Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors

Cathryn Weston; Ian Winfield; Matthew Harris; Rose Hodgson; Archna Shah; Simon J. Dowell; Juan Carlos Mobarec; David A. Woodlock; Christopher A. Reynolds; David R. Poyner; Harriet A. Watkins; Graham Ladds

doi:10.1074/jbc.M116.751362

Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors

Cathryn Weston, Ian Winfield, Matthew Harris, Rose Hodgson, Archna Shah, Simon J. Dowell, Juan Carlos Mobarec, David A. Woodlock, Christopher A. Reynolds, David R. Poyner, Harriet A. Watkins, Graham Ladds^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

The calcitonin gene-related peptide (CGRP) family of G protein- coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in aGα_s-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gα_s and Gα_q but also identify a Gα_i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMPdependent signaling bias among the Gα_s, Gα_i, and Gα_q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.

Original language	English
Pages (from-to)	21925-21944
Number of pages	20
Journal	Journal of Biological Chemistry
Volume	291
Issue number	42
Early online date	26 Aug 2016
DOIs	https://doi.org/10.1074/jbc.M116.751362
Publication status	Published - 14 Oct 2016

Bibliographical note

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Creative Commons CC-BY license.

Funding: National Heart Foundation of New Zealand; University of Auckland seed fund; BBSRC (BB/M00015X/1, BB/M000176/1 and BB/M006883/1 and BBSRC Doctoral Training Partnership Grant BB/JO14540/1); MRC Doctoral Training Partnership MR/J003964/1; Warwick Impact Fund (RD13301) from the Warwick Research Development Fund and the Warwick Undergraduate Research Scholarship Scheme.

This article contains supplemental Movies 1 and 2.

Keywords

G protein-coupled receptor
GPCR
molecular dynamics
molecular modeling
signal transduction
yeast
adrenomedullin
adrenomedullin 2
RAMP
signal bias

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10.1074/jbc.M116.751362Licence: CC BY 3.0

Receptor activity-modifying protein-directed G protein
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Creative Commons CC-BY license.
Final published version, 4.02 MBLicence: CC BY 3.0

Cite this

Weston, C., Winfield, I., Harris, M., Hodgson, R., Shah, A., Dowell, S. J., Mobarec, J. C., Woodlock, D. A., Reynolds, C. A., Poyner, D. R., Watkins, H. A., & Ladds, G. (2016). Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors. Journal of Biological Chemistry, 291(42), 21925-21944. https://doi.org/10.1074/jbc.M116.751362

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abstract = "The calcitonin gene-related peptide (CGRP) family of G protein- coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in aGαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMPdependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.",

keywords = "G protein-coupled receptor, GPCR, molecular dynamics, molecular modeling, signal transduction, yeast, adrenomedullin, adrenomedullin 2, RAMP, signal bias",

author = "Cathryn Weston and Ian Winfield and Matthew Harris and Rose Hodgson and Archna Shah and Dowell, {Simon J.} and Mobarec, {Juan Carlos} and Woodlock, {David A.} and Reynolds, {Christopher A.} and Poyner, {David R.} and Watkins, {Harriet A.} and Graham Ladds",

note = "{\textcopyright} 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Creative Commons CC-BY license. Funding: National Heart Foundation of New Zealand; University of Auckland seed fund; BBSRC (BB/M00015X/1, BB/M000176/1 and BB/M006883/1 and BBSRC Doctoral Training Partnership Grant BB/JO14540/1); MRC Doctoral Training Partnership MR/J003964/1; Warwick Impact Fund (RD13301) from the Warwick Research Development Fund and the Warwick Undergraduate Research Scholarship Scheme. This article contains supplemental Movies 1 and 2.",

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Weston, C, Winfield, I, Harris, M, Hodgson, R, Shah, A, Dowell, SJ, Mobarec, JC, Woodlock, DA, Reynolds, CA, Poyner, DR, Watkins, HA & Ladds, G 2016, 'Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors', Journal of Biological Chemistry, vol. 291, no. 42, pp. 21925-21944. https://doi.org/10.1074/jbc.M116.751362

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T1 - Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors

AU - Weston, Cathryn

AU - Winfield, Ian

AU - Harris, Matthew

AU - Hodgson, Rose

AU - Shah, Archna

AU - Dowell, Simon J.

AU - Mobarec, Juan Carlos

AU - Woodlock, David A.

AU - Reynolds, Christopher A.

AU - Poyner, David R.

AU - Watkins, Harriet A.

AU - Ladds, Graham

N1 - © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Creative Commons CC-BY license. Funding: National Heart Foundation of New Zealand; University of Auckland seed fund; BBSRC (BB/M00015X/1, BB/M000176/1 and BB/M006883/1 and BBSRC Doctoral Training Partnership Grant BB/JO14540/1); MRC Doctoral Training Partnership MR/J003964/1; Warwick Impact Fund (RD13301) from the Warwick Research Development Fund and the Warwick Undergraduate Research Scholarship Scheme. This article contains supplemental Movies 1 and 2.

PY - 2016/10/14

Y1 - 2016/10/14

N2 - The calcitonin gene-related peptide (CGRP) family of G protein- coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in aGαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMPdependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.

AB - The calcitonin gene-related peptide (CGRP) family of G protein- coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in aGαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMPdependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.

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KW - GPCR

KW - molecular dynamics

KW - molecular modeling

KW - signal transduction

KW - yeast

KW - adrenomedullin

KW - adrenomedullin 2

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JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

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ER -

Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors

Abstract

Bibliographical note

Keywords

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