Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a widespread genetic disease with a mortality rate of approximately 50%. Mutations in the cell surface protein polycystin-1 are found in nearly 80% of cases of this malady, and its interaction with the heterotrimeric G protein Gα12 has been implicated as an important event in disease progression. Heterotrimeric G proteins are involved in a myriad of cell signaling processes, including transcriptional activation and apoptosis. Polycystin-1 is important in down regulating Gα12-mediated JNK/Bcl-2 activation of programmed cell death (apoptosis), and abnormal apoptotic activity is a hallmark of ADPKD. Previous work has revealed a 20 amino acid G protein binding domain (GPS domain) within the cytosolic C-terminal tail of polycystin-1. Utilizing PCR directed mutagenesis several single and multiple charge swap mutations of charged amino acid residues within the first half of the GPS domain were developed. Co-precipitation assays of these mutant polycystin-1 constructs reveal the effect on the binding affinity for Gα12. In an attempt to mimic the genetics of the disease by engineering the same polycystin-1 variant found in some disease samples, revealed that a naturally occurring nonsense mutation resulting in a truncated protein severely impaired Gα12 binding. In identifying key amino acids within polycystin-1 that mediate Gα12 binding, the hope is to characterize the structural interface between these two proteins.

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