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Kidney Week

Abstract: FR-PO544

WNT Ligands Drive PKD1-Mediated G Protein Signaling and Receptor Internalization and Downregulation

Session Information

Category: Genetic Diseases of the Kidneys

  • 1201 Genetic Diseases of the Kidneys: Cystic

Authors

  • Hardy, Emily P., The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
  • Tsiokas, Leonidas, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
Background

Autosomal Dominant Polycystic Kidney Disease is a genetic disorder caused by mutations in PKD1 or PKD2, which encode PKD1 and PKD2, respectively. PKD2 is a nonselective cation channel and forms a complex with PKD1 in which the channel is activated upon WNT binding to PKD1. Independently, PKD1 is proposed to act as an atypical G protein-coupled receptor (GPCR) for which the activating ligand is unknown. Here, we investigated the role of WNTs in PKD1-mediated G protein signaling and receptor internalization and downregulation.

Methods

An interaction between PKD1 and Gq has been previously established and was used to investigate PKD1-mediated signaling in response to WNTs. Using wild type and PKD1 knockout IMCD3 cells, single-cell calcium imaging was employed to test whether PKD1-Gq coupling is functional and occurs in response to WNTs. To determine if WNTs induce downstream PKD1 internalization and downregulation, pull down assays in combination with BRET (Bioluminescence Resonance Energy Transfer) were used. HEK293T cells were either singly- or co-transfected with PKD1 and WNT/empty vector, and assayed following co-expression or co-culture. Co-immunoprecipitation and western blot techniques coupled with a BRET titration approach were used to assess GPCR kinase (GRK) and β-arrestin recruitment to PKD1.

Results

Compared to wild type cells, PKD1 knockout IMCD3 cells showed a significant reduction in the rise of intracellular Ca2+ following WNT5A stimulation, suggesting a role for WNT5A in PKD1-dependent downstream Ca2+ transients. Following co-expression/co-culture, our western blot and BRET analysis revealed a significant decrease in whole cell and cell surface levels of PKD1 in cells transfected with WNT5A/9B, but not empty vector. This data suggests requirement for WNT in PKD1 internalization and downregulation. Supporting the notion that this process occurs as GPCR desensitization, GRK2/6 and β-arrestin1/2 were found to interact specifically with PKD1.

Conclusion

Our findings highlight an important role for WNTs in the GPCR-function of PKD1 for both receptor activation and desensitization. WNTs have been shown to interact with PKD1 to activate PKD2 channel activity, andtogether with our findings suggest a model whereby WNT-induced activation of PKD1 leads to intermediary G protein signaling and downstream activation of PKD2 channel activity.

Funding

  • NIDDK Support