Interactions Between RAMP2 And CRF Receptors:The Effect Of Receptor Subtypes, Splice Variants And Cell Context

Sian Bailey; Matthew Harris; Kerry Barkan; Ian Winfield; Matthew Thomas Harper; John Simms; Graham Ladds; Mark Wheatley; David Poyner

doi:10.1016/j.bbamem.2019.02.008

Interactions Between RAMP2 And CRF Receptors:The Effect Of Receptor Subtypes, Splice Variants And Cell Context

Sian Bailey, Matthew Harris, Kerry Barkan, Ian Winfield, Matthew Thomas Harper, John Simms, Graham Ladds, Mark Wheatley, David Poyner

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Abstract

Corticotrophin releasing factor (CRF) acts via two family B G-protein-coupled receptors, CRFR1 and CRFR2. Additional subtypes exist due to alternative splicing. CRFR1α is the most widely expressed subtype and lacks a 29-residue insert in the first intracellular loop that is present in CRFR1β. It has been shown previously that co-expression of CRFR1β with receptor activity modifying protein 2 (RAMP2) in HEK 293S cells increased the cell-surface expression of both proteins suggesting a physical interaction as seen with RAMPs and calcitonin receptor-like receptor (CLR). This study investigated the ability of CRFR1α, CRFR1β and CRFR2β to promote cell-surface expression of FLAG-tagged RAMP2. Four different cell-lines were utilised to investigate the effect of varying cellular context; COS-7, HEK 293T, HEK 293S and [ΔCTR]HEK 293 (which lacks endogenous calcitonin receptor). In all cell-lines, CRFR1α and CRFR1β enhanced RAMP2 cell-surface expression. The magnitude of the effect on RAMP2 was dependent on the cell-line ([ΔCTR]HEK 293 > COS-7 > HEK 293T > HEK 293S). RT-PCR indicated this variation may relate to differences in endogenous RAMP expression between cell types. Furthermore, pre-treatment with CRF resulted in a loss of cell-surface FLAG-RAMP2 when it was co-expressed with CRFR1 subtypes. CRFR2β co-expression had no effect on RAMP2 in any cell-line. Molecular modelling suggests that the potential contact interface between the extracellular domains of RAMP2 and CRF receptor subtypes is smaller than that of RAMP2 and CRL, the canonical receptor:RAMP pairing, assuming a physical interaction. Furthermore, a specific residue difference between CRFR1 subtypes (glutamate) and CRFR2β (histidine) in this interface region may impair CRFR2β:RAMP2 interaction by electrostatic repulsion.

Original language	English
Pages (from-to)	997-1003
Number of pages	7
Journal	BBA - Biomembranes
Volume	1861
Issue number	5
Early online date	28 Feb 2019
DOIs	https://doi.org/10.1016/j.bbamem.2019.02.008
Publication status	Published - 1 May 2019

Bibliographical note

NOTICE: this is the author’s version of a work that was accepted for publication in BBA - Biomembranes. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in BBA – Biomembranes, [1861,] [5], (2019)] DOI: 10.1016/j.bbamem.2019.02.008

© 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/

Keywords

Receptor activity modifying protein (RAMP)
Corticotrophin releasing factor receptor (CRFR)
Family B GPCR
Splice variants
Translocation

Access to Document

10.1016/j.bbamem.2019.02.008

Post-PrintAccepted author manuscript, 1.35 MBLicence: CC BY-NC-ND

http://publications.aston.ac.uk/id/eprint/39956/Licence: CC BY-NC-ND

Mark Wheatley
- Centre for Health and Life Sciences - Professor in Biochemical Pharmacology
Person: Teaching and Research

Cite this

@article{0f24a0c087d649d0b4893c3c8ea228fc,

title = "Interactions Between RAMP2 And CRF Receptors:The Effect Of Receptor Subtypes, Splice Variants And Cell Context",

abstract = "Corticotrophin releasing factor (CRF) acts via two family B G-protein-coupled receptors, CRFR1 and CRFR2. Additional subtypes exist due to alternative splicing. CRFR1α is the most widely expressed subtype and lacks a 29-residue insert in the first intracellular loop that is present in CRFR1β. It has been shown previously that co-expression of CRFR1β with receptor activity modifying protein 2 (RAMP2) in HEK 293S cells increased the cell-surface expression of both proteins suggesting a physical interaction as seen with RAMPs and calcitonin receptor-like receptor (CLR). This study investigated the ability of CRFR1α, CRFR1β and CRFR2β to promote cell-surface expression of FLAG-tagged RAMP2. Four different cell-lines were utilised to investigate the effect of varying cellular context; COS-7, HEK 293T, HEK 293S and [ΔCTR]HEK 293 (which lacks endogenous calcitonin receptor). In all cell-lines, CRFR1α and CRFR1β enhanced RAMP2 cell-surface expression. The magnitude of the effect on RAMP2 was dependent on the cell-line ([ΔCTR]HEK 293 > COS-7 > HEK 293T > HEK 293S). RT-PCR indicated this variation may relate to differences in endogenous RAMP expression between cell types. Furthermore, pre-treatment with CRF resulted in a loss of cell-surface FLAG-RAMP2 when it was co-expressed with CRFR1 subtypes. CRFR2β co-expression had no effect on RAMP2 in any cell-line. Molecular modelling suggests that the potential contact interface between the extracellular domains of RAMP2 and CRF receptor subtypes is smaller than that of RAMP2 and CRL, the canonical receptor:RAMP pairing, assuming a physical interaction. Furthermore, a specific residue difference between CRFR1 subtypes (glutamate) and CRFR2β (histidine) in this interface region may impair CRFR2β:RAMP2 interaction by electrostatic repulsion.",

keywords = "Receptor activity modifying protein (RAMP), Corticotrophin releasing factor receptor (CRFR), Family B GPCR, Splice variants, Translocation",

author = "Sian Bailey and Matthew Harris and Kerry Barkan and Ian Winfield and Harper, {Matthew Thomas} and John Simms and Graham Ladds and Mark Wheatley and David Poyner",

note = "NOTICE: this is the author{\textquoteright}s version of a work that was accepted for publication in BBA - Biomembranes. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in BBA – Biomembranes, [1861,] [5], (2019)] DOI: 10.1016/j.bbamem.2019.02.008 {\textcopyright} 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ ",

