Molecular Mechanisms of Class B GPCR Activation: Insights from Adrenomedullin Receptors

Garelja, Michael L.; Au, Maggie; Brimble, Margaret A.; Gingell, Joseph J.; Hendrikse, Erica R; Lovell, Annie; Prodan, Nicole; Sexton, Patrick M.; Siow, Andrew; Walker, Christopher S.; Watkins, Harriet A.; Williams, Geoffrey M.; Wootten, Denise; Yang, Sung Hyun; Harris, Paul W. R.; Hay, Debbie L.

doi:10.1021/acsptsci.9b00083

Cited by 32 publications

(55 citation statements)

References 106 publications

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“…It should be noted that not all studies reveal pleiotropic signalling for the CGRP family of peptides, either involving direct measurements of G protein coupling in cell-free systems 55 or second messenger generation in cells 12 . We suggest that there is cell membrane (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…To date the cognate receptor for AM2 and its physiological role remain unknown; no receptor shows marked selectivity for it. While these is an abundance of evidence of GPCR signalling bias in recombinant cell systems, and in this case CLR-mediated bias 12,19 , documented examples using natural agonists and endogenously expressed human receptors are currently lacking. We wished to ascertain whether signalling bias at the CLR occurs in primary cells and whether it plays a role in cellular function.…”

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confidence: 99%

“…I) Log potency ratios (as measured by the accumulation of cAMP) calculated as Log (EC50AM2/EC50 agonist). Data are compiled from 12,19 . HUVECs and HUEACs are shown in red and green respectively, HCMs in cyan and RAMP1-HUVECs in blue.…”

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confidence: 99%

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CGRP-receptor family reveals endogenous GPCR agonist bias and its significance in primary human cardiovascular cells

Clark

Mullooly²,

Safitri

et al. 2020

Preprint

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Agonist bias at G protein-coupled receptors has attracted considerable interest, although its relevance for physiologically-produced agonists is not always clear. Here, using primary human cells and gene editing techniques, we demonstrate for the first time, endogenous agonist bias with physiological consequences for the calcitonin-like receptor (CLR). We reveal that by switching the accessory protein: receptor activity-modifying protein (RAMP) associated with CLR we can re-route the physiological pathways activated by the stimulating peptide agonists. These results have revealed a unique role in calcium-mediated nitric oxide signalling for the little-understood peptide adrenomedullin 2 and distinct pro-proliferative effects of calcitonin-gene related peptide (CGRP) and adrenomedullin in cardiovascular cells. This work reveals that CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and its importance in endogenous GPCR signalling.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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CGRP-receptor family reveals endogenous GPCR agonist bias and its significance in primary human cardiovascular cells

Clark

Mullooly²,

Safitri

et al. 2020

Preprint

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“… 4 , 5 Despite their physiological importance and promising therapeutic potential, the small number of full-length ligand-bound structures of class B GPCRs and the limited structural information on druggable binding sites have made the development of compounds that target this GPCR family challenging. 6 , 7 However, a number of structures have been solved recently 8 − 10 due to advances in cryo-EM technology and resolution, so that further developments are now more feasible. Regardless, a number of compounds have been reported in the past decade, including synthetic modulators of glucagon, glucagon-like peptide-1, corticotropin-releasing factor 1, and calcitonin receptor-like receptors.…”

Section: Introductionmentioning

confidence: 99%

Discovery of a First-In-Class Small Molecule Antagonist against the Adrenomedullin-2 Receptor: Structure–Activity Relationships and Optimization

et al. 2021

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Class B G-protein-coupled receptors (GPCRs) remain an underexploited target for drug development. The calcitonin receptor (CTR) family is particularly challenging, as its receptors are heteromers comprising two distinct components: the calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) together with one of three accessory proteins known as receptor activity-modifying proteins (RAMPs). CLR/RAMP1 forms a CGRP receptor, CLR/RAMP2 forms an adrenomedullin-1 (AM 1 ) receptor, and CLR/RAMP3 forms an adrenomedullin-2 (AM 2 ) receptor. The CTR/RAMP complexes form three distinct amylin receptors. While the selective blockade of AM 2 receptors would be therapeutically valuable, inhibition of AM 1 receptors would cause clinically unacceptable increased blood pressure. We report here a systematic study of structure–activity relationships that has led to the development of first-in-class AM 2 receptor antagonists. These compounds exhibit therapeutically valuable properties with 1000-fold selectivity over the AM 1 receptor. These results highlight the therapeutic potential of AM 2 antagonists.

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“…Moreover, the CGRP and AM receptors exhibit pleiotropic Gprotein coupling with the ability to also signal through G q and G i (27), but how the agonists and RAMPs compare in terms of promoting signaling through each G protein remains unresolved. Two groups used cell-based signaling assays in HEK293 or COS-7 cells to examine this issue and found evidence for either dramatic or subtler ligand-and RAMP-dependent G protein signaling bias (28,29). Differences in these reports may be due to the different cell lines used and/or the inherent challenge of studying the signaling bias of these complex heterodimeric receptors in cell-based assays.…”

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confidence: 99%

Biochemical characterization of G protein coupling to calcitonin gene–related peptide and adrenomedullin receptors using a native PAGE assay

Roehrkasse

Warner

Booe

et al. 2020

Journal of Biological Chemistry

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Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) have overlapping and unique functions in the nervous and circulatory systems including vasodilation, cardioprotection, and pain transmission. Their actions are mediated by the class B calcitonin-like G protein–coupled receptor (CLR), which heterodimerizes with three receptor activity–modifying proteins (RAMP1–3) that determine its peptide ligand selectivity. How the three agonists and RAMPs modulate CLR binding to transducer proteins remains poorly understood. Here, we biochemically characterized agonist-promoted G protein coupling to each CLR·RAMP complex. We adapted a native PAGE method to assess the formation and thermostabilities of detergent-solubilized fluorescent protein–tagged CLR·RAMP complexes expressed in mammalian cells. Addition of agonist and the purified Gs protein surrogate mini-Gs (mGs) yielded a mobility-shifted agonist·CLR·RAMP·mGs quaternary complex gel band that was sensitive to antagonists. Measuring the apparent affinities of the agonists for the mGs-coupled receptors and of mGs for the agonist-occupied receptors revealed that both ligand and RAMP control mGs coupling and defined how agonist engagement of the CLR extracellular and transmembrane domains affects transducer recruitment. Using mini-Gsq and -Gsi chimeras, we observed a coupling rank order of mGs > mGsq > mGsi for each receptor. Last, we demonstrated the physiological relevance of the native gel assays by showing that they can predict the cAMP-signaling potencies of AM and AM2/IMD chimeras. These results highlight the power of the native PAGE assay for membrane protein biochemistry and provide a biochemical foundation for understanding the molecular basis of shared and distinct signaling properties of CGRP, AM, and AM2/IMD.

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Molecular Mechanisms of Class B GPCR Activation: Insights from Adrenomedullin Receptors

Cited by 32 publications

References 106 publications

CGRP-receptor family reveals endogenous GPCR agonist bias and its significance in primary human cardiovascular cells

CGRP-receptor family reveals endogenous GPCR agonist bias and its significance in primary human cardiovascular cells

Discovery of a First-In-Class Small Molecule Antagonist against the Adrenomedullin-2 Receptor: Structure–Activity Relationships and Optimization

Biochemical characterization of G protein coupling to calcitonin gene–related peptide and adrenomedullin receptors using a native PAGE assay

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