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Kidney Week

Abstract: FR-PO957

Polycystin-1 Signaling Is Regulated by a Stachel-Like Sequence

Session Information

Category: Genetic Diseases of the Kidney

  • 1001 Genetic Diseases of the Kidney: Cystic

Authors

  • Magenheimer, Brenda S., University of Kansas Medical Center, Kansas City, Kansas, United States
  • Maser, Robin L., University of Kansas Medical Center, Kansas City, Kansas, United States
Background

Polycystin-1 (PC1) is involved in modulation of G protein signaling, but the regulatory mechanism is unknown. Similar to the Adhesion class of GPCRs, PC1 undergoes auto-catalyzed cleavage at a GPS motif present within its conserved GAIN domain that generates noncovalently-associated extracellular N-terminal (NTF) and membrane-embedded C-terminal (CTF) fragments. Adhesion GPCR signaling can be regulated by residues in the extracellular stalk (the so-called ‘Stachel sequence’) of the CTF that are exposed upon removal of the NTF. We hypothesized that PC1-mediated signaling is regulated by a Stachel-like sequence within the N-terminus of its CTF.

Methods

Full-length (FL) or CTF forms of PC1 with wild type or mutant stalk sequences (i.e., deletion, ADPKD-associated missense, or alanine substitution) were transiently expressed in HEK293T cells. Activation of a co-transfected NFAT promoter-luciferase reporter and levels of total and surface-expressed protein were compared between mutant and wild type PC1.

Results

FL PC1 activated the NFAT reporter in a dose-dependent fashion, while the CTF demonstrated an inverse dose relationship possibly dependent on its % surface expression. Deletion of the entire stalk region from CTF eliminated NFAT reporter activation, which was not solely due to reduced total protein or surface expression levels. Alanine-scanning of the CTF stalk sequence resulted in a range of effects on NFAT activity, from abolishment to augmentation, which were not entirely consistent with changes in mutant CTF expression characteristics. ADPKD missense mutations in the CTF stalk abolished NFAT activation with varying effects on total or surface protein expression. The same ADPKD mutations when engineered into FL PC1 also inhibited NFAT reporter activation, but had differing effects on GPS cleavage and cell surface expression.

Conclusion

The CTF may be the 'native form' of PC1 responsible for signaling to the NFAT reporter, whose signaling ability is dependent on the presence of the extracellular stalk. Mutation analyses implicate specific residues within the stalk that are critical for NFAT activation, which are likely involved in intramolecular interactions with membrane-embedded portions of the CTF. These observations are consistent with an adhesion GPCR-like and a stalk/Stachel sequence-dependent mechanism for PC1-mediated signaling.

Funding

  • Other U.S. Government Support