Essential strategies for the detection of constitutive and ligand-dependent Gi-directed activity of 7TM receptors using bioluminescence resonance energy transfer DOI Creative Commons
Sofia Endzhievskaya, Kirti Kandhwal Chahal,

JULIE A. RESNICK

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 9, 2024

Abstract The constitutive (ligand-independent) signaling of G protein-coupled receptors (GPCRs) is being increasingly appreciated as an integral aspect their function; however, it can be technically hard to detect for poorly characterized, e.g. orphan, the cAMP-inhibitory Gi-coupled (GiPCR) family. In this study, we delineate optimal strategies detection such activity across several GiPCRs in two cell lines. As our study examples, chose canonical - constitutively active Smoothened and ligand-activated CXCR4,-and one atypical GPCRs, chemokine receptor ACKR3. We verified applicability three Bioluminescence Resonance Energy Transfer (BRET)-based assays measuring changes intracellular cAMP, another Gβγ/GRK3ct association third Gαi-Gβγ dissociation, assessing both ligand-modulated these receptors. also revealed possible caveats sources false positives, proposed optimization strategies. All types confirmed ligand-dependent CXCR4, controversial protein incompetence ACKR3, Gi-directed SMO, its modulation by PTCH1. demonstrated that PTCH1 promotes SMO localization surface, thus enhancing responsiveness not only agonists but antagonists, which a novel mechanism regulation Class F GiPCR Smoothened.

Language: Английский

Essential strategies for the detection of constitutive and ligand-dependent Gi-directed activity of 7TM receptors using bioluminescence resonance energy transfer DOI Creative Commons
Sofia Endzhievskaya, Kirti Kandhwal Chahal,

JULIE A. RESNICK

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 9, 2024

Abstract The constitutive (ligand-independent) signaling of G protein-coupled receptors (GPCRs) is being increasingly appreciated as an integral aspect their function; however, it can be technically hard to detect for poorly characterized, e.g. orphan, the cAMP-inhibitory Gi-coupled (GiPCR) family. In this study, we delineate optimal strategies detection such activity across several GiPCRs in two cell lines. As our study examples, chose canonical - constitutively active Smoothened and ligand-activated CXCR4,-and one atypical GPCRs, chemokine receptor ACKR3. We verified applicability three Bioluminescence Resonance Energy Transfer (BRET)-based assays measuring changes intracellular cAMP, another Gβγ/GRK3ct association third Gαi-Gβγ dissociation, assessing both ligand-modulated these receptors. also revealed possible caveats sources false positives, proposed optimization strategies. All types confirmed ligand-dependent CXCR4, controversial protein incompetence ACKR3, Gi-directed SMO, its modulation by PTCH1. demonstrated that PTCH1 promotes SMO localization surface, thus enhancing responsiveness not only agonists but antagonists, which a novel mechanism regulation Class F GiPCR Smoothened.

Language: Английский

Citations

0