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Allosteric mechanisms underlie GPCR signaling to SH3-domain proteins through arrestin


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).

Author information

J.-P.S. conceived the idea for allosteric regulatory mechanism of arrestin and its engagement with effectors. J.-Y.W. conceived all chemical biology experiments and synthesis route. J.-P.S. conceived that majority of GPCR members connect to SH3-domain-containing proteins through arrestins. J.-Y.W. brought up the idea for F2Y and KPN. J.-P.S, F. Yang and P.X. designed the key experiments to dissect the allosteric propagation pathways. J.-P.S., J.-Y.W. and X.Y. designed most of the experiments. F. Yang and K.R. collected and analyzed the 19F-NMR data. P.X., Q. Liu. and F. Yang purified β2AR and GRK6 proteins. P.X., C.-X.Q and F. Yang performed SRC kinase activity assays. Q. Liu Synthesized TA-bimane and performed fluorescence spectroscopy assays. Z.-X.L. performed BRET assays. F. Yang, C.-X.Q., C.L., R.-R.L., Y.-M.S. Z.-Y.Y and X.-Z.G. synthesized F2Y and purified F2Y incorporated β-arrestin 1 proteins. F. Yang, C.L. and Q.-T.H. purified GST-SRC-FL, GST-SH3-CPs and GST-clathrin proteins. F. Yang, P.X. C.-X.Q., Q.-T.H. and Q. Li. performed GST-pull down and CO-IP assays. F. Yang and Q.-T.H. performed circular dichroism assays. F. Yang, F. Yi and Q. Li performed Trypsin digestion and MS/MS assays. L.-Y.W., P.X., C.-X.Q., J.-Y.X. and D.-F.H., analyzed the SH3 binding motif (proline region) across GPCR family members. J.-P.S., J.-Y.W. and X.Y. supervised the overall project design and execution. J.-P.S. participated in data analysis and interpretation. J.-P.S. and J.-Y.W. wrote the manuscript. All of the authors have seen and commented on the manuscript.

Correspondence to Jin-peng Sun or Jiangyun Wang.

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Supplementary Figures 1–39

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Supplementary Note 1

Sequence information for the localization of the proline region in the intracellular region of GPCR members; Supplementary methods; Additional Supplemental Figure

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Fig. 1: GPCRs signal to SH3-CPs via the 3 proline regions of β-arrestin 1.
Fig. 2: Residue contacts govern the propagation pathway from phospho-binding site 5 to conformational changes in the P1 and P2 region.
Fig. 3: 19F-NMR spectra and paramagnetic titration revealed structural alterations in the β-arrestin 1 PRs in response to the binding of SRC or different SH3 domains.
Fig. 4: A phospho-receptor/arrestin complex promoted the activation of SRC.
Fig. 5: A phospho-receptor–arrestin complex promoted disassembly of the autoinhibitory domains of SRC.
Fig. 6: Schematic description of the allosteric mechanism underlying arrestin-mediated GPCR functions at two levels.