Characterization of the real-time internalization of nine GPCRs reveals distinct dependence on arrestins and G proteins
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Characterization of the real-time internalization of nine GPCRs reveals distinct dependence on arrestins and G proteins. / Møller, Thor C.; Moo, Ee Von; Inoue, Asuka; Pedersen, Mie F.; Bräuner-Osborne, Hans.
I: Biochimica et Biophysica Acta - Molecular Cell Research, Bind 1871, Nr. 1, 119584, 2024.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - Characterization of the real-time internalization of nine GPCRs reveals distinct dependence on arrestins and G proteins
AU - Møller, Thor C.
AU - Moo, Ee Von
AU - Inoue, Asuka
AU - Pedersen, Mie F.
AU - Bräuner-Osborne, Hans
N1 - Publisher Copyright: © 2023 The Authors
PY - 2024
Y1 - 2024
N2 - 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.
AB - 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.
KW - Arrestins
KW - CRISPR/Cas9
KW - G protein-coupled receptor
KW - Internalization
KW - Knockout
KW - Signaling
U2 - 10.1016/j.bbamcr.2023.119584
DO - 10.1016/j.bbamcr.2023.119584
M3 - Journal article
C2 - 37714305
AN - SCOPUS:85171674261
VL - 1871
JO - B B A - Molecular Cell Research
JF - B B A - Molecular Cell Research
SN - 0167-4889
IS - 1
M1 - 119584
ER -
ID: 378765630