ABSTRACT G-protein-coupled receptors (GPCRs) can initiate unique functional responses depending on the subcellular site of activation. Efforts to uncover the mechanistic basis of

compartmentalized GPCR signaling have concentrated on the biochemical aspect of this regulation. Here we assess the biophysical positioning of receptor-containing endosomes as an alternative

salient mechanism. We devise a strategy to rapidly and selectively redistribute receptor-containing endosomes ‘on command’ in intact cells without perturbing their biochemical composition.

Next, we present two complementary optical readouts that enable robust measurements of bulk- and gene-specific GPCR/cyclic AMP (cAMP)-dependent transcriptional signaling with single-cell

identifying endosome positioning as the principal mediator of spatially selective receptor signaling. Access through your institution Buy or subscribe This is a preview of subscription

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* Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS MOLECULAR MECHANISM OF GPCR SPATIAL ORGANIZATION AT THE PLASMA

MEMBRANE Article 17 July 2023 RAPID WHOLE CELL IMAGING REVEALS A CALCIUM-APPL1-DYNEIN NEXUS THAT REGULATES COHORT TRAFFICKING OF STIMULATED EGF RECEPTORS Article Open access 17 February 2021

SPATIAL DECODING OF ENDOSOMAL CAMP SIGNALS BY A METASTABLE CYTOPLASMIC PKA NETWORK Article 01 March 2021 DATA AVAILABILITY All relevant data supporting the findings are available within the

paper and the Supplementary Data. Source data are provided with the manuscript. Additional information and reagents are available from the corresponding author upon reasonable request.

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PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank members of the Tsvetanova Lab and R. Irannejad (UCSF) for valuable discussions of the project and

feedback on the manuscript. Immunofluorescence microscopy imaging was performed using resources from the Duke Light Microscopy Core Facility, with specific training under L. Cameron, Y. Gao

and B. Carlson. PKARegIIB-mCherry and EGFP-SNX27 were gifts from R. Irannejad (UCSF). SSF-β2-AR, RAB11a-GFP, DynK44E-mCherry, pcDNA3.0, eGFP-C1 and Endo-bPAC were gifts from M. von Zastrow

(UCSF). MK1200 and dCas9-BFP-KRAB were gifts from M. Kampmann (UCSF). pBa.Kif1a 1-396.GFP was a gift from G. Banker and M. Bentley (Addgene plasmid, 45058; http://n2t.net/addgene:45058;

RRID:Addgene_45058). pBa-KIF5C 559-tdTomato-FKBP was a gift from G. Banker and M. Bentley (Addgene plasmid, 64211; http://n2t.net/addgene:64211; RRID:Addgene_64211). pEGFP-FRB was a gift

from K. Hahn (Addgene plasmid, 25919; http://n2t.net/addgene:25919; RRID:Addgene_25919). GFP-EEA1 wt was a gift from S. Corvera (Addgene plasmid, 42307; http://n2t.net/addgene:42307;

RRID:Addgene_42307). pmCherry-N1-GalT was a gift from L. Lu (Addgene plasmid, 87327; http://n2t.net/addgene:87327; RRID:Addgene_87327). Flamindo2 and nlsFlamindo2 were gifts from T.

Kitaguchi (Addgene plasmid, 73938; http://n2t.net/addgene:73938; RRID:Addgene_73938 and Addgene plasmid, 73939; http://n2t.net/addgene:73939; RRID:Addgene_73939). Figures 1a and 6f were

created using BioRender. Research reported in this publication was supported by the National Institutes of Health (R01NS127847 and R35GM142640 to N.G.T., R01DK073368 to J.Z. and F31NS120567

to B.K.A.W.) and the American Heart Association (Predoctoral Fellowship 834472 to J.F.Z.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Pharmacology and Cancer Biology, Duke

University, Durham, NC, USA Blair K. A. Willette & Nikoleta G. Tsvetanova * Department of Pharmacology, University of California San Diego, La Jolla, CA, USA Jin-Fan Zhang & Jin

Zhang * Department of Bioengineering, University of California San Diego, La Jolla, CA, USA Jin-Fan Zhang & Jin Zhang * Department of Chemistry and Biochemistry, University of California

San Diego, La Jolla, CA, USA Jin Zhang Authors * Blair K. A. Willette View author publications You can also search for this author inPubMed Google Scholar * Jin-Fan Zhang View author

publications You can also search for this author inPubMed Google Scholar * Jin Zhang View author publications You can also search for this author inPubMed Google Scholar * Nikoleta G.

Tsvetanova View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS N.G.T. supervised the project. N.G.T. and B.K.A.W. conceived the project and

designed experiments. B.K.A.W. and J.F.Z. performed and analyzed all experiments. J.Z. supervised and coordinated experiments involving the generation and characterization of nuclear

ExRai-AKAR2 sensor. N.G.T., B.K.A.W. and J.F.Z. interpreted results. N.G.T. and B.K.A.W. wrote the manuscript. All authors edited the manuscript. CORRESPONDING AUTHOR Correspondence to

Nikoleta G. Tsvetanova. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Chemical Biology_ thanks Davide

Calebiro and the other, anonymous, reviewers for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–14 and uncropped western blot for

Supplementary Fig. 8c. REPORTING SUMMARY SUPPLEMENTARY DATA Supporting data for Supplementary Figs. 1–14. SUPPLEMENTARY VIDEO 1 Live-cell imaging of HEK293 cells expressing CID components.

The video shows endosome distribution after 5 min of AP21967 (rapalog) treatment. SUPPLEMENTARY VIDEO 2 Live-cell imaging of HEK293 cells expressing Flamindo2-FRB-EEA1 and

Kif1a-tdTomato-FKBP. Cells were pretreated with AP21967 (rapalog) for 30 min. SOURCE DATA SOURCE DATA FIG. 1 Numerical source data and image source data for Fig. 1. SOURCE DATA FIG. 2

Numerical source data and image source data for Fig. 2. SOURCE DATA FIG. 3 Numerical source data and image source data for Fig. 3. SOURCE DATA FIG. 4 Numerical source data and image source

data for Fig. 4. SOURCE DATA FIG. 5 Numerical source data and image source data for Fig. 5. SOURCE DATA FIG. 6 Numerical source data for Fig. 6. RIGHTS AND PERMISSIONS Springer Nature or its

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Willette, B.K.A., Zhang, JF., Zhang, J. _et al._ Endosome positioning coordinates spatially selective GPCR signaling. _Nat Chem Biol_ 20, 151–161 (2024).

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