The relaxin family peptide receptor 3 activates extracellular signal-regulated kinase 1/2 through a protein kinase C-dependent mechanism

Human gene 3 relaxin (H3 relaxin) is a member of the relaxin/insulin family of peptides. It is a neuropeptide involved in mediating behavioural responses to stress and in appetite regulation, however, the cell signalling mechanisms that control these events remain to be identified. The relaxin family peptide receptor 3 (RXFP3, formerly GPCR135 or SALPR) was characterised as the receptor for H3 relaxin, functionally coupled to the inhibition of cAMP. We have identified that RXFP3 stably expressed in CHO-K1 (CHO-RXFP3) and HEK293 (HEK-RXFP3) cells, activates extracellular signal-regulated kinase (ERK) 1/2 when stimulated with H3 relaxin, and a H3 relaxin B-chain (dimer) peptide. Using inhibitors of cellular signalling proteins, we subsequently determined the mechanism of ERK1/2 activation by RXFP3. ERK1/2 phosphorylation requires the activation of Gi/o proteins and receptor internalisation and/or compartmentalisation into lipid-rich environments. ERK1/2 activation also predominantly occurred via activation of a protein kinase C-dependent pathway, although activation of phosphatidylinositol 3-kinase and Src tyrosine kinase were also involved to a lesser extent. The mechanisms underlying ERK1/2 phosphorylation were similar in both CHO-RXFP3 and HEK-RXFP3 cells, although some differences were evident. Phospholipase C beta and the transactivation of endogenous EGF receptors both played a role in RXFP3-mediated ERK1/2 activation in HEK293 cells, however, they were not involved in RXFP3 mediated ERK1/2 activation in the CHO-K1 cell background. The pathways identified in CHO and HEK transfected cells were also utilised in the murine SN56 neuronal cell line, suggesting that these pathways are also important for RXFP3-mediated signalling in the brain.