Nephrocystin-1 Forms a Complex with Polycystin-1 via a Polyproline Motif/SH3 Domain Interaction and Regulates the Apoptotic Response in Mammals

Claas Wodarczyk,Sophie Saunier,Barbara Bricoli,Isaline Rowe,Giovanna Musco,Mordi Muorah,Piero Ricchiuto,Ariel E Casanova,Valeria Mannella,Gianfranco Distefano,Manuela Banzi,Manila Boca,Corinne Antignac,Maddalena Castelli,Massimiliano Gaetani,Monika Pema,Luca Mollica,Andrea Spitaleri,Alessandra Boletta

doi:10.1371/journal.pone.0012719

Abstract

Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation.

Highlights

Autosomal dominant polycystic kidney disease (ADPKD) is a frequent genetic disease affecting 1/1000 people characterized by renal cyst formation
We show that PC-1 interacts with the Src homology domain 3 (SH3) domain of Nephrocystin-1 (NPHP1), the product of the NPHP1 gene mutated in nephronophthisis, an autosomal recessive disease characterized by a small cyst formation at the corticomedullary junction of the kidney [8], [9] [10]
In this study we provide evidence for the first time of the existence of a functional polyproline domain within the C-tail of PC-1 able to interact with SH3 domains

Summary

Introduction

Autosomal dominant polycystic kidney disease (ADPKD) is a frequent genetic disease affecting 1/1000 people characterized by renal cyst formation. Mutations in two genes can cause ADPKD: PKD1 and PKD2 [1]. The first is mutated in the majority of the cases (85%), and it encodes a large (,520 kDa) plasma membrane receptor, Polycystin-1 (PC-1). PC-1 has a very large extracellular domain comprised of a novel combination of protein-protein interaction domains, 11 transmembrane domains and a short intracellular C-terminus containing a coiled-coil motif that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2) [2] [3]. The complex localizes to cell-cell junctions [5], focal adhesions [6] and the primary cilia in renal epithelial cells [7]

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Conclusion