The relaxin peptide family belong to the relaxin/insulin superfamily of peptide hormones. Relaxin peptide hormones have an evolutionarily conserved architecture comprising of two chains linked by three disulfide bonds. The receptors for the relaxin family are however unlike insulin which binds to receptor tyrosine kinases but are instead GPCRs. The relaxin family peptide receptors include four receptors designated RXFP1-4, which all belong to the Class-A family of GPCRs. A comparison of RXFP1-2 and RXFP3-4 however, show distinctly different characteristics. RXFP1-2 are type-C leucine rich repeat containing GPCRs (LGRs) while RXFP3-4 are peptide ligand GPCRs. Interestingly, this difference of GPCR lineage indicates a level of convergent evolution on the part of the receptors, since relaxin peptides share common ancestry. The RXFP receptors and their ligands mediate a wide variety of physiological responses, including cardioprotection, germ cell modulation and appetite regulation. Therefore it is crucial that before development of peptide or chemical modulators toward these receptors, that detailed structural analysis of these receptors take precedent.

Of interest to our lab are the interactions that occur between relaxin peptide hormones and their receptors. GPCRs are notoriously difficult to produce by conventional recombinant methods, so we have chosen to produce these receptors by cell-free protein synthesis (CFPS); a technique capable of producing various GPCRs in sizable quantities. Notably, CF is amenable to manipulation allowing the user to define favourable conditions for solubilisation.

Highlighted here is the successful expression and purification of RXFP3 produced by CFPS. RXFP3 can be expressed directly into detergent micelles or as an insoluble precipitate, capable of refolding into detergent. Methods including EM and CD indicate RXFP3 to be consistent with a folded GPCR. Binding assays however, indicate the receptors are not in a form that is consistent with correct binding. Therefore we are determined to carry out detergent screens to identify conditions suitable for receptor stabilisation, which is necessary for downstream applications.