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 and Poyner, David R (2019). Interactions Between RAMP2 And CRF Receptors:The Effect Of Receptor Subtypes, Splice Variants And Cell Context. BBA -Biomembranes, 1861 (5), pp. 997-1003.

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.

Publication DOI: https://doi.org/10.1016/j.bbamem.2019.02.008
Divisions: Life & Health Sciences
Life & Health Sciences > Pharmacy
Additional Information: © 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.
Uncontrolled Keywords: Corticotrophin releasing factor receptor (CRFR),Family B GPCR,Receptor activity modifying protein (RAMP),Splice variants,Translocation,Biophysics,Biochemistry,Cell Biology
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Related URLs: https://www.sci ... 0471?via%3Dihub (Publisher URL)
http://www.scop ... tnerID=8YFLogxK (Scopus URL)
Published Online Date: 2019-05-01
Published Date: 2019-05-01
Authors: Bailey, Sian
Harris, Matthew
Barkan, Kerry
Winfield, Ian
Thomas-Harper, Matthew
Simms, John W
Ladds, Graham
Wheatley, Mark
Poyner, David R ( 0000-0003-1590-112X)

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Version: Accepted Version

Access Restriction: Restricted to Repository staff only until 28 February 2020.

License: Creative Commons Attribution Non-commercial No Derivatives


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