Determining the Effects of Differential Expression of GRKs and β-arrestins on CLR-RAMP Agonist Bias

Abigail Pearce; Theo Redfern-Nichols; Matthew Harris; David R. Poyner; Mark Wigglesworth; Graham Ladds

Determining the Effects of Differential Expression of GRKs and β-arrestins on CLR-RAMP Agonist Bias

Abigail Pearce, Theo Redfern-Nichols, Matthew Harris, David R. Poyner, Mark Wigglesworth, Graham Ladds^*

^*Corresponding author for this work

Aston Pharmacy School

Research output: Contribution to journal › Article › peer-review

Abstract

Signalling of the calcitonin-like receptor (CLR) is multifaceted, due to its interaction with receptor activity modifying proteins (RAMPs), and three endogenous peptide agonists. Previous studies have focused on the bias of G protein signalling mediated by the receptor and receptor internalisation of the CLR-RAMP complex has been assumed to follow the same pattern as other Class B1 G Protein-Coupled Receptors (GPCRs). Here we sought to measure desensitisation of the three CLR-RAMP complexes in response to the three peptide agonists, through the measurement of β-arrestin recruitment and internalisation. We then delved further into the mechanism of desensitisation through modulation of β-arrestin activity and the expression of GPCR kinases (GRKs), a key component of homologous GPCR desensitisation. First, we have shown that CLR-RAMP1 is capable of potently recruiting β-arrestin1 and 2, subsequently undergoing rapid endocytosis, and that CLR-RAMP2 and -RAMP3 also utilise these pathways, although to a lesser extent. Following this we have shown that agonist-dependent internalisation of CLR is β-arrestin dependent, but not required for full agonism. Overexpression of GRK2-6 was then found to decrease receptor signalling, due to an agonist-independent reduction in surface expression of the CLR-RAMP complex. These results represent the first systematic analysis of the importance of β-arrestins and GRKs in CLR-RAMP signal transduction and pave the way for further investigation regarding other Class B1 GPCRs.

Original language	English
Article number	840763
Number of pages	14
Journal	Frontiers in Physiology
Volume	13
Publication status	Published - 29 Mar 2022

Bibliographical note

© 2022 Pearce, Redfern-Nichols, Harris, Poyner, Wigglesworth and Ladds. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

This work was supported by two UK Biotechnology and Biological Sciences Research Council (BBSRC)-iCase studentships (AP: BB/JO14540/1; TR-N: BB/V509334/1), both co-funded with AstraZeneca.

Keywords

Physiology
GPCRs (G protein-coupled receptors)
signalling bias
CLR
β-arrestins
RAMPs
internalisation
GRK (G protein receptor kinase)

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fphys-13-840763.pdfFinal published version, 3.68 MBLicence: CC BY 3.0
Pearce et al Differential Expression of GRKs and CLR RAMP Agonist Bias
© 2022 Pearce, Redfern-Nichols, Harris, Poyner, Wigglesworth and Ladds. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Final published version, 3.68 MBLicence: CC BY 4.0

Cite this

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title = "Determining the Effects of Differential Expression of GRKs and β-arrestins on CLR-RAMP Agonist Bias",

abstract = "Signalling of the calcitonin-like receptor (CLR) is multifaceted, due to its interaction with receptor activity modifying proteins (RAMPs), and three endogenous peptide agonists. Previous studies have focused on the bias of G protein signalling mediated by the receptor and receptor internalisation of the CLR-RAMP complex has been assumed to follow the same pattern as other Class B1 G Protein-Coupled Receptors (GPCRs). Here we sought to measure desensitisation of the three CLR-RAMP complexes in response to the three peptide agonists, through the measurement of β-arrestin recruitment and internalisation. We then delved further into the mechanism of desensitisation through modulation of β-arrestin activity and the expression of GPCR kinases (GRKs), a key component of homologous GPCR desensitisation. First, we have shown that CLR-RAMP1 is capable of potently recruiting β-arrestin1 and 2, subsequently undergoing rapid endocytosis, and that CLR-RAMP2 and -RAMP3 also utilise these pathways, although to a lesser extent. Following this we have shown that agonist-dependent internalisation of CLR is β-arrestin dependent, but not required for full agonism. Overexpression of GRK2-6 was then found to decrease receptor signalling, due to an agonist-independent reduction in surface expression of the CLR-RAMP complex. These results represent the first systematic analysis of the importance of β-arrestins and GRKs in CLR-RAMP signal transduction and pave the way for further investigation regarding other Class B1 GPCRs.",

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AU - Poyner, David R.

AU - Wigglesworth, Mark

AU - Ladds, Graham

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KW - Physiology

KW - GPCRs (G protein-coupled receptors)

KW - signalling bias

KW - CLR

KW - β-arrestins

KW - RAMPs

KW - internalisation

KW - GRK (G protein receptor kinase)

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M3 - Article

SN - 1664-042X

VL - 13

JO - Frontiers in Physiology

JF - Frontiers in Physiology

M1 - 840763

ER -

Determining the Effects of Differential Expression of GRKs and β-arrestins on CLR-RAMP Agonist Bias

Abstract

Bibliographical note

Keywords

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