Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs

Michael J. Woolley, Christopher A. Reynolds, John Simms, Christopher S. Walker, Juan Carlos Mobarec, Michael L. Garelja, Alex C. Conner, David R. Poyner, Debbie L. Hay

Research output: Contribution to journalArticle

Abstract

Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs.
Original languageEnglish
JournalBiochemical Pharmacology
Volumein press
Early online date11 Jul 2017
DOIs
Publication statusE-pub ahead of print - 11 Jul 2017

Fingerprint

Receptor Activity-Modifying Proteins
Calcitonin Receptor-Like Protein
Adrenomedullin Receptors
Calcitonin Gene-Related Peptide Receptors
Adrenomedullin
Chemical activation
Calcitonin Gene-Related Peptide
Molecular Dynamics Simulation
Molecular dynamics
Mutagenesis
Computer simulation
G-Protein-Coupled Receptors
Hydrophobic and Hydrophilic Interactions
Alanine
Conformations
Binding Sites
Ligands
Peptides
Mutation

Bibliographical note

© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Funding: British Heart Foundation Grant (PG/12/59/29795); Royal Society of New Zealand Marsden Fund; BBSRC (BB/M000176/1 and BB/M006883/1); and University of Auckland Health Doctoral Scholarship.

Keywords

  • Adrenomedullin
  • Calcitonin gene-related peptide
  • GPCR
  • Molecular dynamics
  • Molecular modeling
  • Receptor activity-modifying protein

Cite this

Woolley, Michael J. ; Reynolds, Christopher A. ; Simms, John ; Walker, Christopher S. ; Mobarec, Juan Carlos ; Garelja, Michael L. ; Conner, Alex C. ; Poyner, David R. ; Hay, Debbie L. / Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs. In: Biochemical Pharmacology. 2017 ; Vol. in press.
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abstract = "Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs.",
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Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs. / Woolley, Michael J.; Reynolds, Christopher A.; Simms, John; Walker, Christopher S.; Mobarec, Juan Carlos; Garelja, Michael L.; Conner, Alex C.; Poyner, David R.; Hay, Debbie L.

In: Biochemical Pharmacology, Vol. in press, 11.07.2017.

Research output: Contribution to journalArticle

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T1 - Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs

AU - Woolley, Michael J.

AU - Reynolds, Christopher A.

AU - Simms, John

AU - Walker, Christopher S.

AU - Mobarec, Juan Carlos

AU - Garelja, Michael L.

AU - Conner, Alex C.

AU - Poyner, David R.

AU - Hay, Debbie L.

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N2 - Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs.

AB - Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs.

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KW - Calcitonin gene-related peptide

KW - GPCR

KW - Molecular dynamics

KW - Molecular modeling

KW - Receptor activity-modifying protein

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