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The N terminus of Orai1 couples to the AKAP79 signaling complex to drive NFAT1 activation by local Ca²⁺ entry.

ABSTRACT: 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.

SUBMITTER: Kar P

PROVIDER: S-EPMC8126794 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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The N terminus of Orai1 couples to the AKAP79 signaling complex to drive NFAT1 activation by local Ca2+ entry.

Kar Pulak P Lin Yu-Ping YP Bhardwaj Rajesh R Tucker Charles J CJ Bird Gary S GS Hediger Matthias A MA Monico Carina C Amin Nader N Parekh Anant B AB

Proceedings of the National Academy of Sciences of the United States of America 20210501 19

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 dire ...[more]

PMID: 33941685

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Project description:BackgroundStore-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ channels is an essential signaling pathway in many cell types. Ca2+ release-activated Ca2+ channels are formed by ORAI1, ORAI2, and ORAI3 proteins and activated by stromal interaction molecule (STIM) 1 and STIM2. Mutations in the ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and nonimmunologic symptoms.ObjectiveWe performed molecular and immunologic analysis of patients with CID, anhidrosis, and ectodermal dysplasia of unknown etiology.MethodsWe performed DNA sequencing of the ORAI1 gene, modeling of mutations on ORAI1 crystal structure, analysis of ORAI1 mRNA and protein expression, SOCE measurements, immunologic analysis of peripheral blood lymphocyte populations by using flow cytometry, and histologic and ultrastructural analysis of patient tissues.ResultsWe identified 3 novel autosomal recessive mutations in ORAI1 in unrelated kindreds with CID, autoimmunity, ectodermal dysplasia with anhidrosis, and muscular dysplasia. The patients were homozygous for p.V181SfsX8, p.L194P, and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. In addition to impaired T-cell cytokine production, ORAI1 mutations were associated with strongly reduced numbers of invariant natural killer T and regulatory T (Treg) cells and altered composition of γδ T-cell and natural killer cell subsets.ConclusionORAI1 null mutations are associated with reduced numbers of invariant natural killer T and Treg cells that likely contribute to the patients' immunodeficiency and autoimmunity. ORAI1-deficient patients have dental enamel defects and anhidrosis, representing a new form of anhidrotic ectodermal dysplasia with immunodeficiency that is distinct from previously reported patients with anhidrotic ectodermal dysplasia with immunodeficiency caused by mutations in the nuclear factor κB signaling pathway (IKBKG and NFKBIA).

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The N terminus of Orai1 couples to the AKAP79 signaling complex to drive NFAT1 activation by local Ca²⁺ entry.

Publications

The N terminus of Orai1 couples to the AKAP79 signaling complex to drive NFAT1 activation by local Ca<sup>2+</sup> entry.

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