Multiple Regions of Junctin and Triadin Interact with Calsequestrin 1 and 2 in Skeletal Muscle Cells

Abstract

The release of Ca2+ in skeletal muscle fibers is regulated by several proteins, which are organized in a multi-molecular complex localized at the junctional sarcoplasmic reticulum (jSR). This complex includes the intracellular Ca2+ channel ryanodine receptor (RyRs) and additional proteins, including triadin, junctin and calsequestrin, which have been shown to form a quaternary complex. In FRAP experiments, we found that, in differentiated myotubes, these proteins display specific dynamic properties suggesting the existence of distinctive protein-protein interactions among j-SR proteins. To better define the molecular bases of protein association in the multi-molecular complex assembled around the RyRs, we performed experiments aimed to identify the specific sequences that mediate the interactions among jSR proteins in the lumen of the SR. We expressed GST fusion proteins covering distinct intraluminal domains of triadin and junctin. The purified GST-fusion proteins were incubated with detergent-solubilized SR vesicle from mouse skeletal muscle and probed for their interaction with distinct SR proteins. Experiments were also performed with recombinant proteins expressed in HEK293 cells. We found that different regions in the intraluminal domain of junctin and triadin can bind calsequestrin-1 and/or calsequestrin-2 and that both proteins appear to bind preferentially calsequestrin-1 rather than calsequestrin-2. In addition, calsequestrin-1 and calsequestrin-2 appeared to display distinct binding affinities for junctin and triadin. The correlation between the in vitro protein-protein interactions and the dynamic properties of jSR proteins will be verified.