LGR8 Signal Activation by the Relaxin-like Factor

Abstract

The relaxin-like factor (RLF) is thought to be responsible for the intra-abdominal migration of the testis during mammalian development. Our latest studies of RLF and LGR8 have revealed that the N-terminal region of the A chain is not required for receptor binding but is indispensable for cyclic AMP generation. RLF derivatives with six residues deleted from the N terminus of the A chain are active, whereas further truncation, up to the first A chain cysteine (A-10), yields tightly binding ligands devoid of signaling activity. These derivatives are specific competitive inhibitors (RLFi) of RLF. Although receptor binding is dependent upon B chain residues, the N-terminal region of the A chain is a generic trigger of the trans-membrane signaling activity.The relaxin-like factor (RLF), 1 the product of the Insl3 gene (1), appears to be crucial for testicular descent in human neonates. Insl3Ϫ/Ϫ male mice retain the testes in the body cavity and are infertile (2, 3). The same phenotype was observed when the G-protein coupled receptor (GREAT) was deleted (4). These receptors (GREAT, LGR8) that affect the descent of testis in mice (4, 5) and in humans (LGR8) (6) are RLF-specific (7,8). Thus, an experimental system is now available that invites detailed investigation of the ligand/receptor interaction.RLF, isolated from bovine testes, consists of an A chain comprising 26 residues and a B chain of 40 or 45 residues. The chains are linked by three relaxin-like disulfide bonds (9). This native RLF confirms the structure that has been implied for all synthetic molecules. RLF binds its receptor in part through the B chain residue Trp(B-27) (10) and via additional, yet unidentified, residues that would account for the high receptor binding affinity. However, the molecule can be pared down without loss of binding avidity. Full binding intensity, for example, is observed with RLF ending in Trp(B-27)-amide (11).Our studies suggest that the N-terminal region of the A chain initiates the trans-membrane signal. Sequential shortening of the N-terminal end of the A chain reduces cAMP production, which becomes undetectable when 7 or more residues are removed. A chain truncations did not reduce binding avidity so that Ades-(1-7) RLF is a specific competitive inhibitor of RLF function. In this paper we report that the receptor-binding region of RLF is physically separate from the trans-membrane signal initiation site in the N-terminal region of the A chain. EXPERIMENTAL PROCEDURESMaterials-Fmoc amino acid derivatives were purchased from either Advanced ChemTech (Louisville, KY) or Novabiochem. Reagents and solvents for peptide synthesis were obtained from Advanced ChemTech. The LGR8 cloned into the pcDNA3.1.zeo plasmid was a gift from Dr. Hsueh, Department of Obstetrics and Gynecology, Stanford University School of Medicine. The human embryo kidney cell line 293T/17 was obtained from the American Type Culture Collection (ATCC CRL-11286) and used for LGR8 expression.Peptide Synthesis-All RLF chains were synthesized by Fmoc chemistry, usin...

The Minimal Active Structure of Human Relaxin-2

“…The N terminus of the A-chain of H2 relaxin has no functional role as reported previously (19,20). Shortening of the N terminus of the A-chain of porcine (19,20) or H2 (21) relaxin by up to four residues had no effect on binding or activation of RXFP1 and RXFP2.…”

“…These findings in turn, suggested that no single amino acid in the N-terminal region of the A chain is functionally important, but that the presence of an ␣-helix is required to maintain the overall fold of the protein which is key to the H2-RXFP1 and H2-RXFP2 interactions (21). In contrast, a similar truncation study on INSL3 showed that it can be truncated at the N terminus of its A-chain by up to nine residues without affecting the binding affinity to its receptor RXFP2, while becoming a high affinity antagonist (22). Thus, H2 relaxin is binding to and activating both RXFP1 and RXFP2 receptors in a similar manner but via a mechanism that is different compared with INSL3 (21).…”

“…Shortening of the N terminus of the A-chain of porcine (19,20) or H2 (21) relaxin by up to four residues had no effect on binding or activation of RXFP1 and RXFP2. Further truncation, however, caused both a decrease in affinity and potency and that of structural integrity as assessed by CD and NMR techniques (21).…”

“…1). These included the following: four B-chain N-terminal truncated analogues (H2-(B3-29), H2-(B5-29), H2-(B7-29), and H2-(B9 -29)), six B-chain C-terminal truncated analogues (H2-(B1-28), H2-(B1-27), H2-(B1-26), H2-(B1-25), H2-(B1-24), and H2-(B1-23)), four B-chain Nand C-terminal truncated analogues (H2-(B7-25), H2-(B7-24), H2-(B8 -25), and H2-(B8 -24)), and six A-and B-chain truncated analogues (H2-(A2-24)(B7-24), H2-(A3-24)(B7-24), H2-(A4 -24)(B7-24), H2-(A5-24)(B7-24), H2-(A7-24)(B7-24), and H2-(A9 -24) (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)). All of these H2 analogues were amidated at the C termini of both the A-and B-chains.…”

“…Further truncation, however, caused both a decrease in affinity and potency and that of structural integrity as assessed by CD and NMR techniques (21). The replacement of these deleted residues with alanines capable of adopting the native ␣-helical structure without displaying functionality caused significant biological activity and structure to reappear (20,21). These findings in turn, suggested that no single amino acid in the N-terminal region of the A chain is functionally important, but that the presence of an ␣-helix is required to maintain the overall fold of the protein which is key to the H2-RXFP1 and H2-RXFP2 interactions (21).…”