Kidney Week

Abstract: PO1223

Polycystin 1 Ciliary Localization Is Regulated by Its aGPCR Activity

Session Information

• Cystic Kidney Disease - I
  November 04, 2021 | Location: On-Demand, Virtual Only
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1001 Genetic Diseases of the Kidneys: Cystic

Authors

• Gresko, Nikolay P., Yale University School of Medicine, New Haven, Connecticut, United States

Nikolay P. Gresko, MD, PhD

• Caplan, Michael J., Yale University School of Medicine, New Haven, Connecticut, United States

Michael J. Caplan,

Group or Team Name

• Caplan Lab

Background

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss of function mutations in the PKD1 and PKD2 genes, which encode PC1 and PC2, respectively. PC1 is a 460kD multi-spanning membrane protein that undergoes multiple proteolytic cleavages, one of which occurs at a G Protein Coupled Receptor (GPCR) Proteolytic site near the junction between the large extracellular N terminus (NTF) and the first transmembrane domain. PC1 and 2 both localize to the primary cilium where they may participate in mechano-sensation and other sensory processes. Recent studies demonstrate that PC1 can function as an atypical GPCR and that it binds to Wnt ligands. We have shown that removal of the PC1 NTF exposes a tethered agonist (TA) peptide that activates the PC1 GPCR function. Activation of GPCRs in the cilia induces their trafficking out of the cilium in association with their ligand-induced desensitization.

Methods

Confocal IF, Mechanical stimulation, PC1 mutagenesis.

Results

We find that the localization of PC1 to cilia is similarly regulated by its GPCR activity. A constitutively active PC1 construct that lacks the NTF (PC1^ΔNTF) is absent from the primary cilia when expressed in LLC-PK₁ cells, whereas a constitutively inactive PC1 construct that lacks the NTF and the TA (PC1^ΔNTFΔTA) resides in the primary cilia. Addition of a soluble form of the TA peptide to cells expressing PC1^ΔNTFΔTA results in redistribution of the PC1^ΔNTFΔTA protein out of the cilium. Blocking β-arrestin-mediated receptor desensitization with barbadin prevents the removal of PC1^ΔNTF and of TA peptide-treated PC1^ΔNTFΔTA from the cilium. When full length PC1 is co-expressed with PC1 in LLC-PK₁ cells both proteins localize to the cilium. Extended exposure to the Wnt9b ligand or to mechanical stimuli both lead to reduced quantities of PC1 in the cilium and at the apical cell surface, and this redistribution is blocked by β-arrestin inhibitor barbadin.

Conclusion

Our data demonstrate that PC1 ciliary localization is regulated by physiological stimuli including ligand binding and mechanical stress and, like GPCR regulation, is subject to receptor desensitization processes. Our findings indicate that the GPCR activation state of PC1 can be observed by examining the distribution of PC1, suggesting new approaches through which PC1 activity can be assessed in vivo and in the context of drug discovery.

Funding

• NIDDK Support