Identification Of Polycystin-1 And G Alpha 12 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 G alpha 12. 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 G alpha 12-stimulated apoptosis (Yu et al., J. Biol. Chem. 2010 285(14):10243-51). Herein, a mutational analysis of G alpha 12 and PC1 was undertaken to identify regions required for their interaction and ability to modulate apoptosis. A set of G alpha 12 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 G alpha 12. We identified 3 NAAIRS substitutions in G alpha 12 that completely abrogated binding, and identified a previously described 74 amino acid G alpha i/o binding domain in the PC1 C-terminus as necessary for G alpha 12 interaction. The functional consequences of uncoupling PC1/G alpha 12 binding were studied in apoptosis assays utilizing HEK293 cells with inducible PC1 overexpression. G alpha 12 mutants deficient in PC1 binding were refractory to PC1 inhibition of G alpha 12-stimulated apoptosis. Likewise, deletion of the G alpha 12-interacting sequence from the PC1 cytoplasmic domain abrogated its inhibition of G alpha 12-stimulated apoptosis. Based on the crystal structure of G alpha 12, 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/G alpha 12 signaling in ADPKD. (C) 2010 Elsevier Inc. All rights reserved.