Kidney Week

Abstract: FR-PO546

Cleavage of PC1 Is Not Required for Embryonic Vasculature Development

Session Information

• Genetic Diseases: Cystic - Basic
  November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1201 Genetic Diseases of the Kidneys: Cystic

Authors

• Basquin, Denis, University of Maryland School of Medicine, Baltimore, Maryland, United States

Denis Basquin,

• Outeda, Patricia, University of Maryland School of Medicine, Baltimore, Maryland, United States

Patricia Outeda,

• Watnick, Terry J., University of Maryland School of Medicine, Baltimore, Maryland, United States

Terry J. Watnick,

• Qian, Feng, University of Maryland School of Medicine, Baltimore, Maryland, United States

Feng Qian,

Background

Polycystin 1 (PC1) is a large membrane protein that undergoes an autoproteolytic cleavage at a G protein coupled receptor (GPS) site. PC1 cleavage at the GPS is essential for trafficking of the PC1/PC2 complex to the primary cilium. While homozygous Pkd1 or Pkd2 knock-out mice die at mid-gestation due to vascular and lymphatic abnormalities homozygous Pkd1^V/V mutant embryos, harboring a mutation that abolishes PC1 cleavage, survive embryogenesis, suggesting that uncleaved PC1 (PC1^V) might play a role in embryonic vascular development.

Methods

We analyzed the vascular phenotype of Pkd1^V/V, Pkd1^-/- and control E14.5 embryos including placenta branching morphogenesis and epidermal lymphatic vessel development. Rescue experiments were performed using a transgenic BAC expressing HA-tagged-PC1^V (PC1^V-F/H). We evaluated the impact of PC1 cleavage on cell migration/polarity via wound healing assay. Trafficking of PC1^V and the PC2/PC1^V complex was assessed by N-glycosylation studies and biotinylation assays. Localization of PC1 and PC1^V in the cilia was performed using immunofluorescence in endothelial cells (EC).

Results

Pkd1^V/V embryos lacked the vascular abnormalities observed in Pkd1^-/- embryos including hemorrhage, edema and polyhydramnios. The complexity of the placental labyrinth vasculature and the lymphatic vasculature were similar between Pkd1^V/V and controls but reduced in Pkd1^-/- embryos. Transgenic expression of PC1^V-F/H was sufficient to rescue the vascular phenotype observed in Pkd1^-/- mutants. Using wound-healing assays we observed that Pkd1^V/V cells migrated properly and that front-rear polarity was similar to controls. We confirmed that PC1 and PC2 colocalized in the primary cilia of cultured ECs. In contrast, PC2 was undetectable in cilia from Pkd1^V/V cells. In addition, we showed that the pool of PC2 protein that co-immunoprecipitated with PC1^V was Endo H sensitive, indicating that PC1 cleavage is a pre-requisite for the protein complex to traffic beyond the Golgi in ECs. PC1^V was partially resistant to Endo H and able to reach the cell surface.

Conclusion

Our data suggests that expression and localization of PC1 and/or PC2 in the primary cilia of ECs is not required for a proper vascular/lymphatic vessel development. This is the first time that an extraciliary role for full length PC1 has been identified.

Funding

• NIDDK Support