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

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization. / van Senten, Jeffrey R.; Møller, Thor C.; Moo, Ee Von; Seiersen, Sofie D.; Bräuner-Osborne, Hans.

In: Journal of Biological Chemistry, Vol. 298, No. 10, 102466, 2022.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

van Senten, JR, Møller, TC, Moo, EV, Seiersen, SD & Bräuner-Osborne, H 2022, 'Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization', Journal of Biological Chemistry, vol. 298, no. 10, 102466. https://doi.org/10.1016/j.jbc.2022.102466

APA

van Senten, J. R., Møller, T. C., Moo, E. V., Seiersen, S. D., & Bräuner-Osborne, H. (2022). Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization. Journal of Biological Chemistry, 298(10), [102466]. https://doi.org/10.1016/j.jbc.2022.102466

Vancouver

van Senten JR, Møller TC, Moo EV, Seiersen SD, Bräuner-Osborne H. Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization. Journal of Biological Chemistry. 2022;298(10). 102466. https://doi.org/10.1016/j.jbc.2022.102466

Author

van Senten, Jeffrey R. ; Møller, Thor C. ; Moo, Ee Von ; Seiersen, Sofie D. ; Bräuner-Osborne, Hans. / Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization. In: Journal of Biological Chemistry. 2022 ; Vol. 298, No. 10.

Bibtex

@article{63a1ce0295734dbb93c45ba5a26b5e07,
title = "Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization",
abstract = "The internalization of G protein–coupled receptors (GPCRs) can be regulated by PKC. However, most tools available to study the contribution of PKC isozymes have considerable limitations, including a lack of selectivity. In this study, we generated and characterized human embryonic kidney 293A (HEK293A) cell lines devoid of conventional or novel PKC isozymes (ΔcPKC and ΔnPKC) and employ these to investigate the contribution of PKC isozymes in the internalization of the metabotropic glutamate receptor 5 (mGlu5). Direct activation of PKC and mutation of rat mGlu5a Ser901, a PKC-dependent phosphorylation site in the receptor C-tail, both showed that PKC isozymes facilitate approximately 40% of the receptor internalization. Nonetheless, we determined that mGlu5a internalization was not altered upon the loss of cPKCs or nPKCs. This indicates that isozymes from both classes are involved, compensate for the absence of the other class, and thus fulfill dispensable functions. Additionally, using the Gαq/11 inhibitor YM-254890, GPCR kinase 2 and 3 (GRK2 and GRK3) KO cells, and a receptor containing a mutated putative adaptor protein complex 2 (AP-2) interaction motif, we demonstrate that internalization of rat mGlu5a is mediated by Gαq/11 proteins (77% of the response), GRK2 (27%), and AP-2 (29%), but not GRK3. Our PKC KO cell lines expand the repertoire of KO HEK293A cell lines available to research GPCR pharmacology. Moreover, since pharmacological tools to study PKC isozymes generally lack specificity and/or potency, we present the PKC KO cell lines as more specific research tools to investigate PKC-mediated aspects of cell biology.",
keywords = "CRISPR/cas, G protein-coupled receptor, metabotropic glutamate receptor, mGlu5, PKC, receptor endocytosis, receptor internalization, receptor regulation",
author = "{van Senten}, {Jeffrey R.} and M{\o}ller, {Thor C.} and Moo, {Ee Von} and Seiersen, {Sofie D.} and Hans Br{\"a}uner-Osborne",
note = "Funding Information: We thank Dr Jesper M. Mathiesen and Jens Peter Stenvang for their help with cell sorting at Zealand Pharma and Dr Eric Paul Bennett for his advice on gene-editing and IDAA. Dr Asuka Inoue is acknowledged for providing the HEK293A parental cell line and Mie F. Pedersen for generating the GRK2 and GRK3 knockout HEK293A cell lines. We thank Michel Bouvier for sharing the PKC-c1b biosensor. Domain Therapeutics is the exclusive holder of a license for all commercial uses of the PKC-c1b biosensor. J. R. v. S. T. C. M. and H. B.-O. conceptualization; J. R. v. S. and T. C. M. formal analysis; J. R. v. S. T. C. M. E. V. M. and S. D. S. investigation; J. R. v. S. writing–original draft; J. R. v. S. visualization; T. C. M. and H. B.-O. writing–review and editing; T. C. M. and H. B.-O. funding acquisition. H. B.-O. acknowledges financial support from the Independent Research Fund Denmark | Medical Sciences (4183-00131A and 8020-02308) and the Carlsberg Foundation (CF20-0248). 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. Funding Information: H. B.-O. acknowledges financial support from the Independent Research Fund Denmark | Medical Sciences (4183-00131A and 8020-02308) and the Carlsberg Foundation (CF20-0248). T. C. M. and E. V. M. acknowledges funding from the European Union{\textquoteright}s Horizon2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 797497 and No 846827, respectively. ",
year = "2022",
doi = "10.1016/j.jbc.2022.102466",
language = "English",
volume = "298",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
number = "10",

}

RIS

TY - JOUR

T1 - Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization

AU - van Senten, Jeffrey R.

AU - Møller, Thor C.

