Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy

Maso, Emma Dal; Zhu, Yue; Pham, Vi; Reynolds, Christopher A.; Deganutti, Giuseppe; Hick, Caroline A.; Yang, Dehua; Christopoulos, Arthur; Hay, Debbie L.; Wang, Ming Wei; Sexton, Patrick M.; Furness, Sebastian G.B.; Wootten, Denise

doi:10.1016/j.bcp.2018.02.005

Cited by 25 publications

(50 citation statements)

References 58 publications

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“…We observed no β-arrestin recruitment at the A1R using either BnOCPA, CPA or adenosine ( Supplementary Fig. 3), observations that are consistent with those previously reported for recombinant A1Rs expressing native sequences 30,31,32,33,34 . The lack of β-arrestin recruitment is likely due to the lack of serine and threonine residues in the A1R cytoplasmic tail, which makes the A1R intrinsically biased against β-arrestin signalling 17,35 .…”

Section: Bnocpa Demonstrates Unique Gα Signalling Bias In the Selectisupporting

confidence: 92%

“…Intracellular loop 3 (ICL3) which is missing from PDB ID 6D9H was rebuilt using Modeller 9.19 28,29 . The G protein, with the exception of the C-terminal helix (helix 5) of the G protein alpha subunit (the key region responsible for the receptor TM6 active-like conformation) was removed from the system as in previous work 30,31 . BnOCPA and HOCPA were placed in the extracellular bulk, in two different systems, at least 20 Å from the receptor vestibule.…”

Section: Molecular Dynamics Simulationsmentioning

confidence: 99%

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Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression

Wall

Hill

Huckstepp

et al. 2020

Preprint

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One third of all medicines act at G protein-coupled receptors (GPCRs). However, the identification of new therapeutic GPCR targets is limited by a fundamental property of GPCRs: their propensity to couple to different G protein alpha subunits (Gα) leading to multiple downstream cellular effects. This is especially the case for adenosine A1 receptors (A1Rs), the activation of which results in unwanted cardiorespiratory effects, severely limiting their clinical potential. We have discovered that the novel A1R agonist, BnOCPA, unlike typical A1R agonists, has a unique and highly selective Gα activation preference. Moreover, we found that BnOCPA is a potent and powerful analgesic without causing bradycardia, hypotension or respiratory depression. BnOCPA thus demonstrates a hitherto unknown Gα-selective activation of the native A1R, sheds new light on the fundamentals of GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of biased agonism. Abbreviated summary:We describe the selective activation of an adenosine A1 receptor-mediated intracellular pathway that provides potent analgesia in the absence of cardiorespiratory depression, paving the way for novel medicines based on the far-reaching concept of biased agonism. Wall et al., Main Text and Figures4 Main text: G protein-coupled receptors (GPCRs) are the targets of many FDA-approved drugs (1). However, the promiscuity with which they couple to multiple intracellular signalling cascades leads to unwanted side effects, and limits both the range of GPCRs suitable for drug-targeting, and the number of conditions for which treatments could be developed (2). The four GPCRs for the purine nucleoside adenosine have particularly suffered as drug targets due to their promiscuous coupling, despite their potential for treating many pathological conditions including cancer, cardiovascular, neurological and inflammatory diseases (3-5). For example, activation of the widely-distributed adenosine A1 receptor (A1R) with currently available agonists elicits multiple actions in both the central nervous system (CNS), such as the inhibition of synaptic transmission and neuronal hyperpolarization, and in the cardiorespiratory system through slowing the heart (bradycardia), reducing blood pressure (hypotension) and affecting respiration (dyspnea) (5-8). These multiple effects severely limit the prospects of A1R agonists as life-changing medicines, despite their potential in a wide range of clinical conditions including pain, epilepsy and cerebral ischemia (5,(9)(10)(11).

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Section: Bnocpa Demonstrates Unique Gα Signalling Bias In the Selectisupporting

confidence: 92%

Section: Molecular Dynamics Simulationsmentioning

confidence: 99%

Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression

Wall

Hill

Huckstepp

et al. 2020

Preprint

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“…There was a marked decrease in surface expression of CTR in the AMY 3 R for the R281A, N286A, D287A, C289A, W290A, T295A, L297A, L298A, Y299A, and I300A mutants within ECL2, with decreased expression to a lesser extent also seen with Y284A, F285A, L291A, and S292A mutants within this loop ( Figure 1B,C). In general, the pattern of effect was similar to that seen with the CTR expressed alone 17 ( Figure 1E,F) although greater loss of expression was seen for the L291A and S292A mutants when the receptor was coexpressed with RAMP3. Alanine mutation within ECL3 had less overall impact relative to ECL2, with moderate, significant, decreases in expression for F356A, V357A, P363A, N365A, L286A, D373A, and Y374A, and increases for F359A and P360A ( Figure 1B,C).…”

