Discrete GPCR-triggered endocytic modes enable β-arrestins to flexibly regulate cell signaling
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- Evaluated articles (eLife)
- Cell Biology (eLife)
Abstract
β-arrestins are master regulators of cellular signaling that operate by desensitizing ligand-activated G protein-coupled receptors (GPCRs) at the plasma membrane and promoting their subsequent endocytosis. The endocytic activity of β-arrestins is ligand-dependent, triggered by GPCR binding, and increasingly recognized to have a multitude of downstream signaling and trafficking consequences that are specifically programmed by the bound GPCR. However, only one biochemical ‘mode’ for GPCR-mediated triggering of the endocytic activity is presently known– displacement of the β-arrestin C-terminus (CT) to expose CCP-binding determinants that are masked in the inactive state. Here we revise this view by uncovering a second mode of GPCR-triggered endocytic activity that is independent of the β-arrestin CT and, instead, requires the cytosolic base of the β-arrestin C-lobe (CLB). We further show each of the discrete endocytic modes is triggered in a receptor-specific manner, with GPCRs that bind β-arrestin transiently (‘class A’) primarily triggering the CLB-dependent mode and GPCRs that bind more stably (‘class B’) triggering both the CT and CLB-dependent modes in combination. Moreover, we show that each mode has opposing effects on the net signaling output of receptors– with the CLB-dependent mode promoting rapid signal desensitization and the CT-dependent mode enabling prolonged signaling. Together, these results fundamentally revise understanding of how β-arrestins operate as efficient endocytic adaptors while facilitating diversity and flexibility in the control of cell signaling.
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Author Response
Reviewer #1 (Public Review):
The authors' results revolutionize our understanding of the mechanism of arrestin-mediated GPCR internalization. They identified previously unknown elements on the non-receptor-binding side of arrestins participating in the process. The findings are ground-breaking and very important to the large field of GPCR signaling.
We are pleased that the reviewer appreciates the significance of our findings. We appreciate the important critiques and corrections, and have done our best to address them.
Reviewer #2 (Public Review):
This manuscript from the Von Zastrow laboratory proposes an additional site on Beta-arrestin2 (arrestin 3) to the well characterised Ctail (AP-2+clathrin binding) is responsible in significant part for the downregulation and likely onward signalling from endosomes of a …
Author Response
Reviewer #1 (Public Review):
The authors' results revolutionize our understanding of the mechanism of arrestin-mediated GPCR internalization. They identified previously unknown elements on the non-receptor-binding side of arrestins participating in the process. The findings are ground-breaking and very important to the large field of GPCR signaling.
We are pleased that the reviewer appreciates the significance of our findings. We appreciate the important critiques and corrections, and have done our best to address them.
Reviewer #2 (Public Review):
This manuscript from the Von Zastrow laboratory proposes an additional site on Beta-arrestin2 (arrestin 3) to the well characterised Ctail (AP-2+clathrin binding) is responsible in significant part for the downregulation and likely onward signalling from endosomes of a range of GPCRs. The cell biology appears to me to be thoroughly carried out and data presented in a statistically appropriate manner.
The conclusions made seem appropriate and justified although considerably more information could be extracted with little extra effort I think - including formerly proving that internalisation is by CME by using CME-specific CME inhibitors or inhibitory constructs.
The major weakness is the lack of mechanistic information, most specifically what does the Clobe bind to in order to allow Beta arrestin2 incorporation into CCVs?
The referencing of the relevant literature is sometimes careless or inappropriate, especially with respect to CME.
We are pleased that the reviewer found our conclusions generally appropriate and well-justified. The reviewer is correct that we presently do not know the interaction(s) responsible for CLB activity. We have addressed the reviewer’s critiques with new data and / or changes to the text as follows.
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Evaluation Summary:
This manuscript addresses G protein-coupled receptor signaling and proposes an additional site on Beta-arrestin2 (arrestin 3) as being responsible, in significant part, for the downregulation and likely onward signalling from endosomes of a range of GPCRs. The cell biology appears to be thoroughly carried out and data presented in a statistically appropriate manner. With some textual changes and minor experimental clarification of the route taken and molecules involved, this work will be of broad interest.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors' results revolutionize our understanding of the mechanism of arrestin-mediated GPCR internalization. They identified previously unknown elements on the non-receptor-binding side of arrestins participating in the process. The findings are ground-breaking and very important to the large field of GPCR signaling.
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Reviewer #2 (Public Review):
This manuscript from the Von Zastrow laboratory proposes an additional site on Beta-arrestin2 (arrestin 3) to the well characterised Ctail (AP-2+clathrin binding) is responsible in significant part for the downregulation and likely onward signalling from endosomes of a range of GPCRs. The cell biology appears to me to be thoroughly carried out and data presented in a statistically appropriate manner.
The conclusions made seem appropriate and justified although considerably more information could be extracted with little extra effort I think - including formerly proving that internalisation is by CME by using CME-specific CME inhibitors or inhibitory constructs.
The major weakness is the lack of mechanistic information, most specifically what does the Clobe bind to in order to allow Beta arrestin2 …
Reviewer #2 (Public Review):
This manuscript from the Von Zastrow laboratory proposes an additional site on Beta-arrestin2 (arrestin 3) to the well characterised Ctail (AP-2+clathrin binding) is responsible in significant part for the downregulation and likely onward signalling from endosomes of a range of GPCRs. The cell biology appears to me to be thoroughly carried out and data presented in a statistically appropriate manner.
The conclusions made seem appropriate and justified although considerably more information could be extracted with little extra effort I think - including formerly proving that internalisation is by CME by using CME-specific CME inhibitors or inhibitory constructs.
The major weakness is the lack of mechanistic information, most specifically what does the Clobe bind to in order to allow Beta arrestin2 incorporation into CCVs?
The referencing of the relevant literature is sometimes careless or inappropriate, especially with respect to CME. -
Reviewer #3 (Public Review):
Barsi-Rhyne reports a novel mode of engagement of beta arrestins as endocytic adaptors and associates this novel mode together with the previously known canonical mode to the regulation of endocytosis and signaling by class A versus class B receptors. The manuscript is very well written, very good to read, almost flawless, extremely interesting, and highly relevant to the GPCR field with very well-crafted figures and fantastic microscopy.
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