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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.

Author information

1
From the Departments of Laboratory Medicine, Pharmacology, and yifei.yang@yale.edu.
2
From the Departments of Laboratory Medicine.
3
Pharmacology, and.
4
Pharmacology, and Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven, Connecticut 06520.
5
From the Departments of Laboratory Medicine, michael.hodsdon@yale.edu.

Abstract

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.

KEYWORDS:

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

PMID:
25716316
PMCID:
PMC4400361
DOI:
10.1074/jbc.M115.641803
[Indexed for MEDLINE]
Free PMC Article

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