Spatiotemporally resolved GPCR interactome uncovers unique mediators of receptor agonism

Maria M Shchepinova; Rachel Richardson; Jack W Houghton; Abigail R Walker; Mohammed A Safar; Daniel Conole; Aylin C Hanyaloglu; Edward W Tate

doi:10.1016/j.chembiol.2025.04.006

Spatiotemporally resolved GPCR interactome uncovers unique mediators of receptor agonism

Maria M Shchepinova, Rachel Richardson, Jack W Houghton, Abigail R Walker, Mohammed A Safar, Daniel Conole, Aylin C Hanyaloglu, Edward W Tate

Department of Chemistry

Imperial College London

Research output: Contribution to journal › Article › peer-review

1 Citation (SciVal)

Abstract

Cellular signaling by membrane G protein-coupled receptors (GPCRs) is governed by a complex and diverse array of mechanisms. The dynamics of a GPCR interactome, as it evolves over time and space in response to an agonist, provide a unique perspective on pleiotropic signaling decoding and functional selectivity at the cellular level. In this study, we utilized proximity-based APEX2 proteomics to investigate the interaction network of the luteinizing hormone receptor (LHR) on a minute-to-minute timescale. We developed an analytical approach that integrates quantitative multiplexed proteomics with temporal reference profiles, creating a platform to identify the proteomic environment of APEX2-tagged LHR at the nanometer scale. LHR activity is finely regulated spatially, leading to the identification of putative interactors, including the Ras-related GTPase RAP2B, which modulate both receptor signaling and post-endocytic trafficking. This work provides a valuable resource for spatiotemporal nanodomain mapping of LHR interactors across subcellular compartments.

Original language	English
Pages (from-to)	722-735.e7
Journal	Cell Chemical Biology
Volume	32
Issue number	5
DOIs	https://doi.org/10.1016/j.chembiol.2025.04.006
Publication status	Published - 15 May 2025

Data Availability Statement

-The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD053246.
-This paper does not report original code.
-Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgements

Schematics were created in BioRender.com. We would like to thank Prof. Mark von Zastrow (Univ. California, San Francisco, USA), Prof Gunther Schmalzing (RWTH Aachen Univ., Germany), Prof Ali Tavassoli (Univ. Southampton, UK), and Dr Kim Jonas (King’s College London, UK) for providing certain plasmids. We would like to thank Shabbir Ahmed (Leica Microsystems) and Tracey Williams (Leica Microsystems) for access and assistance with Mica (Zeiss) imaging. We would like to thank Tate group members for assistance and insightful discussions and Erika Bernardini for help with cell culture. We would like to acknowledge Dr Roman Fedoryshchak for his assistance with confocal microscopy and critical reading of this manuscript.

Funding

We acknowledge funding support from the Laboratory for Synthetic Chemistry and Chemical Biology under the Health@InnoHK Program of The Government of Hong Kong Special Administrative Region of the People’s Republic of China and the Biotechnology and Biological Sciences Research Council (BBSRC), grants BB/S001565/1 and BB/V006142/1.

Funders	Funder number
Biotechnology and Biological Sciences Research Council	BB/S001565/1 , BB/V006142/1

Keywords

GPCRs
APEX2
Luteinizing hormone
Proteomics
Signal Transduction
Receptors, G-Protein-Coupled/metabolism
Proximity proteomics
Interactomics
GPCR signalling

ASJC Scopus subject areas

Biochemistry, Genetics and Molecular Biology(all)

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Cite this

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title = "Spatiotemporally resolved GPCR interactome uncovers unique mediators of receptor agonism",

abstract = "Cellular signaling by membrane G protein-coupled receptors (GPCRs) is governed by a complex and diverse array of mechanisms. The dynamics of a GPCR interactome, as it evolves over time and space in response to an agonist, provide a unique perspective on pleiotropic signaling decoding and functional selectivity at the cellular level. In this study, we utilized proximity-based APEX2 proteomics to investigate the interaction network of the luteinizing hormone receptor (LHR) on a minute-to-minute timescale. We developed an analytical approach that integrates quantitative multiplexed proteomics with temporal reference profiles, creating a platform to identify the proteomic environment of APEX2-tagged LHR at the nanometer scale. LHR activity is finely regulated spatially, leading to the identification of putative interactors, including the Ras-related GTPase RAP2B, which modulate both receptor signaling and post-endocytic trafficking. This work provides a valuable resource for spatiotemporal nanodomain mapping of LHR interactors across subcellular compartments.",

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author = "Shchepinova, {Maria M} and Rachel Richardson and Houghton, {Jack W} and Walker, {Abigail R} and Safar, {Mohammed A} and Daniel Conole and Hanyaloglu, {Aylin C} and Tate, {Edward W}",

year = "2025",

month = may,

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doi = "10.1016/j.chembiol.2025.04.006",

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AU - Shchepinova, Maria M

AU - Richardson, Rachel

AU - Houghton, Jack W

AU - Walker, Abigail R

AU - Safar, Mohammed A

AU - Conole, Daniel

AU - Hanyaloglu, Aylin C

AU - Tate, Edward W

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AB - Cellular signaling by membrane G protein-coupled receptors (GPCRs) is governed by a complex and diverse array of mechanisms. The dynamics of a GPCR interactome, as it evolves over time and space in response to an agonist, provide a unique perspective on pleiotropic signaling decoding and functional selectivity at the cellular level. In this study, we utilized proximity-based APEX2 proteomics to investigate the interaction network of the luteinizing hormone receptor (LHR) on a minute-to-minute timescale. We developed an analytical approach that integrates quantitative multiplexed proteomics with temporal reference profiles, creating a platform to identify the proteomic environment of APEX2-tagged LHR at the nanometer scale. LHR activity is finely regulated spatially, leading to the identification of putative interactors, including the Ras-related GTPase RAP2B, which modulate both receptor signaling and post-endocytic trafficking. This work provides a valuable resource for spatiotemporal nanodomain mapping of LHR interactors across subcellular compartments.

KW - GPCRs

KW - APEX2

KW - Luteinizing hormone

KW - Proteomics

KW - Signal Transduction

KW - Receptors, G-Protein-Coupled/metabolism

KW - Proximity proteomics

KW - Interactomics

KW - GPCR signalling

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DO - 10.1016/j.chembiol.2025.04.006

M3 - Article

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SN - 2451-9456

VL - 32

SP - 722-735.e7

JO - Cell Chemical Biology

JF - Cell Chemical Biology

IS - 5

ER -

Spatiotemporally resolved GPCR interactome uncovers unique mediators of receptor agonism

Abstract

Data Availability Statement

Acknowledgements

Funding

Keywords

ASJC Scopus subject areas

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