Optimization of the Heterologous Expression of the Cannabinoid Type-1 (CB1) Receptor
The G protein-coupled type 1 cannabinoid receptor (CB1R) mediates virtually all classic cannabinoid effects, and both its agonists and antagonists hold major therapeutic potential. Heterologous expression of receptors is vital for pharmacological research, however, overexpression of these proteins m...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-10-01
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| Series: | Frontiers in Endocrinology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fendo.2021.740913/full |
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| author | Viktória B. Horváth Eszter Soltész-Katona Eszter Soltész-Katona Éva Wisniewski Anikó Rajki Anikó Rajki Eszter Halász Eszter Halász Balázs Enyedi Balázs Enyedi Balázs Enyedi László Hunyady László Hunyady András Dávid Tóth András Dávid Tóth Gergő Szanda Gergő Szanda |
| author_facet | Viktória B. Horváth Eszter Soltész-Katona Eszter Soltész-Katona Éva Wisniewski Anikó Rajki Anikó Rajki Eszter Halász Eszter Halász Balázs Enyedi Balázs Enyedi Balázs Enyedi László Hunyady László Hunyady András Dávid Tóth András Dávid Tóth Gergő Szanda Gergő Szanda |
| author_sort | Viktória B. Horváth |
| collection | DOAJ |
| description | The G protein-coupled type 1 cannabinoid receptor (CB1R) mediates virtually all classic cannabinoid effects, and both its agonists and antagonists hold major therapeutic potential. Heterologous expression of receptors is vital for pharmacological research, however, overexpression of these proteins may fundamentally alter their localization pattern, change the signalling partner preference and may also spark artificial clustering. Additionally, recombinant CB1Rs are prone to intense proteasomal degradation, which may necessitate substantial modifications, such as N-terminal truncation or signal sequence insertion, for acceptable cell surface expression. We report here that tuning down the expression intensity of the full-length CB1R reduces proteasomal degradation and offers receptor levels that are comparable to those of endogenous CB1 receptors. As opposed to high-efficiency expression with conventional promoters, weak promoter-driven CB1R expression provides ERK 1/2 and p38 MAPK signalling that closely resemble the activity of endogenous CB1Rs. Moreover, weakly expressed CB1R variants exhibit plasma membrane localization, preserve canonical Gi-signalling but prevent CB1R-Gs coupling observed with high-expression variants. Based on these findings, we propose that lowering the expression level of G protein-coupled receptors should always be considered in heterologous expression systems in order to reduce the pressure on the proteasomal machinery and to avoid potential signalling artefacts. |
| first_indexed | 2024-12-21T10:11:33Z |
| format | Article |
| id | doaj.art-64730e0b71c846e1940d57bc165d3272 |
| institution | Directory Open Access Journal |
| issn | 1664-2392 |
| language | English |
| last_indexed | 2024-12-21T10:11:33Z |
| publishDate | 2021-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Endocrinology |
| spelling | doaj.art-64730e0b71c846e1940d57bc165d32722022-12-21T19:07:41ZengFrontiers Media S.A.Frontiers in Endocrinology1664-23922021-10-011210.3389/fendo.2021.740913740913Optimization of the Heterologous Expression of the Cannabinoid Type-1 (CB1) ReceptorViktória B. Horváth0Eszter Soltész-Katona1Eszter Soltész-Katona2Éva Wisniewski3Anikó Rajki4Anikó Rajki5Eszter Halász6Eszter Halász7Balázs Enyedi8Balázs Enyedi9Balázs Enyedi10László Hunyady11László Hunyady12András Dávid Tóth13András Dávid Tóth14Gergő Szanda15Gergő Szanda16Department of Physiology, Semmelweis University, Budapest, HungaryDepartment of Physiology, Semmelweis University, Budapest, HungaryMTA-SE Laboratory of Molecular Physiology, Eötvös Loránd Research Network, Budapest, HungaryDepartment of Physiology, Semmelweis University, Budapest, HungaryDepartment of Physiology, Semmelweis University, Budapest, HungaryMTA-SE Laboratory of