Interactions between RAMP2 and CRF receptors: The effect of receptor subtypes, splice variants and cell context.
Biochim Biophys Acta Biomembr
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Bailey, S., Harris, M., Barkan, K., Winfield, I., Harper, M., Simms, J., Ladds, G., et al. (2019). Interactions between RAMP2 and CRF receptors: The effect of receptor subtypes, splice variants and cell context.. Biochim Biophys Acta Biomembr, 1861 (5), 997-1003. https://doi.org/10.1016/j.bbamem.2019.02.008
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.
COS Cells, Animals, Humans, Receptors, Corticotropin-Releasing Hormone, Alternative Splicing, Models, Molecular, HEK293 Cells, Receptor Activity-Modifying Protein 2, Chlorocebus aethiops
Biotechnology and Biological Sciences Research Council (BB/M00015X/2)
Leverhulme Trust (via University of Essex) (DBG3000)
Biotechnology and Biological Sciences Research Council (1643678)
External DOI: https://doi.org/10.1016/j.bbamem.2019.02.008
This record's URL: https://www.repository.cam.ac.uk/handle/1810/290194