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T1 - Interactions Between RAMP2 And CRF Receptors:The Effect Of Receptor Subtypes, Splice Variants And Cell Context

AU - Bailey, Sian

AU - Harris, Matthew

AU - Barkan, Kerry

AU - Winfield, Ian

AU - Harper, Matthew Thomas

AU - Simms, John

AU - Ladds, Graham

AU - Wheatley, Mark

AU - Poyner, David

N1 - NOTICE: this is the author’s version of a work that was accepted for publication in BBA - Biomembranes. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in BBA – Biomembranes, [1861,] [5], (2019)] DOI: 10.1016/j.bbamem.2019.02.008 © 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/

PY - 2019/5/1

Y1 - 2019/5/1

N2 - Corticotrophin releasing factor (CRF) acts via two family B G-protein-coupled receptors, CRFR1 and CRFR2. Additional subtypes exist due to alternative splicing. CRFR1α is the most widely expressed subtype and lacks a 29-residue insert in the first intracellular loop that is present in CRFR1β. It has been shown previously that co-expression of CRFR1β with receptor activity modifying protein 2 (RAMP2) in HEK 293S cells increased the cell-surface expression of both proteins suggesting a physical interaction as seen with RAMPs and calcitonin receptor-like receptor (CLR). This study investigated the ability of CRFR1α, CRFR1β and CRFR2β to promote cell-surface expression of FLAG-tagged RAMP2. Four different cell-lines were utilised to investigate the effect of varying cellular context; COS-7, HEK 293T, HEK 293S and [ΔCTR]HEK 293 (which lacks endogenous calcitonin receptor). In all cell-lines, CRFR1α and CRFR1β enhanced RAMP2 cell-surface expression. The magnitude of the effect on RAMP2 was dependent on the cell-line ([ΔCTR]HEK 293 > COS-7 > HEK 293T > HEK 293S). RT-PCR indicated this variation may relate to differences in endogenous RAMP expression between cell types. Furthermore, pre-treatment with CRF resulted in a loss of cell-surface FLAG-RAMP2 when it was co-expressed with CRFR1 subtypes. CRFR2β co-expression had no effect on RAMP2 in any cell-line. Molecular modelling suggests that the potential contact interface between the extracellular domains of RAMP2 and CRF receptor subtypes is smaller than that of RAMP2 and CRL, the canonical receptor:RAMP pairing, assuming a physical interaction. Furthermore, a specific residue difference between CRFR1 subtypes (glutamate) and CRFR2β (histidine) in this interface region may impair CRFR2β:RAMP2 interaction by electrostatic repulsion.

AB - Corticotrophin releasing factor (CRF) acts via two family B G-protein-coupled receptors, CRFR1 and CRFR2. Additional subtypes exist due to alternative splicing. CRFR1α is the most widely expressed subtype and lacks a 29-residue insert in the first intracellular loop that is present in CRFR1β. It has been shown previously that co-expression of CRFR1β with receptor activity modifying protein 2 (RAMP2) in HEK 293S cells increased the cell-surface expression of both proteins suggesting a physical interaction as seen with RAMPs and calcitonin receptor-like receptor (CLR). This study investigated the ability of CRFR1α, CRFR1β and CRFR2β to promote cell-surface expression of FLAG-tagged RAMP2. Four different cell-lines were utilised to investigate the effect of varying cellular context; COS-7, HEK 293T, HEK 293S and [ΔCTR]HEK 293 (which lacks endogenous calcitonin receptor). In all cell-lines, CRFR1α and CRFR1β enhanced RAMP2 cell-surface expression. The magnitude of the effect on RAMP2 was dependent on the cell-line ([ΔCTR]HEK 293 > COS-7 > HEK 293T > HEK 293S). RT-PCR indicated this variation may relate to differences in endogenous RAMP expression between cell types. Furthermore, pre-treatment with CRF resulted in a loss of cell-surface FLAG-RAMP2 when it was co-expressed with CRFR1 subtypes. CRFR2β co-expression had no effect on RAMP2 in any cell-line. Molecular modelling suggests that the potential contact interface between the extracellular domains of RAMP2 and CRF receptor subtypes is smaller than that of RAMP2 and CRL, the canonical receptor:RAMP pairing, assuming a physical interaction. Furthermore, a specific residue difference between CRFR1 subtypes (glutamate) and CRFR2β (histidine) in this interface region may impair CRFR2β:RAMP2 interaction by electrostatic repulsion.

KW - Receptor activity modifying protein (RAMP)

KW - Corticotrophin releasing factor receptor (CRFR)

KW - Family B GPCR

KW - Splice variants

KW - Translocation

U2 - 10.1016/j.bbamem.2019.02.008

DO - 10.1016/j.bbamem.2019.02.008

M3 - Article

SN - 0005-2736

VL - 1861

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EP - 1003

JO - BBA - Biomembranes

JF - BBA - Biomembranes

IS - 5

ER -

Interactions Between RAMP2 And CRF Receptors:The Effect Of Receptor Subtypes, Splice Variants And Cell Context

Abstract

Bibliographical note

Keywords

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Mark Wheatley

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