Abstract

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 Gα_s, but this is modulated by inhibition of cAMP through Gα_oB. Over time, RXFP1 recruits coupling to Gα_i3, causing additional cAMP accumulation via a Gα_i3-Gβγ-phosphoinositide 3-kinase (PI3K)-protein kinase C (PKC)ζ pathway. In contrast, INSL3 activation of RXFP2 results in accumulation of cAMP only via Gα_s, modulated by cAMP inhibition through Gα_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 Gα_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 Gα_i3 signaling, and point mutations revealed an obligatory role for Arg⁷⁵². RXFP1-mediated Gα_i3, but not Gα_s or Gα_oB, signaling was also found to be dependent upon membrane rafts, and RXFP1 coupled to Gα_i3 after only 3 min of receptor stimulation. Therefore, RXFP1 coupling to the Gα_i3-Gβγ-PI3K-PKCζ 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 Gα_i3.