Structural basis of modified ligand selectivity from N-terminal PAC1R alternative splicing

Jessica J Lu; Giuseppe Deganutti; Miaomiao Li; Laura J Humphrys; Yandi Li; Theodore J Nettleton; Hariprasad Venugopal; Villy Julita; George Christopoulos; Christopher A Reynolds; Patrick M Sexton; Denise Wootten; Peishen Zhao; Sarah J Piper

doi:10.1073/pnas.2521157122

Structural basis of modified ligand selectivity from N-terminal PAC1R alternative splicing

Jessica J Lu
, Giuseppe Deganutti
, Miaomiao Li
, Laura J Humphrys
, Yandi Li
, Theodore J Nettleton
, Hariprasad Venugopal
, Villy Julita
, George Christopoulos
, Christopher A Reynolds
, Patrick M Sexton
, Denise Wootten
, Peishen Zhao
, Sarah J Piper

Monash University
University of Oxford

Research output: Contribution to journal › Article › peer-review

1 Citation (Scopus)

3 Downloads (Pure)

Abstract

The pituitary adenylate cyclase-activating polypeptide (PACAP) 1 receptor (PAC1R) is a class B1 G protein-coupled receptor activated by the endogenous peptide agonists PACAP and vasoactive intestinal peptide (VIP). Alternate splicing within the receptor extracellular domain (ECD) generates the PAC1R short variant (PAC1sR) that has selectively enhanced VIP function compared to the full-length, PAC1R null variant (PAC1nR). However, to date, a comprehensive pharmacological assessment of the downstream signaling outcomes of PAC1sR activation compared to PAC1nR has not been performed, and little information is available to mechanistically understand how ECD splicing may alter ligand engagement. Here, we demonstrated that VIP, but not PACAP, has globally enhanced activity across a broad range of functional endpoints at PAC1sR compared to PAC1nR. Cryo-EM structures of VIP-bound, stimulatory G protein (G s)-coupled PAC1sR and PAC1nR, supported by molecular dynamics (MD) simulations, demonstrate transient engagement of the null loop in PAC1nR, which is absent in PAC1sR, with residues in extracellular loop 2 (ECL2) and the N-terminal helix of the ECD. These interactions result in differential engagement of VIP with these domains and the top of TM2/ECL1 with PAC1sR and PAC1nR. Moreover, MD simulations predicted differential interactions of the G s protein with the two PAC1R variants when bound by VIP that correlate with a greater allosteric influence of the G s protein on VIP affinity at the PAC1sR, relative to PAC1nR. Our study provides insights into the structural basis and functional consequences of PAC1R ECD splicing, increasing understanding of PAC1R ligand selectivity and signaling.

Original language	English
Article number	e2521157122
Number of pages	11
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	122
Issue number	47
DOIs	https://doi.org/10.1073/pnas.2521157122
Publication status	Published - 25 Nov 2025

Bibliographical note

Copyright © 2025 the Author(s). Published by PNAS.
This article is distributed under Creative Commons
Attribution-NonCommercial-NoDerivatives License 4.0
(CC BY-NC-ND)

Funding

This work was supported by the National Health and Medical Research Council of Australia Senior Principal Research Fellowships (#1154434 to P.M.S.), Senior Research Fellowship (#1155302 to D.W.), Leadership Fellowships (#2025694 to P.M.S.; #2026300 to D.W.), Program grant (#1150083 to P.M.S.), an Australian Research Council Discovery Project (#230102776 to D.W.), an Australian Research Council Discovery Early Career Researcher Award (#DE250100386 to S.J.P.), and a BBSRC project grant (BB/ W016974/1) to C.A.R. and D.G. We would like to thank Yao Lu for generating the expression pellet for PAC1nR-DNGs, Mohammed Akhter Hossain for providing the AF568-PACAP27 fluorescent probe for binding studies as well as Matthew J. Belousoff for assisting with cryo-electron microscopy data collection and data processing support, and Jianjun Cao and Brian P. Cary for assisting with map and model validation. This work was supported by Monash eResearch capabilities, including M3 High-performance computing (https://www.massive.org.au). Cryo-electron microscopy data collection was performed at the Bio21 Ian Holmes Imaging Centre (on the Glacios microscope) and the Monash Ramaciotti Centre for Cryo-Electron Microscopy facilities (on the Krios microscope) (Melbourne, Australia). Molecular graphics and analyses for the cryo-electron microscopy and molecular dynamics simulation datasets were produced with Chimera and ChimeraX from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco. ACKNOWLEDGMENTS. This work was supported by the National Health and Medical Research Council of Australia Senior Principal Research Fellowships (#1154434 to P.M.S.), Senior Research Fellowship (#1155302 to D.W.), Leadership Fellowships (#2025694 to P.M.S.; #2026300 to D.W.), Program