AU - Moo, Ee Von

AU - Seiersen, Sofie D.

AU - Bräuner-Osborne, Hans

N1 - Funding Information: We thank Dr Jesper M. Mathiesen and Jens Peter Stenvang for their help with cell sorting at Zealand Pharma and Dr Eric Paul Bennett for his advice on gene-editing and IDAA. Dr Asuka Inoue is acknowledged for providing the HEK293A parental cell line and Mie F. Pedersen for generating the GRK2 and GRK3 knockout HEK293A cell lines. We thank Michel Bouvier for sharing the PKC-c1b biosensor. Domain Therapeutics is the exclusive holder of a license for all commercial uses of the PKC-c1b biosensor. J. R. v. S. T. C. M. and H. B.-O. conceptualization; J. R. v. S. and T. C. M. formal analysis; J. R. v. S. T. C. M. E. V. M. and S. D. S. investigation; J. R. v. S. writing–original draft; J. R. v. S. visualization; T. C. M. and H. B.-O. writing–review and editing; T. C. M. and H. B.-O. funding acquisition. H. B.-O. acknowledges financial support from the Independent Research Fund Denmark | Medical Sciences (4183-00131A and 8020-02308) and the Carlsberg Foundation (CF20-0248). 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. Funding Information: H. B.-O. acknowledges financial support from the Independent Research Fund Denmark | Medical Sciences (4183-00131A and 8020-02308) and the Carlsberg Foundation (CF20-0248). 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.

PY - 2022

Y1 - 2022

N2 - The internalization of G protein–coupled receptors (GPCRs) can be regulated by PKC. However, most tools available to study the contribution of PKC isozymes have considerable limitations, including a lack of selectivity. In this study, we generated and characterized human embryonic kidney 293A (HEK293A) cell lines devoid of conventional or novel PKC isozymes (ΔcPKC and ΔnPKC) and employ these to investigate the contribution of PKC isozymes in the internalization of the metabotropic glutamate receptor 5 (mGlu5). Direct activation of PKC and mutation of rat mGlu5a Ser901, a PKC-dependent phosphorylation site in the receptor C-tail, both showed that PKC isozymes facilitate approximately 40% of the receptor internalization. Nonetheless, we determined that mGlu5a internalization was not altered upon the loss of cPKCs or nPKCs. This indicates that isozymes from both classes are involved, compensate for the absence of the other class, and thus fulfill dispensable functions. Additionally, using the Gαq/11 inhibitor YM-254890, GPCR kinase 2 and 3 (GRK2 and GRK3) KO cells, and a receptor containing a mutated putative adaptor protein complex 2 (AP-2) interaction motif, we demonstrate that internalization of rat mGlu5a is mediated by Gαq/11 proteins (77% of the response), GRK2 (27%), and AP-2 (29%), but not GRK3. Our PKC KO cell lines expand the repertoire of KO HEK293A cell lines available to research GPCR pharmacology. Moreover, since pharmacological tools to study PKC isozymes generally lack specificity and/or potency, we present the PKC KO cell lines as more specific research tools to investigate PKC-mediated aspects of cell biology.

AB - The internalization of G protein–coupled receptors (GPCRs) can be regulated by PKC. However, most tools available to study the contribution of PKC isozymes have considerable limitations, including a lack of selectivity. In this study, we generated and characterized human embryonic kidney 293A (HEK293A) cell lines devoid of conventional or novel PKC isozymes (ΔcPKC and ΔnPKC) and employ these to investigate the contribution of PKC isozymes in the internalization of the metabotropic glutamate receptor 5 (mGlu5). Direct activation of PKC and mutation of rat mGlu5a Ser901, a PKC-dependent phosphorylation site in the receptor C-tail, both showed that PKC isozymes facilitate approximately 40% of the receptor internalization. Nonetheless, we determined that mGlu5a internalization was not altered upon the loss of cPKCs or nPKCs. This indicates that isozymes from both classes are involved, compensate for the absence of the other class, and thus fulfill dispensable functions. Additionally, using the Gαq/11 inhibitor YM-254890, GPCR kinase 2 and 3 (GRK2 and GRK3) KO cells, and a receptor containing a mutated putative adaptor protein complex 2 (AP-2) interaction motif, we demonstrate that internalization of rat mGlu5a is mediated by Gαq/11 proteins (77% of the response), GRK2 (27%), and AP-2 (29%), but not GRK3. Our PKC KO cell lines expand the repertoire of KO HEK293A cell lines available to research GPCR pharmacology. Moreover, since pharmacological tools to study PKC isozymes generally lack specificity and/or potency, we present the PKC KO cell lines as more specific research tools to investigate PKC-mediated aspects of cell biology.

KW - CRISPR/cas

KW - G protein-coupled receptor

KW - metabotropic glutamate receptor

KW - mGlu5

KW - PKC

KW - receptor endocytosis

KW - receptor internalization

KW - receptor regulation

U2 - 10.1016/j.jbc.2022.102466

DO - 10.1016/j.jbc.2022.102466

M3 - Journal article

C2 - 36087841

AN - SCOPUS:85139012328

VL - 298

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 10

M1 - 102466

ER -

ID: 322784918