Exploring electrostatic interactions of relaxin family peptide receptor 3 and 4 with ligands using a NanoBiT-based binding assay.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely relaxin family peptide receptor 1-4 (RXFP1-4). We recently disclosed electrostatic interactions of the homologous RXFP3 and RXFP4 with some agonists based on activation complementation. However, this activation assay-based approach cannot be applied to antagonists that do not activate receptors. Herein, we propose a general approach suitable for both agonists and antagonists based on our newly-developed NanoBiT-based binding assay. We first validated the binding assay-based approach using the agonist relaxin-3, then applied it to the chimeric antagonist R3(ΔB23-27)R/I5. Three positively charged B-chain Arg residues of the agonist and antagonist were respectively replaced by a negatively charged Glu residue; meanwhile, the negatively charged Glu and Asp residue in the essential WxxExxxD motif of both receptors were respectively replaced by a positively charged Arg residue. Based on binding complementation of mutant ligands towards mutant receptors, we deduced possible electrostatic interactions of the agonist and antagonist with both RXFP3 and RXFP4: their B-chain C-terminal Arg residue interacts with the deeply buried Glu residue in the WxxExxxD motif of both receptors, and one or two of their B-chain central Arg residues interact with the shallowly buried Asp residue in the WxxExxxD motif of both receptors. Our present work shed new light on the interaction mechanism of RXFP3 and RXFP4 with agonists and antagonists, and also provided a novel approach for interaction studies of some plasma membrane receptors with their ligands.

Identifying the binding mechanism of LEAP2 to receptor GHSR1a.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a highly conserved secretory peptide first isolated in 2003. However, its exact biological functions remained elusive until a recent study identified it as an endogenous antagonist for the growth hormone secretagogue receptor (GHSR1a), a G protein-coupled receptor for which the gastric peptide ghrelin is the endogenous agonist. By tuning the ghrelin-GHSR1a system, LEAP2 has an important function in energy metabolism. In the present study, we first demonstrated that LEAP2 and ghrelin actually bound to GHSR1a in a competitive manner, rather than in a non-competitive manner as previously reported, by binding assays and activation assays. Subsequently, we demonstrated that the antagonistic function of LEAP2 was drastically affected by the manner of its addition. LEAP2 primarily affected the maximal activation effect when added before ghrelin, whereas it primarily affected half-maximal effective concentration when added at the same time as ghrelin. Thus, LEAP2 behaved as a competitive antagonist if added at the same time as the agonist and a non-competitive antagonist if added before the agonist. This unusual property of LEAP2 might be caused by its slow dissociation from receptor GHSR1a. We also found that the N-terminal fragment of LEAP2 was important for receptor binding. Our present study revealed an antagonistic mechanism for LEAP2, and will facilitate the design of novel antagonists for receptor GHSR1a in future studies.

Development of a novel ligand binding assay for relaxin family peptide receptor 3 and 4 using NanoLuc complementation.

Relaxin family peptides perform a variety of biological functions by binding and activating relaxin family peptide receptor 1-4 (RXFP1-4), four A-class G protein-coupled receptors. In the present work, we developed a novel ligand binding assay for RXFP3 and RXFP4 based on NanoLuc complementation technology (NanoBiT). A synthetic ligation version of the low-affinity small complementation tag (SmBiT) was efficiently ligated to the A-chain N terminus of recombinant chimeric agonist R3/I5 using recombinant circular sortase A. After the ligation product R3/I5-SmBiT was mixed with human RXFP3 or RXFP4 genetically fused with a secretory large NanoLuc fragment (sLgBiT) at the N terminus, NanoLuc complementation was induced by high-affinity ligand-receptor binding. Binding kinetics and affinities of R3/I5-SmBiT with sLgBiT-fused RXFP3 and RXFP4 were conveniently measured according to the complementation-induced bioluminescence. Using R3/I5-SmBiT and the sLgBiT-fused receptor as a complementation pair, binding potencies of various ligands with RXFP3 and RXFP4 were quantitatively measured without the cumbersome washing step. The novel NanoBiT-based ligand binding assay is convenient for use and suitable for automation, thus will facilitate interaction studies of RXFP3 and RXFP4 with ligands in future. This assay can also be applied to some other plasma membrane receptors for pharmacological characterization of ligands in future studies.

Development of a selective agonist for relaxin family peptide receptor 3.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1-4. Among these receptors, RXFP3 lacks a specific natural or synthetic agonist at present. A previously designed chimeric R3/I5 peptide, consisting of the B-chain of relaxin-3 and the A-chain of INSL5, displays equal activity towards the homologous RXFP3 and RXFP4. To increase its selectivity towards RXFP3, in the present study we conducted extensive mutagenesis around the B-chain C-terminal region of R3/I5. Decreasing or increasing the peptide length around the B23-B25 position dramatically lowered the activation potency of R3/I5 towards both RXFP3 and RXFP4. Substitution of B23Gly with Ala or Ser converted R3/I5 from an efficient agonist to a strong antagonist for RXFP3, but the mutants retained considerable activation potency towards RXFP4. Substitution of B24Gly increased the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. The best mutant, [G(B24)S]R3/I5, displayed 20-fold higher activation potency towards RXFP3 than towards RXFP4, meanwhile retained full activation potency at RXFP3. Thus, [G(B24)S]R3/I5 is the best RXFP3-selective agonist known to date. It is a valuable tool for investigating the physiological functions of RXFP3, and also a suitable template for developing RXFP3-specific agonists in future.

Rapid preparation of bioluminescent tracers for relaxin family peptides using sortase-catalysed ligation.

Relaxin family is a group of peptide hormones with a variety of biological functions by activating G protein-coupled receptors RXFP1-4. We recently developed bioluminescent tracers for their receptor-binding assays by chemical conjugation with the ultrasensitive NanoLuc reporter. To simplify preparation of the bioluminescent tracers, in the present study, we established a sortase-catalysed ligation approach using the chimeric R3/I5 as a model. Following catalysis by recombinant sortase A, a NanoLuc reporter carrying the LPETG sortase recognition motif at the C-terminus was efficiently ligated to an R3/I5 peptide carrying four successive Gly residues at the A-chain N-terminus, via the formation of a peptide bond between the C-terminal LPET sequence of NanoLuc and the A-chain N-terminal Gly residue of R3/I5. Saturation binding assays demonstrated that the NanoLuc-ligated R3/I5 retained high binding affinity to RXFP3 and RXFP4, with the calculated dissociation constants (K d) of 4.34 ± 0.33 nM (n = 3) and 5.66 ± 0.54 nM (n = 3), respectively. Using the NanoLuc-ligated R3/I5 as a tracer in competition binding assays, binding potencies of various ligands towards RXFP3 and RXFP4 were conveniently quantified. This work provides a simple method for rapid preparation of bioluminescent tracers for relaxin family peptides and other protein/peptide hormones for ligand-receptor interaction studies.