Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling

Verena A. Bachmann, Johanna E. Mayrhofer, Ronit Ilouz, Philipp Tschaikner, Philipp Raffeiner, Ruth Röck, Mathieu Courcelles, Federico Apelt, Tsan Wen Lu, George S. Baillie, Pierre Thibault, Pia Aanstad, Ulrich Stelzl, Susan S. Taylor, Eduard Stefan

Research output: Contribution to journalArticlepeer-review

79 Scopus citations

Abstract

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

Original languageEnglish
Pages (from-to)7786-7791
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number28
DOIs
StatePublished - 12 Jul 2016
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2016, National Academy of Sciences. All rights reserved.

Funding

We thank Jim Millonig for providing the Gpr161 cDNAs for cloning, Adi Sandbichler for the help with the imaging platform, Ruth MacLeod for the peptide spotting, Sonja Geisler and Andrea Schraffl for technical support, and Gabi Reiter for management support. This work was supported by Austrian Science Fund Grants P22608, P27606, and SFB-F44 (to E.S.). S.S.T. was funded by NIH Grant DK54441. Proteomics analyses were performed by the Center for Advanced Proteomic Analyses (CAPA), a Node of the Canadian Genomic Innovation Network supported by the Canadian government through Genome Canada.

FundersFunder number
Canadian government through Genome Canada
Center for Advanced Proteomic Analyses
National Institutes of Health
National Institute of Diabetes and Digestive and Kidney DiseasesP01DK054441
Medical Research CouncilMR/J007412/1
Austrian Science FundP22608, SFB-F44, P27606

    Keywords

    • Interaction network
    • Molecular interactions
    • Phosphorylation
    • Primary cilium
    • Scaffolding function

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