Signals from 800 G-protein-coupled receptors (GPCRs) to many SH3 domain-containing proteins (SH3-CPs) regulate important physiological functions. These GPCRs may share a common pathway by signaling to SH3-CPs via agonist-dependent arrestin recruitment rather than through direct interactions. In the present study, 19F-NMR and cellular studies revealed that downstream of GPCR activation engagement of the receptor-phospho-tail with arrestin allosterically regulates the specific conformational states and functional outcomes of remote β-arrestin 1 proline regions (PRs). The observed NMR chemical shifts of arrestin PRs were consistent with the intrinsic efficacy and specificity of SH3 domain recruitment, which was controlled by defined propagation pathways. Moreover, in vitro reconstitution experiments and biophysical results showed that the receptor–arrestin complex promoted SRC kinase activity through an allosteric mechanism. Thus, allosteric regulation of the conformational states of β-arrestin 1 PRs by GPCRs and the allosteric activation of downstream effectors by arrestin are two important mechanisms underlying GPCR-to-SH3-CP signaling.
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We thank D.-S. Li for stimulating discussions and critical reading of the manuscript. We thank S.-S. Zang and X-H. Liu of the Core Facility of Protein Research, Institute of Biophysics, Chinese Academy of Sciences, for their help in the NMR data collection, analysis and valuable discussion. We thank J. Jia and S. Sun for their technical assistance in flow cytometry analysis. We thank X. Ding from the Laboratory of Proteomics, Core Facilities for Protein Science, at the Institute of Biophysics (IBP), Chinese Academy of Sciences (CAS), for her help with the mass spectrometry analysis. We thank R.J. Lefkowitz (Duke University) for giving the constructs of Flag-β2AR, Flag-V2R, Flag-SSTR2, β-arrestin-1-YFP, GRK6-YFP and GRK2-YFP. We thank Z. Yang, Y.-S. He, X.-L. Fu and L. Chen for participating in the collection and analysis of this large sequence library of GPCRs. We thank an anonymous scientist who helped in design of the receptor–arrestin–SRC complex formation strategy and contributed significantly to this project. We acknowledge support from the National Key Basic Research Program of China (2015CB856203 to J.-Y.W.), the National Natural Science Foundation of China (81773704 and 31470789 to J.-P.S., 21325211 to J.-Y.W., 31700692 to P.X.), the Shandong Natural Science Fund for Distinguished Young Scholars (JQ201517 to J.-P.S.), the Fundamental Research Fund of Shandong University (2014JC029 to X.Y.), the National Science Fund for Distinguished Young Scholars (81525005 to F.Y.) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT13028).
The authors declare no competing interests.
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Scientific Reports (2019)
Distinct G protein-coupled receptor phosphorylation motifs modulate arrestin affinity and activation and global conformation
Nature Communications (2019)
World Journal of Biological Chemistry (2018)