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J Biol Chem. 2015 Apr 17;290(16):10544-54. doi: 10.1074/jbc.M115.641803. Epub 2015 Feb 25.

Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties.

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From the Departments of Laboratory Medicine, Pharmacology, and
From the Departments of Laboratory Medicine.
Pharmacology, and.
Pharmacology, and Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven, Connecticut 06520.
From the Departments of Laboratory Medicine,


Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca(2+)-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca(2+)-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca(2+) bound, although TRP channels are believed to be tetramers. We found that there is only one Ca(2+)-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca(2+) binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca(2+) binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca(2+)-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca(2+)-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.


Calcium; Calcium Intracellular Release; Calcium-binding Protein; EF-hand Proteins; Isothermal Titration Calorimetry (ITC); Nuclear Magnetic Resonance (NMR); Polycystic Kidney Disease

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