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ER/K Linked GPCR‐G protein Fusions Systematically Modulate Second Messenger Response in Cells
Author(s) -
Dysthe Matthew,
Malik Rabia U,
Ritt Michael,
Sunahara Roger K,
Sivaramakrishnan Sivaraj
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.619.12
Subject(s) - g protein coupled receptor , förster resonance energy transfer , g protein , receptor , second messenger system , chemistry , signal transduction , microbiology and biotechnology , linker , fusion protein , agonist , biophysics , biology , biochemistry , fluorescence , gene , recombinant dna , physics , quantum mechanics , computer science , operating system
FRET or BRET approaches are well established for detecting ligand induced GPCR‐G protein interactions in cells. However, current FRET/BRET assays rely on co‐expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR‐G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the beta2‐, alpha1‐, and alpha2‐adrenergic receptors, and adenosine type 1 receptor (A 1 R), tethered to Gαs‐XL, Gαi 1 , or Gαq subunits. Agonist stimulation of beta2‐AR and alpha2‐AR increases FRET signal comparable to co‐expressed FRET/BRET sensors. SPASM sensors retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR‐G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist‐induced changes in GPCR‐G protein interactions, and linking these changes to downstream signaling. Support or Funding Information The research conducted here was funded by the American Heart Association Scientist Development Grant (13SDG14270009), the NIH (1DP2 CA186752‐01, 1‐R01‐GM‐105646‐01‐A1) to S.S. and by the American Heart Association Pre‐doctoral Fellowship (14PRE18560010) to R.U.M.