Funders	Funder number
National Health and Medical Research Council	1150083, 1154434, 1155302, 2025694, 2026300
Australian Research Council	230102776
Biotechnology and Biological Sciences Research Council	BB/ W016974/1

Keywords

Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I/genetics
Alternative Splicing
Vasoactive Intestinal Peptide/metabolism
Humans
Ligands
Molecular Dynamics Simulation
Pituitary Adenylate Cyclase-Activating Polypeptide/metabolism
HEK293 Cells
Animals
Cryoelectron Microscopy
Signal Transduction
Protein Binding

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Cite this

Lu, J. J., Deganutti, G., Li, M., Humphrys, L. J., Li, Y., Nettleton, T. J., Venugopal, H., Julita, V., Christopoulos, G., Reynolds, C. A., Sexton, P. M., Wootten, D., Zhao, P., & Piper, S. J. (2025). Structural basis of modified ligand selectivity from N-terminal PAC1R alternative splicing. Proceedings of the National Academy of Sciences of the United States of America, 122(47), Article e2521157122. https://doi.org/10.1073/pnas.2521157122

Lu, JJ, Deganutti, G, Li, M, Humphrys, LJ, Li, Y, Nettleton, TJ, Venugopal, H, Julita, V, Christopoulos, G, Reynolds, CA, Sexton, PM, Wootten, D, Zhao, P & Piper, SJ 2025, 'Structural basis of modified ligand selectivity from N-terminal PAC1R alternative splicing', Proceedings of the National Academy of Sciences of the United States of America, vol. 122, no. 47, e2521157122. https://doi.org/10.1073/pnas.2521157122

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title = "Structural basis of modified ligand selectivity from N-terminal PAC1R alternative splicing",

abstract = "The pituitary adenylate cyclase-activating polypeptide (PACAP) 1 receptor (PAC1R) is a class B1 G protein-coupled receptor activated by the endogenous peptide agonists PACAP and vasoactive intestinal peptide (VIP). Alternate splicing within the receptor extracellular domain (ECD) generates the PAC1R short variant (PAC1sR) that has selectively enhanced VIP function compared to the full-length, PAC1R null variant (PAC1nR). However, to date, a comprehensive pharmacological assessment of the downstream signaling outcomes of PAC1sR activation compared to PAC1nR has not been performed, and little information is available to mechanistically understand how ECD splicing may alter ligand engagement. Here, we demonstrated that VIP, but not PACAP, has globally enhanced activity across a broad range of functional endpoints at PAC1sR compared to PAC1nR. Cryo-EM structures of VIP-bound, stimulatory G protein (G s)-coupled PAC1sR and PAC1nR, supported by molecular dynamics (MD) simulations, demonstrate transient engagement of the null loop in PAC1nR, which is absent in PAC1sR, with residues in extracellular loop 2 (ECL2) and the N-terminal helix of the ECD. These interactions result in differential engagement of VIP with these domains and the top of TM2/ECL1 with PAC1sR and PAC1nR. Moreover, MD simulations predicted differential interactions of the G s protein with the two PAC1R variants when bound by VIP that correlate with a greater allosteric influence of the G s protein on VIP affinity at the PAC1sR, relative to PAC1nR. Our study provides insights into the structural basis and functional consequences of PAC1R ECD splicing, increasing understanding of PAC1R ligand selectivity and signaling. ",

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author = "Lu, \{Jessica J\} and Giuseppe Deganutti and Miaomiao Li and Humphrys, \{Laura J\} and Yandi Li and Nettleton, \{Theodore J\} and Hariprasad Venugopal and Villy Julita and George Christopoulos and Reynolds, \{Christopher A\} and Sexton, \{Patrick M\} and Denise Wootten and Peishen Zhao and Piper, \{Sarah J\}",

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AU - Lu, Jessica J

AU - Deganutti, Giuseppe

AU - Li, Miaomiao

AU - Humphrys, Laura J

AU - Li, Yandi

AU - Nettleton, Theodore J

AU - Venugopal, Hariprasad

AU - Julita, Villy

AU - Christopoulos, George

AU - Reynolds, Christopher A

AU - Sexton, Patrick M

AU - Wootten, Denise

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KW - Pituitary Adenylate Cyclase-Activating Polypeptide/metabolism

KW - HEK293 Cells

KW - Animals

KW - Cryoelectron Microscopy

KW - Signal Transduction

KW - Protein Binding

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DO - 10.1073/pnas.2521157122

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SN - 0027-8424

VL - 122

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

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M1 - e2521157122

ER -

Structural basis of modified ligand selectivity from N-terminal PAC1R alternative splicing

Abstract

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