Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Investigating the structure and function of PAC1R splice isoforms (#146)

Jessica Lu 1 2 , Peishen (Elva) Zhao 1 2 , Sarah J Piper 1 2 , Denise Wootten 1 2 , Patrick Sexton 1 2
  1. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Melbourne, Victoria, Australia
  2. ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Melbourne, Victoria, Australia

The pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) is a promising drug target for the treatment of numerous central nervous system disorders, including migraine and post-traumatic stress disorder where there is significant unmet clinical need [1, 2]. This may, in part, be due to the complexity of signalling downstream of PAC1R activation and ligand selectivity over related receptors, and is further complicated due the existence of multiple PAC1R splice variants, from extensive alternative splicing in the N-terminal extracellular domain (ECD) and intracellular loop 3 (ICL3) regions. Cryo-EM structures of the PAC1R have been determined, though they are currently restricted to the reference splice isoforms, PAC1R null (PAC1nR) or PAC1R short (PAC1sR), with no ICL3 insertions, bound to the high affinity agonists: PACAP38 and maxadilan [3-5]. This research aims to solve select PAC1R splice isoform structures and use this information to understand how these regions have the potential to alter ligand selectivity and the engagement of the PAC1R with G proteins. The pharmacology of the PAC1R splice isoforms was studied using a BRET-based assay to look at G protein subunit dissociation as a measure of G protein activation. The PAC1sR splice isoform, with a truncation N-terminal ECD, was found to improve Gs protein activation induced by the low affinity agonist, VIP. While, the PAC1nR-hip isoform, with a 28 amino acid residue extended ICL3, was found to reduce Gs protein activation induced by a high affinity agonist, PACAP27. Purification of these selected PAC1 receptor splice isoforms bound to PACAP27 (for PAC1nR-hip) or VIP (for PAC1sR) in complex with dominant negative Gs protein was optimised and purity of the samples were verified using SDS-PAGE, size exclusion chromatography, and 2D classifications from negative stain electron microscopy. Obtaining these structures will deepen our understanding of ligand selectivity and G protein activation of the PAC1R splice isoforms. Ultimately, this will provide insights into receptor signalling and drug development towards the PAC1R.

  1. Ressler, K.J., et al., Post-traumatic stress disorder is associated with PACAP and the PAC1 receptor. Nature, 2011. 470(7335): p. 492-497.
  2. Rubio-Beltrán, E., et al., PACAP38 and PAC1 receptor blockade: a new target for headache? The Journal of Headache and Pain, 2018. 19(1): p. 64.
  3. Liang, Y.-L., et al., Toward a Structural Understanding of Class B GPCR Peptide Binding and Activation. Molecular Cell, 2020.
  4. Wang, J., et al., Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism. Cell Research, 2020.
  5. Kobayashi, K., et al., Cryo-EM structure of the human PAC1 receptor coupled to an engineered heterotrimeric G protein. Nature Structural & Molecular Biology, 2020. 27(3): p. 274-280.