The oxytocin and vasopressin 1A (V1A) receptors 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 the low expression levels of these receptors. Thus, the aim of this study was to use our novel mammalian cell directed evolution platform to generate high expressing mutants of the oxytocin and V1A receptors 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, lentivirus carrying a mutant receptor library was created to introduce clones into HEK293F cells. These receptors underwent two rounds of error prone PCR, and fluorescence activated cell sorting in the presence of a fluorescent ligand to enrich the highest binding population.
Individual mutants from this high expressing populations were isolated, sequenced, and assayed for binding, expression and signalling. Expression was enhanced 2-5 fold, with the introduction of only 3-5 point mutations, demonstrated by fluorescent ligand binding and Hibit expression assay. Interestingly there was a tendency towards signalling incompetent receptors.
The highest expressing V1A mutants were cloned into mammalian cell purification vectors, and purified. Small scale purifications of mutant variants confirmed significant increases in expression compared to wild type. Larger scale purifications of the best variant, V1A demonstrated yields of ~1 mg/L. We have thus successfully generated useful variants of these receptors, which will enable us to move forward with further protein characterisation, and structure determination using cryo-EM.