Relaxin family peptide receptors RXFP1 and RXFP2 modulate cAMP signaling by distinct mechanisms

Two orphan leucine-rich repeat-containing G protein-coupled receptors were recently identified as targets for the relaxin family peptides relaxin and insulin-like peptide (INSL) 3. Human gene 2 relaxin is the cognate ligand for relaxin family peptide receptor (RXFP) 1, whereas INSL3 is the ligand for RXFP2. Constitutively active mutants of both receptors when expressed in human embryonic kidney (HEK) 293T cells signal through G alpha(s) to increase cAMP. However, recent studies using cells that endogenously express the receptors revealed greater complexity: cAMP accumulation after activation of RXFP1 involves a time-dependent biphasic pathway with a delayed phase involving phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) zeta, whereas the RXFP2 response involves inhibition of adenylate cyclase via pertussis toxin-sensitive G proteins. The aim of this study was to compare and contrast the cAMP signaling pathways used by these two related receptors. In HEK293T cells stably transfected with RXFP1, preliminary studies confirmed the biphasic cAMP response, with an initial G alpha(s) component and a delayed response involving PI3K and PKC zeta. This delayed pathway was dependent upon G-beta gamma subunits derived from G alpha(i3). An additional inhibitory pathway involving G alpha(oB) affecting cAMP accumulation was also identified. In HEK293T cells stably transfected with RXFP2, the cAMP response involved G alpha(s) and was modulated by inhibition mediated by G alpha(oB) and release of inhibitory G-beta gamma subunits. Thus, initially both RXFP1 and RXFP2 couple to G(alpha)s and an inhibitory G alpha(oB) pathway. Differences in cAMP accumulation stem from the ability of RXFP1 to recruit coupling to G alpha(i3), release G-beta gamma subunits and thus activate a delayed PI3K-PKC zeta pathway to further increase cAMP accumulation.