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Ca2+-Dependent Interaction of Triadin with Histidine-Rich Ca2+-Binding Protein Carboxyl-Terminal Region

Biochemical and Biophysical Research Communications
Publication Date
DOI: 10.1006/bbrc.2001.6126
  • Skeletal Muscle
  • Sarcoplasmic Reticulum
  • Histidine-Rich
  • Ca2+-Binding Protein
  • Triadin
  • Calmodulin Protein Kinase
  • Biology


Abstract A direct binding of HRC (histidine-rich Ca2+-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. Opinions are still divided, however, concerning the triadin domain involved, either the cytoplasmic or the lumenal domain, and the exact role played by Ca2+, in the protein-to-protein interaction. Further support for colocalization of HRC with triadin cytoplasmic domain is provided here by experiments of mild tryptic digestion of tightly sealed TC vesicles. Accordingly, we show that HRC is preferentially phosphorylated by endogenous CaM K II, anchored to SR membrane on the cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate that HRC can be isolated as a complex with triadin, following equilibrium sucrose-density centrifugation in the presence of mM Ca2+. Here, we characterized the COOH-terminal portion of rabbit HRC, expressed and purified as a fusion protein (HRC569–852), with respect to Ca2+-binding properties, and to the interaction with triadin on blots, as a function of the concentration of Ca2+. Our results identify the polyglutamic stretch near the COOH terminus, as the Ca2+-binding site responsible, both for the acceleration in mobility of HRC on SDS–PAGE in the presence of millimolar concentrations of Ca2+, and for the enhancement by high Ca2+ of the interaction between HRC and triadin cytoplasmic segment.

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