Solution Structure and Characterization of the LGR8 Receptor Binding Surface of Insulin-like Peptide 3

Abstract

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein-coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg B16 and Val B19 , with His B12 and Arg B20 playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp B27 , to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor. Insulin-like peptide 3 (INSL3 4; also known as relaxin-like factor and Leydig cell insulin-like peptide) was originally discovered and isolated as a cDNA clone from a boar testis cDNA library (1). It is primarily produced by prenatal and postnatal mature Leydig cells of the testis and the thecal cells of the ovary. Its primary structure showed it to be a bona fide member of the insulin-insulin-like growth factor-relaxin superfamily, which is now known to have a total of ten members in the human. Like insulin, INSL3 is firstly synthesized as a pre-prohormone precursor containing a signal peptide linked to B-C-A domains. It undergoes processing by hitherto unidentified proprotein convertases, resulting in the production of mature INSL3 consisting of a A-B heterodimer that is covalently linked by two interchain disulfide bonds and one intrachain disulfide bond (Fig. 1) (2). These disulfide bonds are essential for maintaining its expected characteristic insulin-like conformation and its unique biological activities (3). The receptor for INSL3, LGR8, is a member of the leucine-rich repeat-containing G-proteincoupled receptor family (4). It is closely related to the relaxin receptor, LGR7, and has been recently classified as relaxin family peptide (RXFP) receptor, RXFP2 (5). Importantly, relaxin peptides from some species can also bind to and activate LGR8, albeit with a lower affinity than INSL3 (5).Male mice homozygous with targeted disrup...

The A-chain of Human Relaxin Family Peptides Has Distinct Roles in the Binding and Activation of the Different Relaxin Family Peptide Receptors

“…Importantly, truncation did not result in an RXFP2 antagonist or partial agonist. A-chain truncation of the H3 relaxin peptide had no effect on (20) and the crystal structure of H2 relaxin (36), it is clear that the peptides have a very similar insulin-like core structure, with the key differences being mainly around the chain termini. For both H2 and H3 relaxin, the A-chains are highly structured with two characteristic ␣-helical segments from A1 to A13 and from A17 to A24.…”

“…The NMR analysis of A-(10 -24) INSL3 did not provide a clear answer as to how much of the structure was disrupted due to the severe signal broadening, but Bullesbach et al (23) showed by CD studies that the overall structure was to a large extent retained after truncations up to A9. It should be noted that INSL3 is a rather dynamic peptide and that significant line broadening was observed in the region around the Cys A10 -Cys A15 disulfide also in the native peptide (20). For H2 and H3 relaxin, we postulated that the loss of ␣-helix structure in the A-chain of H2 and H3 relaxin by removal of the N-terminal residues may have caused an overall loss of structure, resulting in the observed decrease in RXFP1 binding affinity and potency.…”

“…This interaction is determined by the arginine residues at B13 and B17 and an isoleucine or valine at position B20 within the B-chain forming a "relaxin binding cassette" (Arg-X-X-X-Arg-X-X-Ile/Val) (18). Similarly, we have recently demonstrated that the INSL3 B-chain interacts with specific residues in the RXFP2 receptor LRRs (19 (20). Importantly, B-chain-only INSL3 peptides can bind to the primary binding sites in the LRRs but do not activate the receptor (21).…”

The Minimal Active Structure of Human Relaxin-2

“…The identification of the relaxin binding cassette RXXXRXX(I/V) of the H2 relaxin B-chain was made more than a decade ago (14,46). It was subsequently shown that this midregion of the B-chain, which is exposed on the face of a B-chain helix is the primary binding site in other relaxins (47,48). Subsequently, mutations in the LRRs of the ectodomain have identified the corresponding binding site for the cassette in RXFP1.…”

Structure of the R3/I5 Chimeric Relaxin Peptide, a Selective GPCR135 and GPCR142 Agonist

“…We recently determined the NMR solution structures of human relaxin-3 (22) and INSL3 (23). From these structures together with the crystal structure of relaxin-2 and extensive structure-activity studies, a picture of the complex events involved in the recognition of a relaxin by its receptor is starting to emerge.…”