R3(BΔ23-27)R/I5 chimeric peptide, a selective antagonist for GPCR135 and GPCR142 over relaxin receptor LGR7 -: In vitro and in vivo characterization

Both relaxin-3 and its receptor ( GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor ( LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist ( a chimeric relaxin-3/INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(B Delta 23-27) R/I5, consists of the relaxin-3 B-chain with a replacement of Gly(23) to Arg, a truncation at the C terminus ( Gly(24)-Trp(27) deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3( B Delta 23-27) R/I5 binds to human GPCR135 ( IC50 = 0.67 nM) and GPCR142 ( IC50 = 2.29 nM) with high affinity and is a potent functional GPCR135 antagonist ( pA2 = 9.15) but is not a human LGR7 ligand. Furthermore, R3( B Delta 23-27) R/I5 had a similar binding profile at the rat GPCR135 receptor ( IC50 = 0.25 nM, pA2 = 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3( B Delta 23-27) R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3( B Delta 23-27) R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.