Interactions between RAMP2 and CRF receptors: The effect of receptor subtypes, splice variants and cell context

Corticotrophin releasing factor (CRF) acts via two family B G-protein-coupled receptors, CRFR1 and CRFR2. Additional subtypes exist due to alternative splicing. CRFR1 alpha is the most widely expressed subtype and lacks a 29-residue insert in the first intracellular loop that is present in CRFR1 beta. It has been shown previously that co-expression of CRFR1 beta 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 CRFR1 alpha, CRFR1 beta and CRFR2 beta 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 [Delta CTR]HEK 293 (which lacks endogenous calcitonin receptor). In all cell-lines, CRFR1 alpha and CRFR1 beta enhanced RAMP2 cell-surface expression. The magnitude of the effect on RAMP2 was dependent on the cell-line ([Delta CTR]HEK 293 > COS-7 > HEK 293T > HEK 293S). RTPCR 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. CRFR2 beta 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 CRFR2 beta (histidine) in this interface region may impair CRFR2 beta:RAMP2 interaction by electrostatic repulsion.