Identification of polycystin-1 and Ga12 binding regions necessary for regulation of apoptosis

Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations in PKD1, the gene for polycystin-1 (PC1), a transmembrane protein with a cytoplasmic C-terminus that interacts with numerous signaling molecules, including Ga12. The functions of PC1 and the mechanisms of cyst development leading to renal failure are complex. Recently, we reported that PC1 expression levels modulate activity of Ga12-stimulated apoptosis (Yu et al., J. Biol. Chem. 2010 285(14):10243-51). Herein, a mutational analysis of Ga12 and PC1 was undertaken to identify regions required for their interaction and ability to modulate apoptosis. A set of Ga12 mutations with systematic replacement of six amino acids with NAAIRS was tested for binding to the PC1 C-terminus in GST pulldowns. Additionally, a series of deletions within the PC1 C-terminus was examined for binding to Ga12. We identified 3 NAAIRS substitutions in Ga12 that completely abrogated binding, and identified a previously described 74 amino acid Gai/o binding domain in the PC1 C-terminus as necessary for Ga12 interaction. The functional consequences of uncoupling PC1/Ga12 binding were studied in apoptosis assays utilizing HEK293 cells with inducible PC1 overexpression. Ga12 mutants deficient in PC1 binding were refractory to PC1 inhibition of Ga12-stimulated apoptosis. Likewise, deletion of the Ga12-interacting sequence from the PC1 cytoplasmic domain abrogated its inhibition of Ga12-stimulated apoptosis. Based on the crystal structure of Ga12, the PC1 interaction sites are likely to reside on exposed regions within the G protein helical domain. These structural details should facilitate the design of reagents to uncouple PC1/Ga12 signaling in ADPKD.