Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties

Harriet A. Watkins, Madhuri Chakravarthy, Rekhati S. Abhayawardana, Joseph J. Gingell, Michael Garelja, Meenakshi Pardamwar, James M.W.R. McElhinney, Alex Lathbridge, Arran Constantine, Paul W.R. Harris, Tsz-Ying Yuen, Margaret A. Brimble, James Barwell, David R. Poyner, Michael J. Woolley, Alex C. Conner, Augen A. Pioszak, Christopher A. Reynolds, Debbie L. Hay

Research output: Contribution to journalArticle

Abstract

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins (RAMP) 2 and 3, respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMPs 2 and 3 on the activation and conformation of the CLR subunit of AM receptors we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors and determined the effects on cAMP signalling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modelling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket, and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

LanguageEnglish
Pages11657-11675
Number of pages19
JournalJournal of Biological Chemistry
Volume291
Issue number22
Early online date24 Mar 2016
DOIs
Publication statusPublished - 27 May 2016

Fingerprint

Receptor Activity-Modifying Protein 3
Receptor Activity-Modifying Protein 2
Calcitonin Receptor-Like Protein
Adrenomedullin Receptors
Adrenomedullin
G-Protein-Coupled Receptors
Receptor Activity-Modifying Proteins
Chemical activation
Molecular modeling
Peptide Hormones
Accessories
Static Electricity
Blood Vessels
Conformations
Electrostatics
Pharmacology
Ligands
Amino Acids

Bibliographical note

Final version free via Creative Commons CC-BY license.

Funding: BBSRC (BB/M006883/1; BB/M007529/1 and BB/M000176/1)

Supplemental material: http://www.jbc.org/content/suppl/2016/04/13/M115.688218.DC1.htm

Keywords

  • allosteric regulation
  • cardiovascular disease
  • conformational change
  • G protein-coupled receptor
  • GPCR
  • molecular modeling
  • RAMP
  • adrenomedullin
  • extracellular loops
  • receptor activity-modifying protein

Cite this

Watkins, H. A., Chakravarthy, M., Abhayawardana, R. S., Gingell, J. J., Garelja, M., Pardamwar, M., ... Hay, D. L. (2016). Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties. Journal of Biological Chemistry, 291(22), 11657-11675. https://doi.org/10.1074/jbc.M115.688218
Watkins, Harriet A. ; Chakravarthy, Madhuri ; Abhayawardana, Rekhati S. ; Gingell, Joseph J. ; Garelja, Michael ; Pardamwar, Meenakshi ; McElhinney, James M.W.R. ; Lathbridge, Alex ; Constantine, Arran ; Harris, Paul W.R. ; Yuen, Tsz-Ying ; Brimble, Margaret A. ; Barwell, James ; Poyner, David R. ; Woolley, Michael J. ; Conner, Alex C. ; Pioszak, Augen A. ; Reynolds, Christopher A. ; Hay, Debbie L. / Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 22. pp. 11657-11675.
@article{7c9514b69a934059a06d87ffc6f337ca,
title = "Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties",
abstract = "Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins (RAMP) 2 and 3, respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMPs 2 and 3 on the activation and conformation of the CLR subunit of AM receptors we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors and determined the effects on cAMP signalling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modelling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket, and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.",
keywords = "allosteric regulation, cardiovascular disease, conformational change, G protein-coupled receptor, GPCR, molecular modeling, RAMP, adrenomedullin, extracellular loops, receptor activity-modifying protein",
author = "Watkins, {Harriet A.} and Madhuri Chakravarthy and Abhayawardana, {Rekhati S.} and Gingell, {Joseph J.} and Michael Garelja and Meenakshi Pardamwar and McElhinney, {James M.W.R.} and Alex Lathbridge and Arran Constantine and Harris, {Paul W.R.} and Tsz-Ying Yuen and Brimble, {Margaret A.} and James Barwell and Poyner, {David R.} and Woolley, {Michael J.} and Conner, {Alex C.} and Augen A. Pioszak and Reynolds, {Christopher A.} and Hay, {Debbie L.}",
note = "Final version free via Creative Commons CC-BY license. Funding: BBSRC (BB/M006883/1; BB/M007529/1 and BB/M000176/1) Supplemental material: http://www.jbc.org/content/suppl/2016/04/13/M115.688218.DC1.htm",
year = "2016",
month = "5",
day = "27",
doi = "10.1074/jbc.M115.688218",
language = "English",
volume = "291",
pages = "11657--11675",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "22",

