STING is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I Interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER–Golgi intermediate compartment. Here we found that deficiency in the Ca 2+ sensor STIM1 caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient suffering from combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and co-expression of full-length or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1–STING circuit that maintains the resting state of the STING pathway.

STIM1 (stromal interaction molecule 1) mediates SOCE (store-operated Ca²⁺ entry) in skeletal muscle. However, the direct role(s) of STIM1 in skeletal muscle, such as Ca²⁺ release from the SR (sarcoplasmic reticulum) for muscle contraction, have not been identified. The times required for the maximal expression of endogenous STIM1 or Orai1, or for the appearance of puncta during the differentiation of mouse primary skeletal myoblasts to myotubes, were all different, and the formation of puncta was detected with no stimulus during differentiation, suggesting that, in skeletal muscle, the formation of puncta is a part of the differentiation. Wild-type STIM1 and two STIM1 mutants (Triple mutant, missing Ca²⁺-sensing residues but possessing the intact C-terminus; and E136X, missing the C-terminus) were overexpressed in the myotubes. The wild-type STIM1 increased SOCE, whereas neither mutant had an effect on SOCE. It was interesting that increases in the formation of puncta were observed in the Triple mutant as well as in wild-type STIM1, suggesting that SOCE-irrelevant puncta could exist in skeletal muscle. On the other hand, overexpression of wild-type or Triple mutant, but not E136X, attenuated Ca²⁺ releases from the SR in response to KCl [evoking ECC (excitation-contraction coupling) via activating DHPR (dihydropyridine receptor)] in a dominant-negative manner. The attenuation was removed by STIM1 knockdown, and STIM1 was co-immunoprecipitated with DHRP in a Ca²⁺-independent manner. These results suggest that STIM1 negatively regulates Ca²⁺ release from the SR through the direct interaction of the STIM1 C-terminus with DHPR, and that STIM1 is involved in both ECC and SOCE in skeletal muscle.

The expression of TRPC3 (canonical-type transient receptor potential cation channel type 3) is tightly regulated during skeletal muscle cell differentiation, and a functional interaction between TRPC3 and RyR1 [(ryanodine receptor type 1), an SR (sarcoplasmic reticulum) Ca2+-release channel] regulates the gain of SR Ca2+ release during EC (excitation-contraction) coupling. However, it has not been possible to demonstrate direct protein-protein interactions between TRPC3 and RyR1. To identify possible candidate(s) for a linker protein(s) between TRPC3 and RyR1 in skeletal muscle, in the present study we performed MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of a cross-linked triadic protein complex from rabbit skeletal triad vesicles and co-immunoprecipitation assays using primary mouse skeletal myotubes. From these studies, we found that six triadic proteins, that are known to regulate RyR1 function and/or EC coupling [TRPC1, JP2 (junctophilin 2), homer, mitsugumin 29, calreticulin and calmodulin], interacted directly with TRPC3 in a Ca2+-independent manner. However we again found no direct interaction between TRPC3 and RyR1. TRPC1 was identified as a potential physical link between TRPC3 and RyR1, as it interacted with both TRPC3 and RyR1, and JPs showed subtype-specific interactions with both RyR1 and TRPC3 (JP1-RyR1 and JP2-TRPC3). These results support the hypothesis that TRPC3 and RyR1 are functionally engaged via linker proteins in skeletal muscle.

Background: Junctophilin-2 (JP2) contributes to the formation of junctional membrane complexes (JMC) in striated muscle. Results: Different from the S165F mutant of JP2, Y141H induces hypertrophy in skeletal myotubes involving abnormal JMC and altered Ca 2ϩ signaling due to the increased store-operated Ca 2ϩ entry (SOCE) via Orai1. Conclusion: JP2 is linked to muscle hypertrophy via various Ca 2ϩ signaling pathways. Significance: SOCE is a novel factor in understanding muscle hypertrophy.

More than 60 members of the Rab GTPase family exist in the human genome. However, our current understanding is only limited to the role of small Rab GTPases in membrane trafficking. Here we show that CRACR2A encodes a lymphocyte-specific “large Rab GTPase” containing multiple functional domains including EF-hand motifs, proline-rich and Rab GTPase domains with an unconventional prenylation site. We demonstrate its direct role in activation of the Ca2+ and the Jnk signaling pathways upon T cell receptor (TCR) stimulation using gene silencing and transgenic animal models. Mechanistically, vesicles containing this Rab GTPase translocate from the Golgi into the immunological synapse (IS) to activate these signaling pathways. The interaction between proline-rich domain of this Rab GTPase and a guanidine nucleotide exchange factor/scaffold protein Vav1 is essential for accumulation of these vesicles at the IS. Furthermore, we demonstrate that GTP binding and prenylation are closely linked to membrane association, stability, and thereby activation of downstream signaling by this large GTPase. Our findings reveal a novel function of a large Rab GTPase in TCR signaling pathways, which is potentially shared by other GTPases with similar domain architecture.