Abstract
Beta-arrestins (βarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of βarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of βarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted βarr–clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting βarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.
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Acknowledgements
The research program in the Department of Biological Science and Bioengineering is supported by the Indian Institute of Technology Kanpur (IITK/BSBE/2014011), Department of Biotechnology (DBT) (BT/08/IYBA/2014/03), Council of Scientific and Industrial Research (37(1637)/14/EMR-II) and the Wellcome Trust DBT India Alliance (IA/I/14/1/501285). A.K. Shukla is an Intermediate Fellow of the Wellcome Trust/DBT India Alliance (IA/I/14/1/501285). We thank L. Traub, M. Scott, T. Pucadyil, R. Shaw and R. Davis for the plasmids that encode the clathrin terminal domain, βarr2–mCherry, GST-β2 adaptin, hTfr1 (Addgene no. 69610) and JNK3 (Addgene no. 15748), respectively. We also acknowledge the help from C. Gupta and P. Gupta in the early stages of this work, and S. Pandey for help in the protein purification.
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E.G. designed, optimized and performed the endocytosis and ERK activation experiments with the ScFv5 intrabody and the cross-linking experiment with ScFv5, and assisted in the ERK assays. A.S. performed the ELISA based assessment of clathrin and ERK interaction with βarr, and assisted in the endocytosis and ERK assays. M.B. performed the confocal microscopy using the ScFv5-YFP intrabody and assisted in the subcloning and endocytosis experiments. P.K. carried out the ELISA based selectivity test for βarr2 Fabs, and the endocytosis and ERK assays for M2R and β2V2R together with M.B. H.D. carried out the selectivity assays for Fabs by coIP together with A.S., the ELISA-based selectivity assays for Fab5 and ScFv5 and the mapping experiment for ScFv5. R.R. converted the Fabs into intrabodies for expression, and assisted in subcloning the various constructs and endocytosis experiments. K.N. performed the initial phase of intrabody expression, functional validation and their effect on receptor endocytosis and ERK activation. S.D. and P.N.Y. assisted in the βarr knockdown. S.K., A.K. and S.S.S. provided the phage display libraries. A.K.S. carried out the phage display screening, wrote the manuscript and supervised the overall project design and execution. All the authors approved the final draft of the manuscript.
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Ghosh, E., Srivastava, A., Baidya, M. et al. A synthetic intrabody-based selective and generic inhibitor of GPCR endocytosis. Nature Nanotech 12, 1190–1198 (2017). https://doi.org/10.1038/nnano.2017.188
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DOI: https://doi.org/10.1038/nnano.2017.188
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