Molecular Physiology, Eötvös Loránd Research Network, Budapest, HungaryDepartment of Physiology, Semmelweis University, Budapest, HungaryMTA-SE Laboratory of Molecular Physiology, Eötvös Loránd Research Network, Budapest, HungaryDepartment of Physiology, Semmelweis University, Budapest, HungaryMTA-SE Lendület Tissue Damage Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, HungaryHCEMM-SE Inflammatory Signaling Research Group, Department of Physiology, Semmelweis University, Budapest, HungaryDepartment of Physiology, Semmelweis University, Budapest, HungaryMTA-SE Laboratory of Molecular Physiology, Eötvös Loránd Research Network, Budapest, HungaryMTA-SE Laboratory of Molecular Physiology, Eötvös Loránd Research Network, Budapest, HungaryDepartment of Internal Medicine and Haematology, Semmelweis University, Budapest, HungaryDepartment of Physiology, Semmelweis University, Budapest, HungaryMTA-SE Laboratory of Molecular Physiology, Eötvös Loránd Research Network, Budapest, HungaryThe G protein-coupled type 1 cannabinoid receptor (CB1R) mediates virtually all classic cannabinoid effects, and both its agonists and antagonists hold major therapeutic potential. Heterologous expression of receptors is vital for pharmacological research, however, overexpression of these proteins may fundamentally alter their localization pattern, change the signalling partner preference and may also spark artificial clustering. Additionally, recombinant CB1Rs are prone to intense proteasomal degradation, which may necessitate substantial modifications, such as N-terminal truncation or signal sequence insertion, for acceptable cell surface expression. We report here that tuning down the expression intensity of the full-length CB1R reduces proteasomal degradation and offers receptor levels that are comparable to those of endogenous CB1 receptors. As opposed to high-efficiency expression with conventional promoters, weak promoter-driven CB1R expression provides ERK 1/2 and p38 MAPK signalling that closely resemble the activity of endogenous CB1Rs. Moreover, weakly expressed CB1R variants exhibit plasma membrane localization, preserve canonical Gi-signalling but prevent CB1R-Gs coupling observed with high-expression variants. Based on these findings, we propose that lowering the expression level of G protein-coupled receptors should always be considered in heterologous expression systems in order to reduce the pressure on the proteasomal machinery and to avoid potential signalling artefacts.https://www.frontiersin.org/articles/10.3389/fendo.2021.740913/fullCB1 receptorreceptor degradationcannabinoidsweak promotersheterologous expressionnon-canonical signaling |
| spellingShingle | Viktória B. Horváth Eszter Soltész-Katona Eszter Soltész-Katona Éva Wisniewski Anikó Rajki Anikó Rajki Eszter Halász Eszter Halász Balázs Enyedi Balázs Enyedi Balázs Enyedi László Hunyady László Hunyady András Dávid Tóth András Dávid Tóth Gergő Szanda Gergő Szanda Optimization of the Heterologous Expression of the Cannabinoid Type-1 (CB1) Receptor Frontiers in Endocrinology CB1 receptor receptor degradation cannabinoids weak promoters heterologous expression non-canonical signaling |
| title | Optimization of the Heterologous Expression of the Cannabinoid Type-1 (CB1) Receptor |
| title_full | Optimization of the Heterologous Expression of the Cannabinoid Type-1 (CB1) Receptor |
| title_fullStr | Optimization of the Heterologous Expression of the Cannabinoid Type-1 (CB1) Receptor |
| title_full_unstemmed | Optimization of the Heterologous Expression of the Cannabinoid Type-1 (CB1) Receptor |
| title_short | Optimization of the Heterologous Expression of the Cannabinoid Type-1 (CB1) Receptor |
| title_sort | optimization of the heterologous expression of the cannabinoid type 1 cb1 receptor |
| topic | CB1 receptor receptor degradation cannabinoids weak promoters heterologous expression non-canonical signaling |
| url | https://www.frontiersin.org/articles/10.3389/fendo.2021.740913/full |
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