The relaxin family peptide receptors (RXFP) 1 and 2 are targets for the relaxin family peptides relaxin and insulin-like peptide 3 (INSL3), respectively. Although both receptors and peptides share a high degree of sequence identity, the cAMP signaling pathways activated by the two systems are quite distinct. Relaxin activation of RXFP1 initially results in accumulation of cAMP via G alpha(s), but this is modulated by inhibition of cAMP through G alpha(oB). Over time, RXFP1 recruits coupling to G alpha(i3), causing additional cAMP accumulation via a G alpha(i3) - G beta gamma-phosphoinositide 3-kinase (PI3K)-protein kinase C(PKC)zeta pathway. In contrast, INSL3 activation of RXFP2 results in accumulation of cAMP only via G alpha(s), modulated by cAMP inhibition through G alpha(oB). Thus, the aim of this study was to identify the cause of differential G-protein coupling between these highly similar receptors. Construction of chimeric receptors revealed that G alpha(i3) coupling is dependent upon the transmembrane region of RXFP1 and independent of the receptor ectodomain or ligand bound. Generation of C-terminal truncated receptors identified the terminal 10 amino acids of the RXFP1 C terminus as essential for G alpha(i3) signaling, and point mutations revealed an obligatory role for Arg(752). RXFP1-mediated G alpha(i3), but not G alpha(s) or G alpha(oB), signaling was also found to be dependent upon membrane rafts, and RXFP1 coupled to G alpha(i3) after only 3 min of receptor stimulation. Therefore, RXFP1 coupling to the G alpha(i3) G beta gamma-PI3K-PKC zeta pathway requires the terminal 10 amino acids of the RXFP1 C terminus and membrane raft localization, and the observed delay in this pathway occurs downstream of G alpha(i3).
