Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization

Møller, Thor C.; Moo, Ee Von; Seiersen, Sofie D.; Bräuner‐Osborne, Hans

doi:10.1016/j.jbc.2022.102466

Cited by 6 publications

(4 citation statements)

References 60 publications

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“…Another prominent family of kinases involved in GPCR phosphorylation and regulation is the PKCs [45]. Our group recently established and validated a conventional PKC (absence of α/β 1 /β 2 /γ) and novel PKC (absence of δ/ε/η/θ) knockout cell line using CRISPR/Cas9 genome editing [40]. Utilizing the knockout cell lines in the internalization experiment, we showed that the deletion of conventional or novel PKC subtypes did not significantly affect GPR15 internalization (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Dynamin and caveolin-inhibited HEK293A cells were achieved by transiently expression of the dominant-negative mutant dynamin K44A DNA and caveolin P132L DNA, respectively. The Ga s/olf/z/q/11/12/13 knockout (DGa s/olf/z/q/11/12/13 ) cells [35], the arrestin-2/3 knockout (DArrestin-2/3) cells [37], the GRK2/3 knockout (DGRK2/3) cells [38], the GRK5/6 knockout (DGRK5/6) cells [39], the GRK2/3/5/6 knockout (DGRK2/3/5/6) cells [39], the PKC-a/b 1 /b 2 /c knockout (DPKC-a/b 1 /b 2 /c) cells, and the PKC-d/e/g/h knockout (DPKC-d/e/g/h) cells were established with by CRISPR/ Cas9 genome editing [40].…”

Section: Modified Cell Conditionsmentioning

confidence: 99%

“…The parental HEK293A, DArrestin-2/3, DGa s/olf/z/q/11/12/13 cells, DGRK5/6 and DGRK2/3/5/6 cells were previously generated and validated by A. Inoue [35,37,39]. The DPKC-a/b 1 /b 2 /c, DPKC-d/e/g/h, and DGRK2/3 cells were generated and validated in our lab as described previously [38,40]. All the cells were grown with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified 37 °C cell incubator with a 5% CO 2 atmosphere.…”

Section: Cell Lines and Culturingmentioning

confidence: 99%

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Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms

et al. 2023

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GPR15 is a G protein‐coupled receptor involved in immune disorders such as human immunodeficiency virus‐induced enteropathy, multiple sclerosis, and colitis. Yet, the important endocytosis mechanism of GPR15 remained unclear. This study determined the participation of endocytic machinery proteins, including Gα proteins, G protein‐coupled receptor kinases (GRKs), protein kinase C, arrestins, clathrin, caveolin, and dynamin in GPR15 internalization. The results demonstrate that GPR15 internalization is moderately dependent on GRKs and clathrin, and highly dependent on caveolin and dynamin. Moreover, a bystander arrestin recruitment assay showed that GPR15 recruits arrestin‐3 to the cell membrane upon agonist stimulation, although GPR15 internalizes in an arrestin‐independent manner. Overall, our study provides novel insights into β‐arrestin recruitment and receptor internalization mechanisms for the recently deorphanized GPR15.

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Section: Resultsmentioning

confidence: 99%

Section: Modified Cell Conditionsmentioning

confidence: 99%

Section: Cell Lines and Culturingmentioning

confidence: 99%

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Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms

et al. 2023

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“…However, as with GRKs, most reports to date have used pharmacological inhibitors or knock-down approaches. With the recent generation of typical and atypical PKC isoform CRISPR/Cas9 knock out cells (van Senten et al, 2022), the relative contribution to MOR signalling of GRK and PKC isoforms upon morphine stimulation can be further investigated in the future.…”

Section: Discussionmentioning

confidence: 99%

Key phosphorylation sites for robust β-arrestin2 binding at the MOR revisited

Underwood,

Fritzwanker,

Glenn

et al. 2023

Preprint

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FigureDesensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, we and others have shown that phosphorylation is important for β-arrestin recruitment to the receptor, and that ligands of different efficacies induce distinct patterns, or barcodes, of receptor phosphorylation. Within the MOR C-tail, the370TREHPSTANT379motif harbours Ser/Thr residues important for these regulatory functions.375Ser acts as a primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking370Thr,376Thr and379Thr residues can get subsequently phosphorylated. Here we used HEK293 GRK KO cells, in combination with phosphosite specific antibodies and site-directed mutagenesis of the MOR, to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and β-arrestin2 recruitment. We show that both GRK subfamilies (GRK2/3 and GRK5/6) promote phosphorylation of Thr370and Ser375. However, only GRK2/3 induce phosphorylation of Thr376and Thr379, which is required to promote robust β-arrestin recruitment to the receptor. Moreover, while DAMGO and fentanyl can engage all kinases to promote Thr370and Ser375phosphorylation, under endogenous GRK expression conditions, morphine-induced phosphorylation of these residues is specifically mediated by GRK5/6. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of the different GRK subfamilies plays an important role in determining the tissue responses of distinct opioid agonists.

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Characterization of the real-time internalization of nine GPCRs reveals distinct dependence on arrestins and G proteins

Møller,

Moo,

Inoue

et al. 2024

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization

Cited by 6 publications

References 60 publications

Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms

Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms

Key phosphorylation sites for robust β-arrestin2 binding at the MOR revisited

Characterization of the real-time internalization of nine GPCRs reveals distinct dependence on arrestins and G proteins

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