of an endosomal signaling GPCR–G protein–β-arrestin megacomplex

Nguyen, Anthony H.; Thomsen, A.; Cahill, Thomas J.; Huang, Rick; Huang, Li; Marcink, Tara C.; Clarke, Oliver; Heissel, Søren; Masoudi, Ali; Ben-Hail, Danya; Samaan, Fadi; Dandey, Venkata P.; Tan, Yong Zi; Hong, Chuan; Mahoney, Jacob P.; Triest, Sarah; Little, John R.; Chen, Xin; Sunahara, Roger K.; Steyaert, Jan; Molina, Henrik; Yu, Zhiheng; Georges, Amédée des; Lefkowitz, Robert J.

doi:10.1038/s41594-019-0330-y

Cited by 149 publications

(146 citation statements)

References 62 publications

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“…There are multiple mechanisms by which this could occur. First, the D1R-arrestin-3-ERK1/2 signaling complex could directly incorporate Gs into a super-complex, as was described for a chimeric b2-adrenergic receptor containing the V2 vasopressin receptor C-terminus (63,64). Second, Gs could compete with arrestin-3 for binding to active D1R, resulting in a mixed population of D1R-Gs and D1R-arrestin-3 complexes with cross-talk enhancing ERK1/2 activation.…”

Section: Resultsmentioning

confidence: 97%

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: Resultsmentioning

confidence: 97%

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Kaya

Perry

Gurevich

et al. 2020

Preprint

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“…Indeed, it has been already been successful in helping to resolve a number of interesting new structures. Examples include a structure of FACT manipulating the nucleasome [19], multiple conformations of an ABC exporter [14], mTORC1 docked on the lysosome [24], the respiratory syncytial virus polymerase complex [9], a GPCR-G protein-β-arrestin megacomplex [20], and MERS-CoV/SARS-CoV with neutralizing antibodies [32]. An updated non-uniform refinement algorithm implementation, as described in this work, will be released in an upcoming version of cryoSPARC (www.cryosparc.com).…”

Section: Discussionmentioning

confidence: 99%

Non-uniform refinement: Adaptive regularization improves single particle cryo-EM reconstruction

Punjani

Zhang

Fleet

2019

Preprint

277

329

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Single particle cryo-EM is a powerful method for studying proteins and other biological macromolecules. Many of these molecules comprise regions with varying structural properties including disorder, flexibility, and partial occupancy. These traits make computational 3D reconstruction from 2D images challenging. Detergent micelles and lipid nanodiscs, used to keep membrane proteins in solution, are common examples of locally disordered structures that can negatively affect existing iterative refinement algorithms which assume rigidity (or spatial uniformity). We introduce a cross-validation approach to derive non-uniform refinement, an algorithm that automatically regularizes 3D density maps during iterative refinement to account for spatial variability, yielding dramatically improved resolution and 3D map quality. We find that in common iterative refinement methods, regularization using spatially uniform filtering operations can simultaneously over-and under-regularize local regions of a 3D map. In contrast, non-uniform refinement removes noise in disordered regions while retaining signal useful for aligning particle images. Our results include state-of-the-art resolution 3D reconstructions of multiple membrane proteins with molecular weight as low as 90kDa. These results demonstrate that higher resolutions and improved 3D density map quality can be achieved even for small membrane proteins, an important use case for single particle cryo-EM, both in structural biology and drug discovery. Non-uniform refinement is implemented in the cryoSPARC software package and has already been used successfully in several notable structural studies.

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“…The half map sphericity values of all the three cryo-EM maps in this study are all over 0.8, suggesting no concerns on the preferred orientation problem. Guided by the previously described procedure [53], the map-to-model sphericity values and 3D-FSCs were also calculated using Chimera [46], Relion 3 [54], and the 3D-FSC server at an FSC threshold of 0.5. The statistics of cryo-EM data collection, 3D reconstruction, and model refinement were shown in S1 Table. All the figures were created using Chimera [46].…”

Section: Structural Modelling and Refinementmentioning

confidence: 99%

Visualization of two architectures in class-II CAP-dependent transcription activation

et al. 2020

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Transcription activation by cyclic AMP (cAMP) receptor protein (CAP) is the classic paradigm of transcription regulation in bacteria. CAP was suggested to activate transcription on class-II promoters via a recruitment and isomerization mechanism. However, whether and how it modifies RNA polymerase (RNAP) to initiate transcription remains unclear. Here, we report cryo-electron microscopy (cryo-EM) structures of an intact Escherichia coli class-II CAP-dependent transcription activation complex (CAP-TAC) with and without de novo RNA transcript. The structures reveal two distinct architectures of TAC and raise the possibility that CAP binding may induce substantial conformational changes in all the subunits of RNAP and transiently widen the main cleft of RNAP to facilitate DNA promoter entering and formation of the initiation open complex. These structural changes vanish during further RNA transcript synthesis. The observations in this study may reveal a possible on-pathway intermediate and suggest a possibility that CAP activates transcription by inducing intermediate state, in addition to the previously proposed stabilization mechanism.

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Structure of an endosomal signaling GPCR–G protein–β-arrestin megacomplex

Cited by 149 publications

References 62 publications

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Non-uniform refinement: Adaptive regularization improves single particle cryo-EM reconstruction

Visualization of two architectures in class-II CAP-dependent transcription activation

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