Cell-free protein synthesis as a tool to study RXFP3- Relaxin-3 protein interactions

With the discovery of the relaxin family peptide receptors there is interest in obtaining a clearer understanding of the structure of these proteins and the molecular mechanism of receptor-ligand interaction. As G-protein coupled receptors, obtaining milligram quantities for structural investigations is hampered by the inherent instability of these integral membrane proteins. In the current context, understanding of GPCR structural biology has increased dramatically with crystal structures of several inactive and now active forms solved. In addition, the first nuclear magnetic resonance structure of a GPCR was obtained which is of crucial importance to studying these receptors in a more “biologically relevant” setting. However despite this expansion in the field, most structures have been solved on modified systems so as to increase stability and are not necessarily representative of the native receptors. In relation to the relaxin family peptide receptors, we chose to investigate relaxin-family peptide receptor-3 expressed by cellfree protein synthesis. In contrast to in-vivo expression, cell-free was capable of producing large amounts of native receptor which makes it amenable to demanding structural studies

Relaxin family peptide receptors in GtoPdb v.2023.1

Relaxin family peptide receptors (RXFP, nomenclature as agreed by the NC-IUPHAR Subcommittee on Relaxin family peptide receptors [23, 119]) may be divided into two pairs, RXFP1/2 and RXFP3/4. Endogenous agonists at these receptors are heterodimeric peptide hormones structurally related to insulin: relaxin-1, relaxin, relaxin-3 (also known as INSL7), insulin-like peptide 3 (INSL3) and INSL5. Species homologues of relaxin have distinct pharmacology and relaxin interacts with RXFP1, RXFP2 and RXFP3, whereas mouse and rat relaxin selectively bind to and activate RXFP1 [260]. relaxin-3 is the ligand for RXFP3 but it also binds to RXFP1 and RXFP4 and has differential affinity for RXFP2 between species [259]. INSL5 is the ligand for RXFP4 but is a weak antagonist of RXFP3. relaxin and INSL3 have multiple complex binding interactions with RXFP1 [267] and RXFP2 [132] which direct the N-terminal LDLa modules of the receptors together with a linker domain to act as a tethered ligand to direct receptor signaling [262]. INSL5 and relaxin-3 interact with their receptors using distinct residues in their B-chains for binding, and activation, respectively [321, 152]

Development of a scaffold displaying exoloops of RXFP1

Relaxin family peptide receptor 1 (RXFP1), the cognate receptor for relaxin, is a G-protein coupled receptor (GPCR) possessing a unique extracellular region consisting of a domain of 10 leucine rich repeats (LRRs) linked to an N-terminal low density lipoprotein Class A module. Relaxin binds to its receptor primarily by a high affinity interaction with the LRRs. An additional low-affinity interaction has been proposed to occur between relaxin and the the exoloops (ELs) of the transmembrane domain, however the molecular detail of this interaction remains undefined. While site directed mutagenesis and subsequent functional characterisation of these mutants traditionally allows identification of residues contributing to receptor function, in this case results are complicated by the presence of the high affinity binding site in the LRRs. To create a tool to investigate the low-affinity interaction, a protein scaffold system displaying exoloops 1 and 2 from RXFP1 was designed. This was achieved by inserting RXFP1 exoloops 1 and 2 into the native loops of a thermostabilised 6 kDa GB1 protein creating EL1/EL2-GB1. This protein has been expressed and purified in milligram quantities and used in conjunction with biophysical techniques such as NMR to explore relaxin binding to the exoloops of RXFP1

Activation of relaxin family receptor 1 from different mammalian species by relaxin peptide and small-molecule agonist ML290

Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit–human and guinea pig–human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing

Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization

Relaxin, a heterodimeric polypeptide hormone, is a key regulator of collagen metabolism and multiple vascular control pathways in humans and rodents. Its actions are mediated via its cognate G-protein-coupled receptor, RXFP1 although it also "pharmacologically" activates RXFP2, the receptor for the related, insulin-like peptide 3 (INSL3), which has specific actions on reproduction and bone metabolism. Therefore, experimental tools to facilitate insights into the distinct biological actions of relaxin and INSL3 are required, particularly for studies of tissues containing both RXFP1 and RXFP2. Here, we chemically functionalized human (H2) relaxin, the RXFP1-selective relaxin analog H2:A(4-24)(F23A), and INSL3 to accommodate a fluorophore without marked reduction in binding or activation propensity. Chemical synthesis of the two chains for each peptide was followed by sequential regioselective formation of their three disulfide bonds. Click chemistry conjugation of Cy5.5 at the B-chain N-terminus, with conservation of the disulfide bonds, yielded analogs displaying appropriate selective binding affinity and ability to activate RXFP1 and/or RXFP2 in vitro. The in vivo biological activity of Cy5.5-H2 relaxin and Cy5.5-H2:A(4-24)(F23A) was confirmed in mice, as acute intracerebroventricular (icv) infusion of these peptides (but not Cy5.5-INSL3) stimulated water drinking, an established behavioral response elicited by central RXFP1 activation. The central distribution of Cy5.5-conjugated peptides was examined in mice killed 30 min after infusion, revealing higher fluorescence within brain tissue near-adjacent to the cerebral ventricle walls relative to deeper brain areas. Production of fluorophore-conjugated relaxin family peptides will facilitate future pharmacological studies to probe the function of H2 relaxin/RXFP1 and INSL3/RXFP2 signaling in vivo while tracking their distribution following central or peripheral administration

A Chemogenetic Tool that Enables Functional Neural Circuit Analysis

Chemogenetics enables manipulation of neuronal activity in experimental animals. While providing information about the transduced neuron expressing a ligand-activated molecule, chemogenetics does not provide understanding about the antecedent circuit that drives that neuron's activity. For current approaches, this is not feasible, because the activating molecules are not genetically encoded. The insect allatostatin/allatostatin receptor system, a highly specific, powerful inhibitory chemogenetic approach, has this advantage, because the ligand, being a peptide, is genetically encoded. We developed viral vector-based systems to express biologically active allatostatin in neurons in vivo and allatostatin receptors in subpopulations of postsynaptic neurons. We demonstrate that activity-dependent release of allatostatin induces inhibition of allatostatin receptor-expressing neurons. We validate the approach in the vagal viscerosensory system where inhibitory, rather than the usual excitatory, viscerosensory input leads to sustained decreases in baroreceptor reflex sensitivity and bodyweight

Viral-mediated delivery of an RXFP3 agonist into brain promotes arousal in mice

Anatomical and functional studies of central relaxin-3/RXFP3 systems suggest they constitute an ascending arousal network. For example, relaxin-3 knockout mice display circadian hypoactivity compared to wild type littermate controls. In studies to explore the effect of chronic RXFP3 activation on behaviour, we engineered a lentiviral construct to constitutively secrete the RXFP3 agonist, R3/I5, and express a green fluorescent protein (GFP) marker in transduced cells. Intracerebroventricular injection of the lenti-R3/I5-GFP virus (~10^8 genomic copies in 2 μl) in adult C57BL/6J mice resulted in GFP expression within cells of the ventricle walls and choroid plexus over a period of 1-4 weeks, suggesting likely chronic R3/I5 secretion and RXFP3 activation in brain regions proximal to the ventricular system. Subsequent testing in automated locomotor cells on day 8 and 9 post-injection revealed that lenti-R3/I5-GFP treated mice displayed prolonged, elevated locomotor activity (~18% higher over the last 15 min on day 8, and over the entire 30 min test on day 9) compared to mice injected with a control lenti-GFP virus, which habituated normally to the novel environment (n=18/12 respectively, p<0.05). These findings are consistent with an earlier report of increased activity scores in rats acutely injected centrally with R3/I5, and further suggest a role for relaxin-3/RXFP3 signalling in promoting behavioural arousal