PT - JOURNAL ARTICLE AU - Michelle L. Halls AU - Emma T. van der Westhuizen AU - John D. Wade AU - Bronwyn A. Evans AU - Ross A. D. Bathgate AU - Roger J. Summers TI - Relaxin Family Peptide Receptor (RXFP1) Coupling to Gα<sub>i3</sub> Involves the C-Terminal Arg<sup>752</sup> and Localization within Membrane Raft Microdomains AID - 10.1124/mol.108.051227 DP - 2009 Feb 01 TA - Molecular Pharmacology PG - 415--428 VI - 75 IP - 2 4099 - http://molpharm.aspetjournals.org/content/75/2/415.short 4100 - http://molpharm.aspetjournals.org/content/75/2/415.full SO - Mol Pharmacol2009 Feb 01; 75 AB - 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 Gαs, but this is modulated by inhibition of cAMP through GαoB. Over time, RXFP1 recruits coupling to Gαi3, causing additional cAMP accumulation via a Gαi3-Gβγ-phosphoinositide 3-kinase (PI3K)-protein kinase C (PKC)ζ pathway. In contrast, INSL3 activation of RXFP2 results in accumulation of cAMP only via Gαs, modulated by cAMP inhibition through GαoB. 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 Gαi3 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 Gαi3 signaling, and point mutations revealed an obligatory role for Arg752. RXFP1-mediated Gαi3, but not Gαs or GαoB, signaling was also found to be dependent upon membrane rafts, and RXFP1 coupled to Gαi3 after only 3 min of receptor stimulation. Therefore, RXFP1 coupling to the Gαi3-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 Gαi3. The American Society for Pharmacology and Experimental Therapeutics