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SummaryProtein isoforms are widely expressed in biological systems. How isoforms that co-exist within the same sub-cellular domain are differentially activated remains unclear. Here, we compare the regulatory mechanism of two closely related transcription factor isoforms, NFAT1 and NFAT4, that migrate from the cytoplasm to the nucleus following the increase in intracellular Ca2+ that accompanies the opening of store-operated Orai1/CRAC channels. We demonstrate that NFAT1 has a private line of communication with Orai1, activating in response to Ca2+ microdomains near the open channels. By contrast, NFAT4 stimulation requires both local Ca2+ entry and a nuclear Ca2+ rise. We mapped differences in nuclear location to amino acids within the SP-3 motif of the NFAT regulatory domain. The different Ca2+ dependencies enable agonists to recruit different isoform combinations as stimulus strength increases. Our study uncovers a mechanism whereby co-existing cytoplasmic transcription factor isoforms are differentially activated by distinct sub-cellular Ca2+ signals.

SummaryNFAT-dependent gene expression is essential for the development and function of the nervous, immune, and cardiovascular systems and kidney, bone, and skeletal muscle [1]. Most NFAT protein resides in the cytoplasm because of extensive phosphorylation, which masks a nuclear localization sequence. Dephosphorylation by the Ca2+-calmodulin-activated protein phosphatase calcineurin triggers NFAT migration into the nucleus [2, 3]. In some cell types, NFAT can be activated by Ca2+ nanodomains near open store-operated Orai1 and voltage-gated Ca2+ channels in the plasma membrane [4, 5]. How local Ca2+ near Orai1 is detected and whether other Orai channels utilize a similar mechanism remain unclear. Here, we report that the paralog Orai3 fails to activate NFAT. Orai1 is effective in activating gene expression via Ca2+ nanodomains because it participates in a membrane-delimited signaling complex that forms after store depletion and brings calcineurin, via the scaffolding protein AKAP79, to calmodulin tethered to Orai1. By contrast, Orai3 interacts less well with AKAP79 after store depletion, rendering it ineffective in activating NFAT. A channel chimera of Orai3 with the N terminus of Orai1 was able to couple local Ca2+ entry to NFAT activation, identifying the N-terminal domain of Orai1 as central to Ca2+ nanodomain-transcription coupling. The formation of a store-dependent signaling complex at the plasma membrane provides for selective activation of a fundamental downstream response by Orai1.

Stimulation of cells with physiological concentrations of calciummobilizing agonists often results in the generation of repetitive cytoplasmic Ca 2+ oscillations. Although oscillations arise from regenerative Ca 2+ release, they are sustained by store-operated Ca 2+ entry through Ca 2+ release-activated Ca 2+ (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca 2+ oscillations, CRAC channel activity, and downstream Ca 2+ -dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca 2+ sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca 2+ oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca 2+ signals and downstream responses.excitation-transcription coupling | transcription S timulation of cell-surface receptors that couple to the phospholipase C pathway with physiological concentrations of agonist generally evokes repetitive cytoplasmic Ca 2+ oscillations (1). Oscillatory Ca 2+ signals enable cytoplasmic Ca 2+ to reach high levels transiently, thereby avoiding the deleterious effects of a prolonged, elevated Ca 2+ rise. Information is encoded in the oscillatory amplitude and frequency (2) and the spatial profile of the Ca 2+ signal (3), each of which can be deciphered by cells to drive selective downstream responses.Ca 2+ oscillations are triggered by inositol trisphosphate (InsP 3 )-mediated Ca 2+ release from intracellular Ca 2+ stores, primarily the endoplasmic reticulum (ER) (2). The resulting fall in Ca 2+ within the stores opens store-operated CRAC channels in the plasma membrane (4, 5). Ca 2+ entry through these channels refills the stores and, thus, sustains InsP 3 -dependent Ca 2+ oscillations (6). In addition to this supportive role, local Ca 2+ entry through Ca 2+ release-activated Ca 2+ (CRAC) channels during oscillatory responses in mast cells, and not the oscillations per se, signals to the nucleus to regulate Ca 2+ -dependent gene expression (7).The two main molecular components of store-operated Ca 2+ entry are the stromal interaction molecule (STIM) and Orai proteins (reviewed in refs. 8-10). The transmembrane ER proteins STIM1 and STIM2 detect ER Ca 2+ content through an EF-hand domain in their respective N-termini, which face the lumen of the store. Loss of luminal Ca 2+ leads to STIM aggregation within the ER followed by migration to ER-plasma membrane junctions located just below the plasma membrane. Here, they bind to and activate Orai1, a four transmembrane domain spanning plasma membrane protein, which forms the CRAC channel.Despite significant homology between STIM1 and STIM2, there are some important differences between them. First, they differ in their respective abilities to activate Orai1. STIM2 activates Ca 2+ ...

To avoid conflicting and deleterious outcomes, eukaryotic cells often confine second messengers to spatially restricted subcompartments. The smallest signaling unit is the Ca2+ nanodomain, which forms when Ca2+ channels open. Ca2+ nanodomains arising from store-operated Orai1 Ca2+ channels stimulate the protein phosphatase calcineurin to activate the transcription factor nuclear factor of activated T cells (NFAT). Here, we show that NFAT1 tethered directly to the scaffolding protein AKAP79 (A-kinase anchoring protein 79) is activated by local Ca2+ entry, providing a mechanism to selectively recruit a transcription factor. We identify the region on the N terminus of Orai1 that interacts with AKAP79 and demonstrate that this site is essential for physiological excitation–transcription coupling. NMR structural analysis of the AKAP binding domain reveals a compact shape with several proline-driven turns. Orai2 and Orai3, isoforms of Orai1, lack this region and therefore are less able to engage AKAP79 and activate NFAT. A shorter, naturally occurring Orai1 protein that arises from alternative translation initiation also lacks the AKAP79-interaction site and fails to activate NFAT1. Interfering with Orai1–AKAP79 interaction suppresses cytokine production, leaving other Ca2+ channel functions intact. Our results reveal the mechanistic basis for how a subtype of a widely expressed Ca2+ channel is able to activate a vital transcription pathway and identify an approach for generation of immunosuppressant drugs.