}

Watkins, HA, Chakravarthy, M, Abhayawardana, RS, Gingell, JJ, Garelja, M, Pardamwar, M, McElhinney, JMWR, Lathbridge, A, Constantine, A, Harris, PWR, Yuen, T-Y, Brimble, MA, Barwell, J, Poyner, DR, Woolley, MJ, Conner, AC, Pioszak, AA, Reynolds, CA & Hay, DL 2016, 'Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties' Journal of Biological Chemistry, vol. 291, no. 22, pp. 11657-11675. https://doi.org/10.1074/jbc.M115.688218

Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties. / Watkins, Harriet A.; Chakravarthy, Madhuri; Abhayawardana, Rekhati S.; Gingell, Joseph J.; Garelja, Michael; Pardamwar, Meenakshi; McElhinney, James M.W.R.; Lathbridge, Alex; Constantine, Arran; Harris, Paul W.R.; Yuen, Tsz-Ying; Brimble, Margaret A.; Barwell, James; Poyner, David R.; Woolley, Michael J.; Conner, Alex C.; Pioszak, Augen A.; Reynolds, Christopher A.; Hay, Debbie L.

In: Journal of Biological Chemistry, Vol. 291, No. 22, 27.05.2016, p. 11657-11675.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties

AU - Watkins, Harriet A.

AU - Chakravarthy, Madhuri

AU - Abhayawardana, Rekhati S.

AU - Gingell, Joseph J.

AU - Garelja, Michael

AU - Pardamwar, Meenakshi

AU - McElhinney, James M.W.R.

AU - Lathbridge, Alex

AU - Constantine, Arran

AU - Harris, Paul W.R.

AU - Yuen, Tsz-Ying

AU - Brimble, Margaret A.

AU - Barwell, James

AU - Poyner, David R.

AU - Woolley, Michael J.

AU - Conner, Alex C.

AU - Pioszak, Augen A.

AU - Reynolds, Christopher A.

AU - Hay, Debbie L.

N1 - Final version free via Creative Commons CC-BY license. Funding: BBSRC (BB/M006883/1; BB/M007529/1 and BB/M000176/1) Supplemental material: http://www.jbc.org/content/suppl/2016/04/13/M115.688218.DC1.htm

PY - 2016/5/27

Y1 - 2016/5/27

N2 - Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins (RAMP) 2 and 3, respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMPs 2 and 3 on the activation and conformation of the CLR subunit of AM receptors we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors and determined the effects on cAMP signalling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modelling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket, and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

AB - Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins (RAMP) 2 and 3, respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMPs 2 and 3 on the activation and conformation of the CLR subunit of AM receptors we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors and determined the effects on cAMP signalling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modelling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket, and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

KW - allosteric regulation

KW - cardiovascular disease

KW - conformational change

KW - G protein-coupled receptor

KW - GPCR

KW - molecular modeling

KW - RAMP

KW - adrenomedullin

KW - extracellular loops

KW - receptor activity-modifying protein

UR - http://www.scopus.com/inward/record.url?scp=84971290136&partnerID=8YFLogxK

U2 - 10.1074/jbc.M115.688218

DO - 10.1074/jbc.M115.688218

M3 - Article

VL - 291

SP - 11657

EP - 11675

JO - Journal of Biological Chemistry

T2 - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 22

ER -