Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor

RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor

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

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY3 receptors (AMY3R) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY3R 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 AMY3Rs, 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 AMY3R, 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.
Original languageEnglish
Pages (from-to)183-197
Number of pages15
JournalACS Pharmacology and Translational Sciences
Volume2
Issue number3
DOIs
Publication statusPublished - 18 Mar 2019

Cite this

@article{2a0b3eed4c6a4a248a93a70512b70fbc,
title = "Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor",
abstract = "Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY3 receptors (AMY3R) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY3R 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 AMY3Rs, 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 AMY3R, 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.",
author = "Vi Pham and Yue Zhu and {Dal Maso}, Emma and Reynolds, {Christopher A.} and Giuseppe Deganutti and Silvia Atanasio and Hick, {Caroline A.} and Dehua Yang and Arthur Christopoulos and Hay, {Debbie L.} and Furness, {Sebastian G. B.} and Ming-Wei Wang and Denise Wootten and Sexton, {Patrick M.}",
year = "2019",
month = "3",
day = "18",
doi = "10.1021/acsptsci.9b00010",
language = "English",
volume = "2",
pages = "183--197",
journal = "ACS Pharmacology and Translational Sciences",
issn = "2575-9108",
publisher = "American Chemical Society",
number = "3",

}

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

In: ACS Pharmacology and Translational Sciences, Vol. 2, No. 3, 18.03.2019, p. 183-197.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor

T2 - RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor

AU - Pham, Vi

AU - Zhu, Yue

AU - Dal Maso, Emma

AU - Reynolds, Christopher A.

AU - Deganutti, Giuseppe

AU - Atanasio, Silvia

AU - Hick, Caroline A.

AU - Yang, Dehua

AU - Christopoulos, Arthur

AU - Hay, Debbie L.

AU - Furness, Sebastian G. B.

AU - Wang, Ming-Wei

AU - Wootten, Denise

AU - Sexton, Patrick M.

PY - 2019/3/18

Y1 - 2019/3/18

N2 - Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY3 receptors (AMY3R) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY3R 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 AMY3Rs, 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 AMY3R, 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.

AB - Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY3 receptors (AMY3R) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY3R 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 AMY3Rs, 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 AMY3R, 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.

U2 - 10.1021/acsptsci.9b00010

DO - 10.1021/acsptsci.9b00010

M3 - Article

VL - 2

SP - 183

EP - 197

JO - ACS Pharmacology and Translational Sciences

JF - ACS Pharmacology and Translational Sciences

SN - 2575-9108

IS - 3

ER -