Development of an ERK1/2 activation assay to determine relaxin-3/RXFP3 activation

OBJECTIVE To develop ERK1/2 activation assays to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS Plated HEK-RXFP3,CHO-RXFP3,HEK293 Tand CHO-K1 cells in poly-L-lysine coated well plates.The cells were serum starved and treated with either human relaxin-3(H3relaxin)(10nmol·L-1),R3B1-22R(10μmol·L-1)and pertussis toxin(PTX,100ng·mL-1).The cells were lysed and the ERK1/2 activation was determined by SDS-PAGE followed by immunoblotting for phosphorylated ERK1/2(pERK1/2)and total ERK1/2(tERK1/2)for the lysates.RESULTSThe quantification of the data revealed that the peak of ERK1/2activation can be detected precisely at 10 min post stimulation with 10nmol·L-1 H3 relaxin in both HEK-RXFP3 and CHO-RXFP3 cell lines in all three trials compared to the cells treated with vehicle(P<0.05).However,HEK-RXFP3 cells demonstrated a transient activation of ERK1/2and CHORXFP3 cells demonstrated a continuous activation of ERK1/2which was inhibited by the Gi inhibitor,PTX.Activation of ERK1/2was significantly inhibited by pre-treating the cells with RXFP3 antagonist R3B1-22 Rin HEK-RXFP3 cells.ERK1/2 activation was observed neither in wild type HEK293 Tnor in CHO-K1 cells.CONCLUSION The developed assay can detect RXFP3 activation or inhibition by agonists and antagonists via the detection of pERK1/2 in multiple cell lines.This assay will also be useful to detect signaling pathways upstream to ERK1/2 activation mediated by RXFP3.Activation of ERK1/2 in CHO-RXFP3 cells was mediated by Gi proteins at 10 min as well as at 25-35 min time points.