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Agonist-induced extracellular vesicles contribute to the transfer of functional bombesin receptor-subtype 3 to recipient cells

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Abstract

Extracellular vesicles (EVs) are important carriers for biomolecules in the microenvironment that greatly promote intercellular and extracellular communications. However, it is unclear whether bombesin receptor-subtype 3 (BRS-3), an orphan G-protein coupled receptor, can be packed into EVs and functionally transferred to recipient cells. In this study, we applied the synthetic agonist and antagonist to activate and inhibit the BRS-3 in HEK293-BRS-3 cells, whose EVs release was BRS-3 activation dependent. The presence of BRS-3 in harvested EVs was further confirmed by an enhanced green fluorescent protein tag. After recipient cells were co-cultured with these EVs, the presence of BRS-3 in the recipient cells was discovered, whose function was experimentally validated. Quantitative proteomics approach was utilized to decipher the proteome of the EVs derived from HEK293-BRS-3 cells after different stimulations. More than 900 proteins were identified, including 51 systematically dysregulated EVs proteins. The Ingenuity Pathway Analysis (IPA) revealed that RhoA signaling pathway was as an essential player for the secretion of EVs. Selective inhibition of RhoA signaling pathway after BRS-3 activation dramatically reversed the increased secretion of EVs. Our data, collectively, demonstrated that EVs contributed to the transfer of functional BRS-3 to the recipient cells, whose secretion was partially regulated by RhoA signaling pathway.

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Data availability

The mass spectrometry proteomics data and the search data by MaxQuant (version 1.6.1.0) have been deposited to the ProteomeXchange Consortium (http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository with the dataset identifier PXD027980.

Code availability

Not applicable.

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Funding

This work was supported by grants from the Natural Science Foundation of Shanghai (No. 21ZR1433200, No. 19ZR1427800), the National Key Research and Development Program of China (No. 2017YFC1200204), the National Natural Science Foundation of China (No. 21675110), and the Key Scientific Project of Shanghai Jiao Tong University (No. TMSK-2020–130, No. YG2017MS80).

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  1. Zeyuan Wang and Lehao Wu contributed equally as first authors to this work.

Authors and Affiliations

  1. State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China

    Zeyuan Wang, Lehao Wu, Huiyu Wang & Hua Xiao

  2. School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China

    Yan Zhang

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  1. Zeyuan Wang
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ZYW, LHW, and HYW performed the experiments and analyzed the data. YZ and HX directed the project and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Yan Zhang or Hua Xiao.

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Wang, Z., Wu, L., Wang, H. et al. Agonist-induced extracellular vesicles contribute to the transfer of functional bombesin receptor-subtype 3 to recipient cells. Cell. Mol. Life Sci. 79, 72 (2022). https://doi.org/10.1007/s00018-021-04114-z

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