Abstract
CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor’s expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.
Highlights
The myeloid C-type lectin-like inhibitory receptor 12A (CLEC12A) regulates immune responses in various pathological contexts, including gout, rheumatoid arthritis, and viral infection [1,2,3,4,5]
We previously reported that the addition of this tag does not interfere with CLEC12A expression and that CLEC12A-HA can be cross-linked with an anti-HA antibody to induced signaling in HEK-293T cells [17]
In addition to the C-type lectin-like domain (CTLD), some C-type lectin receptors (CLRs) such as CLEC12A habour cysteine residues in their stalk domain that are postulated to be essential for receptor oligomerisation [24]
Summary
The myeloid C-type lectin-like inhibitory receptor 12A (CLEC12A) regulates immune responses in various pathological contexts, including gout, rheumatoid arthritis, and viral infection [1,2,3,4,5]. In knock-out mouse models of gout and rheumatoid arthritis, a diminution in CLEC12A expression enhances inflammation and disease severity [6,7]. Low levels of CLEC12A expression by circulating neutrophils and monocytes from early rheumatoid arthritis patients correlate with higher disease activity [8]. CLEC12A has an extracellular C-type lectin-like domain (CTLD), the defining feature of C-type lectin receptors (CLRs) [10,11,12,13]. CLEC12A CTLD is linked to the transmembrane domain by a stalk region that mediates receptor oligomerisation in other CLRs [5] The short cytoplasmic tail of CLEC12A comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM) through which it regulates intracellular signaling pathways
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