Endosome positioning coordinates spatially selective GPCR signaling

Willette, Blair K. A.; Tsvetanova, Nikoleta G.

doi:10.1101/2022.07.26.501572

Cited by 4 publications

(3 citation statements)

References 49 publications

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“…(Figure 4). GPCRs influence cellular functioning in large part through transcriptional outputs, which has been shown to be related to the subcellular localization of signaling (Willette et al, 2023). While GPCR activation can trigger immediate and transient cellular responses via signaling involving intracellular second messengers, the process of transcription is more intricate, complex, and slower in nature.…”

Section: Discussionmentioning

confidence: 99%

Semi‐synthetic nanobody‐ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor‐1

Braga Emidio,

Cheloha

2024

Protein Science

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Antibodies have proven highly valuable for therapeutic development; however, they are typically poor candidates for applications that require activation of G protein‐coupled receptors (GPCRs), the largest collection of targets for clinically approved drugs. Nanobodies (Nbs), the smallest antibody fragments retaining full antigen‐binding capacity, have emerged as promising tools for pharmacologic applications, including GPCR modulation. Past work has shown that conjugation of Nbs with ligands can provide GPCR agonists that exhibit improved activity and selectivity compared to their parent ligands. The neurokinin‐1 receptor (NK1R), a GPCR targeted for the treatment of pain, is activated by peptide agonists such as Substance P (SP) and neurokinin A (NKA), which induce signaling through multiple pathways (Gs, Gq and β‐arrestin). In this study, we investigated whether conjugating NK1R ligands with Nbs that bind to a separate location on the receptor would provide chimeric compounds with distinctive signaling properties. We employed sortase A‐mediated ligation to generate several conjugates consisting of Nbs linked to NK1R ligands. Many of these conjugates exhibited divergent and unexpected signaling properties and transcriptional outputs. For example, some Nb‐NKA conjugates showed enhanced receptor binding capacity, high potency partial agonism, prolonged cAMP production, and an increase in transcriptional output associated with Gs signaling; whereas other conjugates were virtually inactive. Nanobody conjugation caused only minor alterations in ligand‐induced upstream Gq signaling with unexpected enhancements in transcriptional (downstream) responses. Our findings underscore the potential of nanobody conjugation for providing compounds with advantageous properties such as biased agonism, prolonged duration of action, and enhanced transcriptional responses. These compounds hold promise not only for facilitating fundamental research on GPCR signal transduction mechanisms but also for the development of more potent and enduring therapeutics.This article is protected by copyright. All rights reserved.

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Section: Discussionmentioning

confidence: 99%

Semi‐synthetic nanobody‐ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor‐1

Braga Emidio,

Cheloha

2024

Protein Science

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“…2h-i), which are expected to abolish compartmentalized signaling. 17,43,44 Future studies that apply cAMP and PKA biosensors targeted to distinct subcellular organelles would be invaluable in further defining local signaling in RBM12 knockouts.…”

Section: Discussionmentioning

confidence: 99%

The psychosis risk factorRBM12encodes a novel repressor of GPCR/cAMP signal transduction

Semesta

Garces

Tsvetanova

2023

Preprint

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RBM12 is a high-penetrance risk factor for familial schizophrenia and psychosis, yet its precise cellular functions and the pathways to which it belongs are not known. We utilize two complementary models, HEK293 cells and human iPSC-derived neurons, and delineate RBM12 as a novel repressor of the G protein-coupled receptor/cyclic AMP/protein kinase A (GPCR/cAMP/PKA) signaling axis. We establish that loss of RBM12 leads to hyperactive cAMP production and increased PKA activity as well as altered neuronal transcriptional responses to GPCR stimulation. Notably, the cAMP and transcriptional signaling steps are subject to discrete RBM12-dependent regulation. We further demonstrate that the two RBM12 truncating variants linked to familial psychosis impact this interplay, as the mutants fail to rescue GPCR/cAMP signaling hyperactivity in cells depleted of RBM12. Lastly, we present a mechanism underlying the impaired signaling phenotypes. In agreement with its activity as an RNA-binding protein, loss of RBM12 leads to altered gene expression, including that of multiple effectors of established significance within the receptor pathway. Specifically, the abundance of adenylyl cyclases, phosphodiesterase isoforms, and PKA regulatory and catalytic subunits is impacted by RBM12 depletion. We note that these expression changes are fully consistent with the entire gamut of hyperactive signaling outputs. In summary, the current study identifies a previously unappreciated role for RBM12 in the context of the GPCR/cAMP pathway that could be explored further as a tentative molecular mechanism underlying the functions of this factor in neuronal physiology and pathophysiology.

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“…Live cells are complex self-sustained machines that actively respond to different types of external and internal stimuli in different ways, including cell-type switching, senescence, proliferation, and death. 64 These responses may influence their delivery performances when used as delivery vehicles. 65 Therefore, the effects of FSS, which is a stimulus to which cells are constantly exposed in the blood, should be investigated.…”

mentioning

confidence: 99%

Reducing Cholesterol Level in Live Macrophages Improves Delivery Performance by Enhancing Blood Shear Stress Adaptation

Zhang,

Li,

et al. 2023

Nano Lett.

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In recent years, live-cell-based drug delivery systems have gained considerable attention. However, shear stress, which accompanies blood flow, may cause cell death and weaken the delivery performance. In this study, we found that reducing cholesterol in macrophage plasma membranes enhanced their tumor targeting ability by more than 2-fold. Our study demonstrates that the reduced cholesterol level deactivated the mammalian target of rapamycin (mTOR) and consequently promoted the nuclear translocation of transcription factor EB (TFEB), which in turn enhanced the expression of superoxide dismutase (SOD) to reduce reactive oxygen species (ROS) induced by shear stress. A proof-of-concept system using low cholesterol macrophages attached to MXene (e.g., l-RX) was fabricated. In a melanoma mouse model, l-RX and laser irradiation treatments eliminated tumors with no recurrences observed in mice. Therefore, cholesterol reduction is a simple and effective way to enhance the targeting performance of macrophage-based drug delivery systems.

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Endosome positioning coordinates spatially selective GPCR signaling

Cited by 4 publications

References 49 publications

Semi‐synthetic nanobody‐ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor‐1

Semi‐synthetic nanobody‐ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor‐1

The psychosis risk factorRBM12encodes a novel repressor of GPCR/cAMP signal transduction

Reducing Cholesterol Level in Live Macrophages Improves Delivery Performance by Enhancing Blood Shear Stress Adaptation

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