Co-expression of MG29 and Ryanodine Receptor Leads to Apoptotic Cell Death

Abstract

Perturbation of intracellular Ca2؉ homeostasis has been shown to regulate the process of cell proliferation and apoptosis. Our previous studies show that mitsugumin 29 (MG29), a synaptophysin-related protein localized in the triad junction of skeletal muscle, serves an essential role in muscle Ca 2؉ signaling by regulating the process of store-operated Ca 2؉ entry. Here we report a functional interaction between MG29 and the ryanodine receptor (RyR)/Ca 2؉ release channel. The purified MG29 protein enhances activity of the RyR/Ca 2؉ release channel incorporated into the lipid bilayer membrane. Coexpression of MG29 and RyR in Chinese hamster ovary cells leads to apoptotic cell death resulting from depletion of intracellular Ca 2؉ stores, despite neither protein expression alone exhibits any significant effect on cell viability. In transient expression studies, the presence of RyR in the endoplasmic reticulum leads to retention of MG29 from the plasma membrane into the intracellular organelles. This functional interaction between MG29 and RyR could have important implications in the Ca 2؉ signaling processes of muscle cells. Our data also show that perturbation of intracellular Ca 2؉ homeostasis can serve as a key signal in the initiation of apoptosis. Ca2ϩ as a second messenger plays important roles in a variety of cellular processes, ranging from cell motility to enzymatic control, and to gene regulation and apoptosis. The ryanodine receptors (RyR) 1 /Ca 2ϩ release channels are present in the endo/(sarco)plasmic reticulum of virtually every cell types, providing a pathway for fast Ca 2ϩ release from intracellular stores (1-4). Mitsugumin29 (MG29) is a novel membrane protein localized specifically to the triad junction of skeletal muscle (5). The primary amino acid sequence of MG29 shares ϳ45% homology to synaptophysin, a family of proteins with presumed roles in secretion and neurotransmitter release (6, 7). The MG29 protein appears to participate in the excitation-contraction coupling process of skeletal muscle, since mutant mice lacking the MG29 gene exhibit apparent reduction in contractile force, with altered structure of the triad junction (8), and increased susceptibility to fatigue of the skeletal muscle (9, 10).Several studies have suggested a central role for the participation of endoplasmic reticulum (ER) and intracellular Ca 2ϩ release in the initiation of apoptosis (11-13). Our previous studies have shown that depletion of intracellular Ca 2ϩ stores via activation of the RyR/Ca 2ϩ release channel could induce apoptosis (14, 15). Here we tested the hypothesis that overexpression of MG29 can influence the intracellular Ca 2ϩ homeostasis through changes of the RyR/Ca 2ϩ release function. We show that co-expression of MG29 and RyR in cultured cells leads to apoptotic cell death resulting from depletion of intracellular Ca 2ϩ stores, and the purified MG29 protein can enhance activity of the RyR/Ca 2ϩ release channel incorporated into the lipid bilayer membrane. Our data provide evidence for a functional ...

Immuno-proteomic approach to excitation–contraction coupling in skeletal and cardiac muscle: Molecular insights revealed by the mitsugumins

“…In further studies, we discovered a dysfunctional property of store-operated Ca 2+ entry (SOCE) in the mg29(−/−) muscle and established a functional role of SOCE in muscle fatigue [22,27,28]. We also identified a functional interaction between MG29 and RyR, through co-expression of MG29 and RyR in heterologous cells and reconstitutional studies of RyR in lipid bilayer membranes [29].…”

Altered Ca2+ sparks in aging skeletal and cardiac muscle

“…2B). A series of studies on the role of MG29 in muscle Ca 2+ signaling (Pan et al, 2002;Brotto et al, 2004;Pan et al, 2004) have provided intriguing observations that allowed us to formulate the hypothesis that MG29 could act as a sentinel, shielding muscle from aging induced alterations in Ca 2+ homeostasis. There were two important observations in these initial studies that suggest this is the case.…”

“…Assuming MG29 does act as a sentinel against aging effect on skeletal muscle, an important investigation would be to examine the how decreased MG29 modulates cellular Ca 2+ handling. MG29 can function in triad junction organization and can also directly modulate RyR function (Pan et al, 2004). One approach to examine the role of MG29 in aging skeletal muscle would be to rescue the expression of MG29 in aged mouse muscle through transgenic and molecular approaches and assay if compromised Ca 2+ spark signaling, the segregated Ca 2+ store and alterations to triad junction structure are restored to a level similar to the young wild type fibers.…”

Mitsugumin 29 regulates t-tubule architecture in the failing heart

“…S1). Since Mg29 has been described as both a t-tubule and SR-resident protein in skeletal muscle 11 , and it was previously identified as a potential regulator of RyR1 activity 24 , we measured Ca 2+ spark frequency and found no effect with Mg29 expression (Fig. 1e).…”