The receptors for the peptide hormones relaxin and insulinlike peptide 3 (INSL3) are the leucine-rich repeat-containing G-protein-coupled receptors LGR7 and LGR8 recently renamed as the relaxin family peptide (RXFP) receptors, RXFP1 and RXFP2, respectively. These receptors differ from other LGRs by the addition of an N-terminal low density lipoprotein receptor class A (LDLa) module and are the only human G-protein-coupled receptors to contain such a domain. Recently it was shown that the LDLa

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... dule of the RXFP1 and RXFP2 receptors is essential for ligand-stimulated cAMP signaling. The mechanism by which the LDLa module modulates receptor signaling is unknown; however, it represents a unique paradigm in understanding G-protein-coupled receptor signaling. Here we present the structure of the RXFP1 receptor LDLa module determined by solution NMR spectroscopy. The structure is similar to other LDLa modules but shows small differences in side chain orientations and inter-residue packing. Interchange of the module with the second ligand binding domain of the LDL receptor, LB2, results in a receptor that binds relaxin with full affinity but is unable to signal. Furthermore, we demonstrate via structural studies on mutated LDLa modules and functional studies on mutated full-length receptors that a hydrophobic surface within the N-terminal region of the module is essential for activation of RXFP1 receptor signal in response to relaxin stimulation. This study has highlighted the necessity to understand the structural effects of single amino acid mutations on the LDLa module to fully interpret the effects of these mutations on receptor activity. Relaxin and INSL3 (insulin-like peptide 3) are small twochain peptide hormones that belong to the relaxin-insulin superfamily. Relaxin was observed in the 1920s to induce the widening of the birth canal (1) and subsequently has been studied for the roles that it plays during pregnancy. More recently it has become apparent that relaxin is a pleiotropic hormone involved in numerous nonreproductive processes such as vasodilatation and neoangiogenesis, embryo implantation (2), collagen turnover (3, 4), and cancer progression (5). INSL3 was more recently discovered in the 1990s (6 -8), and although understanding of this hormone is still ongoing, in rodents it is clearly involved in testis descent (9, 10) and involved in oocyte maturation and male germ cell survival (11). The receptors for relaxin and INSL3 are LGR7 and LGR8, recently renamed the relaxin family peptide 1 (RXFP1) and the relaxin family peptide 2 (RXFP2) receptors, respectively (12) (13) (14) . These receptors are leucine-rich repeat-containing G-protein-coupled receptors (LGR) 3 and thus belong to the LGR family. The discovery of the RXFP1 and RXFP2 receptors resulted in the addition of a third class of LGRs (15), and at present there are three subclasses of LGRs, type A, B, and C. Type A LGRs include receptors for the glycoprotein hormones follicle-stimulating hormone, thyrotropin, and luteinizing hormone and consist of a rhodopsin-like seven transmembrane region, and the hallmark large ␣/␤ ectodomain between 5 and 7 leucinerich repeats (LRRs). The three type B LGRs are currently orphan receptors. Apart from containing between 11 and 17 LRRs, they are of similar structural architecture to the type A receptors. The RXFP1 (LGR7) and RXFP2 (LGR8) receptors differ from type A and type B LGRs by having 10 LRRs and the addition of an N-terminal low density lipoprotein receptor class A (LDLa) module. RXFP1 and RXFP2 receptor orthologs are found in all mammals and are the only GPCRs with LDLa modules. Homologous LGR type C receptors have been identified in the fly (16) and snail (17), although there are no relaxin peptides in these species. Intriguingly, the pond snail LGR contains 12 3 The abbreviations used are: LGR, leucine-rich repeat-containing G-proteincoupled receptor; LDLa, low density lipoprotein receptor class A; GPCR, G-protein-coupled receptors; LRR, leucine-rich repeats; PBS, phosphatebuffered saline; BSA, bovine serum albumin; HEK, human embryonic kidney; r.m.s.d., root mean square deviation; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; RP-HPLC, reversed-phase-high pressure liquid chromatography.