Background/Purpose: The Tyrosine Activation Motif (ITAM)-containing FcRg chain, encoded by FCER1G, non-covalently couples with the immunoglobulin binding receptors, — FcγRI (CD64), FcγRIIIa (CD16), and FcaRI (CD89), — to initiate intracellular signals and mediate phagocytosis, cytokine production and other functions. While functionally impactful variants have been identified in the ligand-binding alpha-chains of receptors associated with FcRg, variants in the signal-generating FcRg have not been explored. Signal modulation through structural variants at the level of FcRγ would have implications for all FcRγ-associated receptors and their role in the pathogenesis of immune-mediated diseases.

Methods: We carried out in silico analyses of whole genome DNA sequence databases based on short read sequencing (eg, gnomAD with ~79,000 genomes) and long-read sequencing (eg, >600 genomes from All of Us, the Arab and Chinese Pangenomes, and local long-read sequenced cohorts). We also assessed whole exome sequences (eg, gnomAD, UK Biobank and Regeneron totally >1M participants) as well as RNA transcript databases. We used Sanger sequencing of cDNAs derived from FcRg mRNAs isolated from local participants to confirm ESTs (eg, BG548748) in circulating immune cells. We used hg38 reference genome to identify genomic DNA and cDNA structural variants at the single nucleotide and larger variant levels.

Results: In the human genome, insertions, deletions and rearrangements of FCER1G are absent, and with the exception of very rare single nucleotide variants (allele frequency < 0.01), the FcRγ protein is monomorphic. Surprisingly, we confirmed that in addition to the canonical FcRγ transcript encoding the classically recognized FcRγ protein, an alternatively spliced (AS) form of FcRg message encodes a translated protein (AS-FcRg) which is expressed in human myeloid cells. AS-FcRγ is able to pair with ligand binding α-chains. The incorporation of the alternate exon 5’ in AS-FcRγ creates a novel cytoplasmic domain with a disrupted ITAM signaling motif (Figure 1). The AS-FcRg mRNA is expressed in cell lines (U937, THP-1, HL-60) and in circulating neutrophils, monocytes and platelets, but not in lymphocytes, from normal donors. Co-immunoprecipitation experiments show that AS-FcRg associates with FcaRI in circulating neutrophils and mononuclear cells (Figure 2). An engineered chimeric molecule consisting of the extracellular domain of FcaRI fused to the cytoplasmic domain of AS-FcRg fails to stimulate cellular degranulation and cytokine production (Figure 3)

Conclusion: The AS-FcRg variant is not signaling competent and may represent a novel inhibitory mechanism limiting the ability of Fc receptors to stimulate cellular signaling in myeloid cells with important implications for host defense and antibody-mediated autoimmune diseases, including SLE and RA.

Figure 1. Sequence of FcRγ isoforms. The partial amino acid sequences of WT-FcRγ and AS-FcRγ are shown. Exon 5’ in AS-FcRγ replaces exon 5 and encodes a novel cytoplasmic domain with no traditional signaling motifs. The ITAM signaling motif [ YxxL(x7)YxxL ] present in the WT-FcRγ is highlighted.

Figure 2. Western blot analysis of FcαR1 immunoprecipitates showing that AS-FcRγ protein pairs with and co-precipitates with FcαR1 in monocytes (MNC) and neutrophils (PMN).

Figure 3. Stimulation of AS-FcRγ chimeric construct. A) Degranulation of β-Hexosaminidase activity. RBL-2H3 cells stably expressing vector alone or a chimeric receptor consisting of the extracellular domain of FcαRI fused to the cytoplasmic domain of WT-FcRγ or AS-FcRγ were stimulated with anti-FcαRI antibody A59 F(ab’)2 for the times indicated. Cell degranulation was measured as a percent of total β-Hexosaminidase released by triton X-100 treated cells. B) IL-4 cytokine production. RBL-2H3 cells stably expressing the chimeric receptors were stimulated as outlined above for the indicated times. Cell culture media was then assayed by ELISA for the presence of IL-4 cytokine.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-a-novel-expressed-alternatively-spliced-fcer1g-protein-that-inhibits-receptor-function/