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Functional communication between IP₃R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE.

ABSTRACT: Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca²⁺] ([Ca²⁺]_ER) and cluster in ER-plasma membrane (ER-PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca²⁺]_ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca²⁺ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER-PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER-PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP₃R) function and local [Ca²⁺]_ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP₃R function at ambient [IP₃] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca²⁺]_ER mediated by IP₃R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP₂-PLC activity of the cell determines IP₃R function, immobilization of STIM2, and basal Ca²⁺ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER-PM junctions, a first response to ER-Ca²⁺ store depletion, is facilitated by the juxtaposition of IP₃R and marks a checkpoint for initiation of Ca²⁺ entry.

SUBMITTER: Ahmad M

PROVIDER: S-EPMC8784118 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Anti-apoptotic Bcl-2 proteins are upregulated in different cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), enabling survival by inhibiting pro-apoptotic Bcl-2-family members and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-mediated Ca2+-signaling. A peptide tool (Bcl-2/IP3R Disruptor-2; BIRD-2) was developed to abrogate the interaction of Bcl-2 with IP3Rs by targeting Bcl-2's BH4 domain. BIRD-2 triggers cell death in primary CLL cells and in DLBCL cell lines. Particularly, DLBCL cells with high levels of IP3R2 were sensitive to BIRD-2. Here, we report that BIRD-2-induced cell death in DLBCL cells does not only depend on high IP3R2-expression levels, but also on constitutive IP3 signaling, downstream of the tonically active B-cell receptor. The basal Ca2+ level in SU-DHL-4 DLBCL cells was significantly elevated due to the constitutive IP3 production. This constitutive IP3 signaling fulfilled a pro-survival role, since inhibition of phospholipase C (PLC) using U73122 (2.5?µM) caused cell death in SU-DHL-4 cells. Milder inhibition of IP3 signaling using a lower U73122 concentration (1?µM) or expression of an IP3 sponge suppressed both BIRD-2-induced Ca2+ elevation and apoptosis in SU-DHL-4 cells. Basal PLC/IP3 signaling also fulfilled a pro-survival role in other DLBCL cell lines, including Karpas 422, RI-1 and SU-DHL-6 cells, whereas PLC inhibition protected these cells against BIRD-2-evoked apoptosis. Finally, U73122 treatment also suppressed BIRD-2-induced cell death in primary CLL, both in unsupported systems and in co-cultures with CD40L-expressing fibroblasts. Thus, constitutive IP3 signaling in lymphoma and leukemia cells is not only important for cancer cell survival, but also represents a vulnerability, rendering cancer cells dependent on Bcl-2 to limit IP3R activity. BIRD-2 seems to switch constitutive IP3 signaling from pro-survival into pro-death, presenting a plausible therapeutic strategy.

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Functional communication between IP₃R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Functional communication between IP3R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Functional communication between IP₃R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE.