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 W Simms, Graham Ladds, Mark Wheatley, David R Poyner

Research output: Contribution to journalArticle

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 languageEnglish
Pages (from-to)997-1003
Number of pages7
JournalBBA -Biomembranes
Volume1861
Issue number5
Early online date28 Feb 2019
DOIs
Publication statusPublished - 1 May 2019

Fingerprint

Corticotropin-Releasing Hormone Receptors
Corticotropin-Releasing Hormone
Calcitonin Receptors
Cell Line
Histidine
Glutamic Acid
Proteins
HEK293 Cells
Polymerase Chain Reaction

Bibliographical note

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

Funding: BBSRC - BB/M00015X/2, BB/M007529/1, BBSRC Doctoral Training Partnership Grant BB/JO14540/1, BBSRC-MIBTP award, and The Leverhulme Trust Grant number DBG3000.

Keywords

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

Cite this

Bailey, Sian ; Harris, Matthew ; Barkan, Kerry ; Winfield, Ian ; Thomas-Harper, Matthew ; Simms, John W ; Ladds, Graham ; Wheatley, Mark ; Poyner, David R. / Interactions Between RAMP2 And CRF Receptors : The Effect Of Receptor Subtypes, Splice Variants And Cell Context. In: BBA -Biomembranes. 2019 ; Vol. 1861, No. 5. pp. 997-1003.
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Bailey, S, Harris, M, Barkan, K, Winfield, I, Thomas-Harper, M, Simms, JW, Ladds, G, Wheatley, M & Poyner, DR 2019, 'Interactions Between RAMP2 And CRF Receptors: The Effect Of Receptor Subtypes, Splice Variants And Cell Context', BBA -Biomembranes, vol. 1861, no. 5, pp. 997-1003. https://doi.org/10.1016/j.bbamem.2019.02.008

Interactions Between RAMP2 And CRF Receptors : The Effect Of Receptor Subtypes, Splice Variants And Cell Context. / Bailey, Sian; Harris, Matthew; Barkan, Kerry; Winfield, Ian; Thomas-Harper, Matthew; Simms, John W; Ladds, Graham; Wheatley, Mark; Poyner, David R.

In: BBA -Biomembranes, Vol. 1861, No. 5, 01.05.2019, p. 997-1003.

Research output: Contribution to journalArticle

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T1 - Interactions Between RAMP2 And CRF Receptors

T2 - The Effect Of Receptor Subtypes, Splice Variants And Cell Context

AU - Bailey, Sian

AU - Harris, Matthew

AU - Barkan, Kerry

AU - Winfield, Ian

AU - Thomas-Harper, Matthew

AU - Simms, John W

AU - Ladds, Graham

AU - Wheatley, Mark

AU - Poyner, David R

N1 - © 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ Funding: BBSRC - BB/M00015X/2, BB/M007529/1, BBSRC Doctoral Training Partnership Grant BB/JO14540/1, BBSRC-MIBTP award, and The Leverhulme Trust Grant number DBG3000.

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 - Corticotrophin releasing factor receptor (CRFR)

KW - Family B GPCR

KW - Receptor activity modifying protein (RAMP)

KW - Splice variants

KW - Translocation

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