Evolving Peptide G Protein-Coupled Receptors (GPCRs) to Facilitate Structural Studies Using a Novel Lentiviral Based Method of Directed Evolution — ASN Events
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  3. Evolving Peptide G Protein-Coupled Receptors (GPCRs) to Facilitate Structural Studies Using a Novel Lentiviral Based Method of Directed Evolution

Evolving Peptide G Protein-Coupled Receptors (GPCRs) to Facilitate Structural Studies Using a Novel Lentiviral Based Method of Directed Evolution (#205)

Jonathan Siah 1 , Riley Cridge 1 , Lisa Williams 1 , Ross Bathgate 1 2 , Daniel Scott 1 2
  1. The Florey Institute of Neuroscience and Mental Health, Parkville, VIC, Australia
  2. Department of Biochemistry and Molecular BIology, The University of Melbourne, Parkville, VIC, Australia

Peptide GPCRs are membrane proteins that, upon peptide binding, trigger cellular responses via intracellular signaling cascades. Because of their vital physiological roles, many peptide GPCRs are considered pharmacological targets to treat numerous diseases. However, only 18% of Class A peptide GPCRs are targeted by approved drugs versus the 97% of aminergic GPCRs. This disparity is due to the complexity of peptide mediated receptor activation. To understand this activation mechanism, structures of these receptors would aid greatly. There are currently limited peptide receptor structures, particularly with peptide bound. The lack of structures highlights the technical difficulties involved in studying GPCR structure, including low native expression and detergent instability.

A novel form of directed evolution (CHESS)1 was developed to select for detergent stable GPCRs. This enabled the stabilization of NTS1, the receptor for the neurotensin peptide, and the solution of two crystal structures of NTS1 bound to peptide. CHESS used a bacterial system, however many peptide GPCRs cannot be expressed in bacterial systems due to requiring post-translational modifications. Thus, highlighting the need for a mammalian-based system to evolve peptide receptors.

Here, we developed a lentiviral-based system of directed evolution in mammalian cells to evolve high expression. This involved generating a library of random receptor mutants. Mutants are transduced into HEK293F cells, where we use a fluorescent ligand and fluorescence-activated cell sorting to screen for high fluorescence (high expression). This is repeated until we evolve our desired trait. We applied this to the vasopressin receptor, V1a and produced mutants exhibiting 5-6x cell surface expression versus WT. Crucially, these mutants displayed similar binding and signaling profiles to WT.

These results highlight the systems utility in GPCR engineering, it is being used concurrently on relaxin and oxytocin peptide receptors, RXFP1 and OTR, along with evolving receptor specific nanobodies and signaling sensors. Resulting mutants would facilitate structural studies of these receptors, thus enabling rational drug design.

1Scott, DJ., Plückthun, A. J. Mol. Biol (2013).