Abstract
G protein-coupled receptors (GPCRs) are seven transmembrane receptors that respond to external stimuli and undergo conformational changes to activate G proteins and modulate cellular processes leading to biological outcomes. To prevent overstimulation and prolonged exposure to stimuli, GPCRs are regulated by internalization. While the canonical GPCR internalization mechanism in mammalian cells is arrestin-dependent, clathrin-mediated endocytosis, more diverse GPCR internalization mechanisms have been described over the years. However, there is a lack of consistent methods used in the literature making it complicated to determine a receptor's internalization pathway. Here, we utilized a highly efficient time-resolved Förster resonance energy transfer (TR-FRET) internalization assay to determine the internalization profile of nine distinct GPCRs representing the GPCR classes A, B and C and with different G protein coupling profiles. This technique, coupled with clustered regularly interspaced palindromic repeats (CRISPR) engineered knockout cells allows us to effectively study the involvement of heterotrimeric G proteins and non-visual arrestins. We found that all the nine receptors internalized upon agonist stimulation in a concentration-dependent manner and six receptors showed basal internalization. Yet, there is no correlation between the receptor class and primary G protein coupling to the arrestin and G protein dependence for GPCR internalization. Overall, this study presents a platform for studying internalization that is applicable to most GPCRs and may even be extended to other membrane proteins. This method can be easily applicable to other endocytic machinery of interest and ultimately will lend itself towards the construction of comprehensive receptor internalization profiles.
Originalsprog | Engelsk |
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Artikelnummer | 119584 |
Tidsskrift | Biochimica et Biophysica Acta - Molecular Cell Research |
Vol/bind | 1871 |
Udgave nummer | 1 |
Antal sider | 12 |
ISSN | 0167-4889 |
DOI | |
Status | Udgivet - 2024 |
Bibliografisk note
Funding Information:H.B.-O. acknowledges financial support from the Independent Research Fund Denmark | Medical Sciences ( 4183-00131A and 8020-02308 ). T.C.M. and E.V.M. acknowledges funding from the European Union's Horizon2020 Research and Innovation Program under the Marie Sklodowska-Curie grant agreement No 797497 and No 846827 , respectively. A.I. was funded by Japan Society for the Promotion of Science (JSPS) KAKENHI grants 21H04791 , 21H05113 , JPJSBP120213501 and JPJSBP120218801 ; Japan Science and Technology Agency (JST) grants JPMJFR215T and JPMJMS2023 ; The Uehara Memorial Foundation ; and Daiichi Sankyo Foundation of Life Science .
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