G protein-regulated endocytic trafficking of adenylyl cyclase type 9

Lazar, André M; Irannejad, Roshanak; Baldwin, Tanya A.; Sundaram, Aparna; Gutkind, J. Silvio; lnoue, Asuka; Dessauer, Carmen W.; Zastrow, Mark von

doi:10.1101/2020.04.23.057026

Cited by 5 publications

(6 citation statements)

References 71 publications

(99 reference statements)

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“…Our results shed more light onto the signaling role of long-lived clathrin structures such as plaques by suggesting that they play an important role in regulating local cAMP dynamics. The sustained cAMP elevations within clathrin plaques are likely achieved by the exclusion or inhibition of PDEs and by the recruitment of ACs with the endocytosed receptor, consistent with previous reports of G s and AC cointernalization with GPCRs (44,50,60). Notably, our studies were performed in HEK293T cells, and the unique cAMP dynamics we observed within these clathrin structures may differ in other cell types depending on the expression of specific ACs isoforms and other cAMP effectors in this microdomain (61,62).…”

Section: Discussionsupporting

confidence: 90%

“…Local variations in cAMP accumulation within the cell can be controlled by the spatial organization of ACs and PDEs, which synthesize and degrade cAMP, respectively (22), and the compartmentalized cAMP dynamics observed using clathrin-targeted FluoSTEP-ICUE may be due to the differential distribution of these enzymes. For instance, AC3 and AC9 have been shown to undergo internalization after GPCR stimulation and traffic to endosomes containing the receptors (44,50). Thus, to test the role of these enzymes in regulating the sustained cAMP accumulation detected by clathrin-targeted FluoSTEP-ICUE, we treated isoproterenol-stimulated CLTA-FP11 cells expressing FluoSTEP-ICUE or PM-ICUE3 with IBMX (100 M) to acutely inhibit PDE activity or 2′,3′-dideoxyadenosine (ddAdo; 100 M) to acutely inhibit transmembrane AC activity.…”

Section: Transmembrane Acs Regulate Sustained Camp Production At Long-lived Clathrin Microdomains Following -Ar Stimulationmentioning

confidence: 99%

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FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains

et al. 2021

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Growing evidence suggests that many essential intracellular signaling events are compartmentalized within kinetically distinct microdomains in cells. Genetically encoded fluorescent biosensors are powerful tools to dissect compartmentalized signaling, but current approaches to probe these microdomains typically rely on biosensor fusion and overexpression of critical regulatory elements. Here, we present a novel class of biosensors named FluoSTEPs (fluorescent sensors targeted to endogenous proteins) that combine self-complementing split green fluorescent protein, CRISPR-mediated knock-in, and fluorescence resonance energy transfer biosensor technology to probe compartmentalized signaling dynamics in situ. We designed FluoSTEPs for simultaneously highlighting endogenous microdomains and reporting domain-specific, real-time signaling events including kinase activities, guanosine triphosphatase activation, and second messenger dynamics in live cells. A FluoSTEP for 3′,5′-cyclic adenosine monophosphate (cAMP) revealed distinct cAMP dynamics within clathrin microdomains in response to stimulation of G protein–coupled receptors, showcasing the utility of FluoSTEPs in probing spatiotemporal regulation within endogenous signaling architectures.

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Section: Discussionsupporting

confidence: 90%

Section: Transmembrane Acs Regulate Sustained Camp Production At Long-lived Clathrin Microdomains Following -Ar Stimulationmentioning

confidence: 99%

FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains

et al. 2021

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“…Much remains to be discovered regarding the role of adenylyl cyclase in sustained signaling. A recent study has shown that endosomal cAMP may potentially arise from only certain subtypes of adenylyl cyclases that have been selectively trafficked to endosomes [31]. Subtypes of adenylyl cyclases have been known to be differentially compartmentalized, and it is tempting to speculate that these subtypes may play a different role or lead to different cellular responses with respect to compartmentalized G protein signaling.…”

Section: Concluding Remarks and Perspectivementioning

confidence: 99%

Signaling at the endosome: cryo‐EM structure of a GPCR–G protein–beta‐arrestin megacomplex

Nguyen

Lefkowitz

2021

The FEBS Journal

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G-protein coupled receptors (GPCRs) are a large class of cell-surface receptor involved in cellular signaling that are currently the target of over one third of all clinically approved therapeutics. Classically, an agonist-bound, active GPCR couples to and activates G proteins through the receptor intracellular core. To attenuate G protein signaling, the GPCR is phosphorylated at its C-terminal tail and/or relevant intracellular loops, allowing for the recruitment of βarrestins (βarrs). βarrs then couple to the receptor intracellular core in order to mediate receptor desensitization and internalization. However, our lab and others have observed that some GPCRs are capable of continuously signaling through G protein even after internalization. This mode of sustained signaling stands in contrast with our previous understanding of GPCR signaling, and its molecular mechanism is still not well understood. Recently, we have solved the structure of a GPCR-G protein-βarr megacomplex by cryo-electron microscopy (cryo-EM). This 'megaplex' structure illustrates the independent and simultaneous coupling of a G protein to the receptor intracellular core, and binding of a βarr to a phosphorylated receptor C-terminal tail, with all three components maintaining their respective canonically active conformations. The structure provides evidence for the ability of a GPCR to activate G protein even while being bound to and internalized by βarr. It also reveals that the binding of G protein and βarr to the same GPCR is not mutually exclusive, and raises a number of future questions to be answered regarding the mechanism of sustained signaling.

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“…Termination of Gα s activity is believed to happen in seconds 29,30 and AC activity is strictly dependent on GTP-loaded Gα s . However, receptor inactivation is much slower and, in most cases, not complete: a small proportion of receptors is thought to recycle to the plasma membrane where they can become reactivated by the agonist and continue to signal 31 .…”

Section: Resultsmentioning

confidence: 99%

Dynamic FRET-FLIM based screens of signal transduction pathways: a feasibility study

Harkes

Kukk

Mukherjee

et al. 2021

Preprint

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Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protein-protein interactions and frequently used to record the outcome of signal transduction events. With new highly sensitive and photon efficient FLIM instrumentation, the technique also becomes attractive to screen, with high temporal resolution, for fast changes in Forster Resonance Energy Transfer (FRET), such as those occurring upon activation of cell signaling. We studied the effects of siRNA-mediated individual knockdown of an extensive set of 22 different phosphodiesterases (PDEs) on baseline levels and agonist-induced changes of the second messenger cAMP. Using HeLa cells stably expressing our FRET-FLIM sensor we imaged many hundreds of cells at 5 second intervals for each condition. Following segmentation of cells by the deep-learning implementation Cellpose, FLIM time traces were calculated and fitted for dynamic analysis with custom-made Python scripts. Taking advantage of the quantitative FLIM data, we found very limited effects of PDE knockdown on baseline and agonist-induced peak levels of cAMP. However, cAMP breakdown in the decay phase was significantly slower when PDE3A and, to a lesser amount, PDE10A were knocked down, identifying these isoforms as dominant in HeLa cells. In conclusion, we present a robust platform that combines photon-efficient FLIM instrumentation with systematic gene knockdown and an automated open-source analysis pipeline. Our quantitative platform provides detailed kinetic analysis of cellular signals in individual cells with unprecedented throughput.

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G protein-regulated endocytic trafficking of adenylyl cyclase type 9

Cited by 5 publications

References 71 publications

FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains

FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains

Signaling at the endosome: cryo‐EM structure of a GPCR–G protein–beta‐arrestin megacomplex

Dynamic FRET-FLIM based screens of signal transduction pathways: a feasibility study

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