Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells

2019 ◽  
Vol 14 (6) ◽  
pp. 1141-1149 ◽  
Author(s):  
Robert Serfling ◽  
Lisa Seidel ◽  
Andreas Bock ◽  
Martin J. Lohse ◽  
Paolo Annibale ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
Bioorthogonal Labeling ◽  
G Protein Coupled

scholarly journals Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells

2017 ◽  
Vol 139 (38) ◽  
pp. 13376-13386 ◽  
Author(s):  
Carlo P. Ramil ◽  
Maoqing Dong ◽  
Peng An ◽  
Tracey M. Lewandowski ◽  
Zhipeng Yu ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
Bioorthogonal Labeling ◽  
Class B ◽  
G Protein Coupled ◽  
Tetrazine Ligation

scholarly journals Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells

2018 ◽  
Vol 293 (19) ◽  
pp. 7466-7473 ◽  
Author(s):  
Qingwen Wan ◽  
Najeah Okashah ◽  
Asuka Inoue ◽  
Rony Nehmé ◽  
Byron Carpenter ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
G Protein Coupled

scholarly journals The Ciliary Lumen Accommodates Passive Diffusion and Vesicle Trafficking in Cytoplasmic-Ciliary Transport

2019 ◽  
Author(s):  
Andrew Ruba ◽  
Wangxi Luo ◽  
Jingjie Yu ◽  
Daisuke Takao ◽  
Athanasios Evangelou ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
G Protein ◽  
Single Molecule ◽  
Primary Cilium ◽  
Intraflagellar Transport ◽  
Vesicle Transport ◽  
Passive Diffusion ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
G Protein Coupled

AbstractTransport of membrane and cytosolic proteins into the primary cilium is essential for its role in cellular signaling. Using single molecule microscopy, we mapped the movement of membrane and soluble proteins at the base of the primary cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary cilium - passive diffusional and vesicle transport routes - that are adopted by proteins for cytoplasmic-cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins.

CypHer 5: A Generic Approach for Measuring the Activation and Trafficking of G Protein-Coupled Receptors in Live Cells

2003 ◽  
Vol 1 (2) ◽  
pp. 251-259 ◽  
Author(s):  
Elaine J. Adie ◽  
Michael J. Francis ◽  
June Davies ◽  
Lynne Smith ◽  
Angela Marenghi ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
G Protein Coupled

scholarly journals Genetically encoded intrabody sensors report the interaction and trafficking of β-arrestin 1 upon activation of G-protein–coupled receptors

2020 ◽  
Vol 295 (30) ◽  
pp. 10153-10167 ◽  
Author(s):  
Mithu Baidya ◽  
Punita Kumari ◽  
Hemlata Dwivedi-Agnihotri ◽  
Shubhi Pandey ◽  
Badr Sokrat ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
Single Chain ◽  
Downstream Signaling ◽  
C Terminus ◽  
V2 Receptor ◽  
Intracellular Vesicles ◽  
G Protein Coupled

Agonist stimulation of G-protein–coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called β-arrestins (βarrs). The GPCR–βarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR–βarr complex formation can be used as a generic readout of GPCR and βarr activation. Although several methods are currently available to monitor GPCR–βarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated βarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR–βarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments and used them to monitor the localization and trafficking of βarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of βarr1, and the intrabodies co-localized with βarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report βarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for βarr1 recruitment and trafficking expands currently available approaches to visualize GPCR–βarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.

scholarly journals Label-Free Imaging of Histamine Mediated G Protein-Coupled Receptors Activation in Live Cells

2016 ◽  
Vol 88 (23) ◽  
pp. 11498-11503 ◽  
Author(s):  
Jin Lu ◽  
Yunze Yang ◽  
Wei Wang ◽  
Jinghong Li ◽  
Nongjian Tao ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
Label Free ◽  
G Protein Coupled
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