The oxytocin receptor (OTR) and vasopressin 1A receptor (V1A) are emerging therapeutic targets for social disorders such as schizophrenia and autism. The native ligands of these GPCRs are closely related nonapeptides oxytocin and arginine vasopressin. Despite therapeutic interest, there is a lack of understanding about how these peptides bind and activate their receptors. This is due to the limited structural information available of these receptors, and absence of any peptide bound structures. This can be attributed to their low recombinant expression levels. Thus, the aim of this study was to use our novel mammalian cell directed evolution platform to generate high expressing mutants of the OTR and V1A to enable structure determination and elucidation of peptide binding modes.
We sought to introduce and select for mutations that would enhance protein expression levels, and protein stability using directed evolution. To achieve this, OTR and V1a genes underwent error-prone PCR, and lentivirus carrying mutant receptor library was used to introduce clones into HEK293F cells. These clones were selected using fluorescence activated cell sorting in the presence of a fluorescent ligand to enrich the highest binding population.
Individual mutants from these high expressing populations were isolated, sequenced, and assayed for binding, expression, and signalling. Expression was enhanced 5-fold, with the introduction of only 3-5 point mutations, demonstrated with saturation ligand binding assays, and protein purification. These high expressing mutants have enabled previously impossible purifications of OTR and V1a receptor, with yields of ~1 mg/L.
Using this novel method we have thus successfully generated useful variants of these receptors, which will enable us to move forward with further biophysical characterisation, animal immunisations and structure determination using cryo-EM. These methods will aid in the understanding of the mechanism of peptide binding and activation, and the drivers of selectivity between these very homologous proteins.