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Identification of polycystin-1 and G?12 binding regions necessary for regulation of apoptosis.

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

SUBMITTER: Yu W

PROVIDER: S-EPMC2998059 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Identification of polycystin-1 and Gα12 binding regions necessary for regulation of apoptosis.

Yu Wanfeng W Ritchie Benjamin J BJ Su Xuefeng X Zhou Jing J Meigs Thomas E TE Denker Bradley M BM

Cellular signalling 20100917 1

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 Gα12. 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 Gα12-stimulated apoptosis (Yu et al., J. Biol. Chem. 2010 285(14):10243-51). Here ...[more]

PMID: 20837139

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Dataset Information

Identification of polycystin-1 and G?12 binding regions necessary for regulation of apoptosis.

Publications

Identification of polycystin-1 and Gα12 binding regions necessary for regulation of apoptosis.

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