ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: PO1239

Allosteric Mechanism of PC1 Tethered Agonist-Mediated Signaling

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Pawnikar, Shristi, University of Kansas College of Liberal Arts and Sciences, Lawrence, Kansas, United States
  • Magenheimer, Brenda S., University of Kansas Medical Center, Kansas City, Kansas, United States
  • Nevarez Munoz, Ericka, University of Kansas Medical Center, Kansas City, Kansas, United States
  • Maser, Robin L., University of Kansas Medical Center, Kansas City, Kansas, United States
  • Miao, Yinglong, University of Kansas College of Liberal Arts and Sciences, Lawrence, Kansas, United States
Background

Polycystin-1 (PC1) is the 11-transmembrane protein product of the human autosomal dominant polycystic kidney disease (ADPKD) gene PKD1. PC1 functions as an atypical GPCR and shares multiple features with the Adhesion GPCRs, including a GPCR autoproteolysis-inducing (GAIN) domain that catalyzes cis-cleavage of the receptors into extracellular N-terminal (NTF) and membrane-embedded C-terminal (CTF) fragments. We previously reported that CTF-mediated signaling to an NFAT promoter-luciferase reporter is dependent on the presence of the stalk, is reduced by ADPKD-associated missense mutations within the stalk, and can be rescued by synthetic, stalk-derived peptides, supporting a tethered ligand mechanism of PC1-G protein signaling (JASN 2018;29:671, JASN 2019;30:882).

Methods

We have combined highly complementary experiments and computer simulations to investigate mechanism of the tethered agonist-mediated signaling of PC1 CTF. A computer model of the human PC1 CTF was generated using the cryo-EM structure of the PC1-PC2 complex with important missing regions added through I-TASSER homology modeling. All-atom enhanced simulations (1000 ns) using a robust Gaussian accelerated molecular dynamics (GaMD) technique were performed, followed by calculations of residue correlation matrices and free energy profiles. GaMD simulation-predicted residue interactions important for WT stalk-mediated activation of PC1 CTF were further investigated by mutagenesis and cellular assay experiments.

Results

GaMD simulations were consistent with experimental signaling data obtained with PC1 CTF expression constructs encoding wild type and stalk variants of the PC1 CTF. Correlation matrices revealed regions of highly correlated residue motions involving the stalk, TOP and putative pore loop domains. Key residue interactions predicted from the GaMD simulations were validated with newly designed mutation experiments.

Conclusion

Complementary experiments and simulations studies support the function of the PC1 CTF stalk region as a tethered agonist and suggest a mechanism whereby it can induce TOP-pore loop interactions which can be further translated to the C-tail for G protein activation. This in-depth knowledge is expected to facilitate future drug design efforts targeting this function of PC1 for more effective treatments of ADPKD.

Funding

  • NIDDK Support