Section: ■ Results and Discussionsupporting

confidence: 57%

“…Alanine mutants that selectively increased cAMP functional affinity for pCT clustered away from the peptide binding site and were located on the periphery of the receptor transmembrane domain. This region would be predicted to interact with the membrane bilayer, suggesting that these candidate receptor-membrane interactions constrain the receptor in a way that limits pCT functional affinity when RAMP3 is present, as the effect of mutation was not seen with mutants of CTR alone 17 ( Figure 6C versus Figure 6H). For the other peptides, there was limited effect of loop mutation on functional affinity for either pathway, with the quantifiable effects primarily occurring within residues involved in packing of the ECL2 in the active structures ( Figure 5).…”

Section: Acs Pharmacology and Translational Sciencementioning

confidence: 99%

Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor

Pham

Zhu

Maso

et al. 2019

ACS Pharmacol. Transl. Sci.

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Amylin is coexpressed with insulin in pancreatic islet βcells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY 3 receptors (AMY 3 R) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY 3 R is thus important for peptide drug targeting of this receptor. We have previously used alanine scanning mutagenesis to study the contribution of the extracellular surface of the CTR to binding and signaling initiated by calcitonin (CT) and related peptides (Dal Maso, E., et al. (2019) The molecular control of calcitonin receptor signaling. ACS Pharmacol. Transl. Sci. 2, 31−51). That work revealed ligand-and pathway-specific effects of mutation, with extracellular loops (ECLs) 2 and 3 particularly important in the distinct propagation of signaling mediated by individual peptides. In the current study, we have used equivalent alanine scanning of ECL2 and ECL3 of the CTR in the context of coexpression with RAMP3 to form AMY 3 Rs, to examine functional affinity and efficacy of peptides in cAMP accumulation and extracellular signal-regulated kinase (ERK) phosphorylation (pERK). The effect of mutation was determined on representatives of the three major distinct classes of CT peptide, salmon CT (sCT), human CT (hCT), and porcine CT (pCT), as well as rat amylin (rAmy) or human α-CGRP (calcitonin gene-related peptide, hCGRP) whose potency is enhanced by RAMP interaction. We demonstrate that the dynamic nature of CTR ECL2 and ECL3 in propagation of signaling is fundamentally altered when complexed with RAMP3 to form the AMY 3 R, despite only having predicted direct interactions with ECL2. Moreover, the work shows that the role of these loops in receptor signaling is highly peptide dependent, illustrating that even subtle changes to peptide sequence may change signaling output downstream of the receptor.

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“…it was crucial for signaling of all three peptides. In the active agonist-bound calcitonin receptor, there is a high degree of overlap in the structural organization of ECL2 despite considerable sequence variation; Arg-281 that is positionally equivalent to Lys-288 of the GLP-1R appears to play a similar coordinating role in maintenance of this structure (19,25). The organization of the ECL2 network is also required for calcium signaling, although it does not appear as important for peptidemediated pERK (Figs.…”

Section: Structural Insights Into Glp-1r Biased Agonismmentioning

confidence: 99%

Two distinct domains of the glucagon-like peptide-1 receptor control peptide-mediated biased agonism

Lei

Clydesdale

Dai

et al. 2018

Journal of Biological Chemistry

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G protein-coupled receptors (GPCRs) can be differentially activated by ligands to generate multiple and distinct downstream signaling profiles, a phenomenon termed biased agonism. The glucagon-like peptide-1 receptor (GLP-1R) is a class B GPCR and a key drug target for managing metabolic disorders; however, its peptide agonists display biased signaling that affects their relative efficacies. In this study, we combined mutagenesis experiments and mapping of surface mutations onto recently described GLP-1R structures, which revealed two major domains in the GLP-1/GLP-1R/G protein active structure that are differentially important for both receptor quiescence and ligand-specific initiation and propagation of biased agonism. Changes to the conformation of transmembrane helix (TM) 5 and TM 6 and reordering of extracellular loop 2 were essential for the propagation of signaling linked to cAMP formation and intracellular calcium mobilization, whereas ordering and packing of residues in TMs 1 and 7 were critical for extracellular signal-regulated kinase 1/2 (pERK) activity. On the basis of these findings, we propose a model of distinct peptide-receptor interactions that selectively control how these different signaling pathways are engaged. This work provides important structural insight into class B GPCR activation and biased agonism.

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Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy

Abstract: Graphical abstract

Cited by 25 publications

References 58 publications

Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression

Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression

Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor

Two distinct domains of the glucagon-like peptide-1 receptor control peptide-mediated biased agonism

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