Abstract: SA-PO464
Peptides Derived from the Stalk Region of Polycystin 1 Function as Ligands to Activate Signaling by the C-Terminal Fragment and to Ameliorate Cystogenesis
Session Information
- Cystic Kidney Diseases: Basic/Translational
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1001 Genetic Diseases of the Kidneys: 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) modulates G protein signaling by as yet unknown regulatory mechanisms. Like the Adhesion class of GPCRs, PC1 undergoes auto-catalyzed cleavage at a GPS motif, which generates a large extracellular, N-terminal fragment and a membrane-embedded, C-terminal fragment (CTF) composed of 11 transmembrane domains preceded by an N-terminal extracellular stalk of 25 residues. We previously reported that PC1 CTF-mediated signaling to an NFAT reporter is dependent on the presence of the stalk, and is reduced by specific amino acid substitutions within its sequence. To determine if this stalk-dependent mechanism contributes to the inhibition of cyst formation, the ability of stalk-derived peptides to rescue signaling by a ‘stalk-less’ CTF expression construct (ΔCTF) and to ameliorate cAMP-driven cystogenesis was determined.
Methods
HEK293T cells transiently transfected with the ΔCTF construct lacking the first 21 residues of the stalk region were treated with soluble peptides (P) ranging from 7 to 21 residues in length whose sequences were derived from the stalk region. Activation of a co-transfected NFAT promoter-luciferase reporter was compared between ΔCTF- and empty expression vector control-transfected cells. Metanephric organ cultures derived from E15.5 Pkd1RC/RC fetal mice were stimulated with 8-Br-cAMP, and were treated with stalk peptides for up to 4 days in culture. The cystic area was compared between non-treated and peptide-treated kidneys.
Results
All of the stalk peptides (P7-P21) enhanced signaling from ΔCTF to the NFAT reporter, albeit to varying degrees, possibly due to differences in peptide structure. Peptides P7, P9, P13, P15 and P17 significantly reduced the cystic index of treated embryonic kidneys to different extents (e.g., ~25% for P7 to ~90% for P17), while P11 had no ameliorating effect. Treatment with P19 or P21 prevented kidney growth and decreased kidney survival. A mutant peptide containing a human ADPKD-associated missense mutation was also detrimental in organ culture unlike its wild type parental peptide.
Conclusion
These results support an Adhesion GPCR-like, stalk-dependent mechanism for regulation of signaling by PC1, implicate a physiological and disease-relevant role for this mechanism in renal tubulogenesis, and suggest a novel therapeutic avenue for ADPKD.
Funding
- Other U.S. Government Support