A novel G protein-biased agonist at the μ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase

Sam Groom; Nina K Blum; Alexandra E Conibear; Alexander Disney; Rob Hill; Stephen M Husbands; Yangmei Li; Lawrence Toll; Andrea Kliewer; Stefan Schulz; Graeme Henderson; Eamonn Kelly; Chris P Bailey

doi:10.1111/bph.15334

A novel G protein-biased agonist at the μ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase

Sam Groom, Nina K Blum, Alexandra E Conibear, Alexander Disney, Rob Hill, Stephen M Husbands, Yangmei Li, Lawrence Toll, Andrea Kliewer, Stefan Schulz, Graeme Henderson, Eamonn Kelly, Chris P Bailey

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Abstract

Background and Purpose: G protein-biased μ opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased μ agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. Experimental Approach: The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced μ receptor desensitisation was probed using kinase inhibitors. Key Results: Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased μ agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular μ receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid μ receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. Conclusion and Implications: Compound 1 is a novel G protein-biased μ agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Original language	English
Pages (from-to)	943-957
Number of pages	15
Journal	British Journal of Pharmacology
Volume	180
Issue number	7
Early online date	27 Nov 2020
DOIs	https://doi.org/10.1111/bph.15334
Publication status	Published - 30 Apr 2023

Bibliographical note

Funding Information:
S.G. was funded by an A.J. Clark studentship from the British Pharmacological Society (BPS). This work was supported by the following grants:- National Institute of Health (NIH, USA) Grant RO1DA036975 to G.H. and National Institute on Drug Abuse (NIDA, USA) Grant RO1DA07315 to S.M.H. The funders of the study had no role in the study design, data collection, data analysis, data interpretation or writing of the report.

Funding Information:
S.G. was funded by an A.J. Clark studentship from the British Pharmacological Society (BPS). This work was supported by the following grants:‐ National Institute of Health (NIH, USA) Grant RO1DA036975 to G.H. and National Institute on Drug Abuse (NIDA, USA) Grant RO1DA07315 to S.M.H. The funders of the study had no role in the study design, data collection, data analysis, data interpretation or writing of the report.

Publisher Copyright:
© 2020 The British Pharmacological Society.

Keywords

G protein-coupled receptor kinases
arrestins
biased agonism
electrophysiology
opiates
opioids
receptor desensitisation

ASJC Scopus subject areas

Pharmacology

Access to Document

10.1111/bph.15334

Groom et al - Accepted version inc figures.PDF
This is the peer reviewed version of the following article: Groom, S, Blum, NK, Conibear, AE, et al. A novel G protein‐biased agonist at the μ opioid receptor induces substantial receptor desensitization through G protein‐coupled receptor kinase. Br J Pharmacol. 2020. Accepted Author Manuscript, which has been published in final form at https://doi.org/10.1111/bph.15334. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
Accepted author manuscript, 1.56 MB

Cite this

Groom, S., Blum, N. K., Conibear, A. E., Disney, A., Hill, R., Husbands, S. M., Li, Y., Toll, L., Kliewer, A., Schulz, S., Henderson, G., Kelly, E., & Bailey, C. P. (2023). A novel G protein-biased agonist at the μ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase. British Journal of Pharmacology, 180(7), 943-957. https://doi.org/10.1111/bph.15334

Groom, S, Blum, NK, Conibear, AE, Disney, A, Hill, R, Husbands, SM, Li, Y, Toll, L, Kliewer, A, Schulz, S, Henderson, G, Kelly, E & Bailey, CP 2023, 'A novel G protein-biased agonist at the μ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase', British Journal of Pharmacology, vol. 180, no. 7, pp. 943-957. https://doi.org/10.1111/bph.15334

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abstract = "Background and Purpose: G protein-biased μ opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased μ agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. Experimental Approach: The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced μ receptor desensitisation was probed using kinase inhibitors. Key Results: Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased μ agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular μ receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid μ receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. Conclusion and Implications: Compound 1 is a novel G protein-biased μ agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.",

keywords = "G protein-coupled receptor kinases, arrestins, biased agonism, electrophysiology, opiates, opioids, receptor desensitisation",

author = "Sam Groom and Blum, {Nina K} and Conibear, {Alexandra E} and Alexander Disney and Rob Hill and Husbands, {Stephen M} and Yangmei Li and Lawrence Toll and Andrea Kliewer and Stefan Schulz and Graeme Henderson and Eamonn Kelly and Bailey, {Chris P}",

note = "Funding Information: S.G. was funded by an A.J. Clark studentship from the British Pharmacological Society (BPS). This work was supported by the following grants:- National Institute of Health (NIH, USA) Grant RO1DA036975 to G.H. and National Institute on Drug Abuse (NIDA, USA) Grant RO1DA07315 to S.M.H. The funders of the study had no role in the study design, data collection, data analysis, data interpretation or writing of the report. Funding Information: S.G. was funded by an A.J. Clark studentship from the British Pharmacological Society (BPS). This work was supported by the following grants:‐ National Institute of Health (NIH, USA) Grant RO1DA036975 to G.H. and National Institute on Drug Abuse (NIDA, USA) Grant RO1DA07315 to S.M.H. The funders of the study had no role in the study design, data collection, data analysis, data interpretation or writing of the report. Publisher Copyright: {\textcopyright} 2020 The British Pharmacological Society.",

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T1 - A novel G protein-biased agonist at the μ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase

AU - Groom, Sam

AU - Blum, Nina K

AU - Conibear, Alexandra E

AU - Disney, Alexander

AU - Hill, Rob

AU - Husbands, Stephen M

AU - Li, Yangmei

AU - Toll, Lawrence

AU - Kliewer, Andrea

AU - Schulz, Stefan

AU - Henderson, Graeme

AU - Kelly, Eamonn

AU - Bailey, Chris P

N1 - Funding Information: S.G. was funded by an A.J. Clark studentship from the British Pharmacological Society (BPS). This work was supported by the following grants:- National Institute of Health (NIH, USA) Grant RO1DA036975 to G.H. and National Institute on Drug Abuse (NIDA, USA) Grant RO1DA07315 to S.M.H. The funders of the study had no role in the study design, data collection, data analysis, data interpretation or writing of the report. Funding Information: S.G. was funded by an A.J. Clark studentship from the British Pharmacological Society (BPS). This work was supported by the following grants:‐ National Institute of Health (NIH, USA) Grant RO1DA036975 to G.H. and National Institute on Drug Abuse (NIDA, USA) Grant RO1DA07315 to S.M.H. The funders of the study had no role in the study design, data collection, data analysis, data interpretation or writing of the report. Publisher Copyright: © 2020 The British Pharmacological Society.

PY - 2023/4/30

Y1 - 2023/4/30

N2 - Background and Purpose: G protein-biased μ opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased μ agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. Experimental Approach: The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced μ receptor desensitisation was probed using kinase inhibitors. Key Results: Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased μ agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular μ receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid μ receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. Conclusion and Implications: Compound 1 is a novel G protein-biased μ agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

AB - Background and Purpose: G protein-biased μ opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased μ agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. Experimental Approach: The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced μ receptor desensitisation was probed using kinase inhibitors. Key Results: Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased μ agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular μ receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid μ receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. Conclusion and Implications: Compound 1 is a novel G protein-biased μ agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

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A novel G protein-biased agonist at the μ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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