Identification of a novel, expressed, alternatively spliced FCER1G protein that inhibits receptor function

The ITAM-containing FcRγ chain, encoded by FCER1G, non-covalently couples with the immunoglobulin-binding receptors, -- FcγRI (CD64), FcγRIIIa (CD16), FcϵRI and FcαRI (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 FcRγ, variants in the signal-generating FcRγ have not been explored. Signal modulation through structural FcRγ variants would have implications for FcRγ-associated receptors and their role in immune-mediated diseases.

We carried out in silico analyses of whole genome DNA sequence databases based on short-read sequencing of ~76, 000 genomes, long-read sequencing of 1132 genomes, whole exome sequences of ~800, 000 participants and RNA transcript databases. We used the hg38 reference genome to identify genomic DNA and cDNA structural variants and Sanger sequencing of cDNAs derived from FcRγ mRNAs isolated from local participants to confirm ESTs in circulating immune cells. To detect a putative novel alternatively spliced translated protein (AS-FcRγ), we developed isoform-specific monoclonal antibodies to confirm AS-FcRγ co-immunoprecipitation with and support of receptor α-chain expression in transfection systems and primary cells. We assessed the function of the AS-FcRγ isoform by stimulus-induced cytokine production and degranulation.

In the human genome, insertions, deletions and rearrangements of FCER1G are absent, and with the exception of very rare single nucleotide variants, the FcRγ protein is monomorphic. Surprisingly, we confirmed that an alternatively spliced (AS) form of FcRγ message encodes a translated protein (AS-FcRγ) expressed in human myeloid cells. AS-FcRγ pairs with ligand binding FcR α-chains. The alternate exon 5' in AS-FcRγ creates a novel cytoplasmic domain with a disrupted ITAM motif. Co-immunoprecipitation showed that AS-FcRγ associates with FcαRI in circulating neutrophils and mononuclear cells. A chimeric molecule consisting of the extracellular domain of FcαRI fused to the cytoplasmic domain of AS-FcRγ fails to stimulate cellular degranulation and cytokine production.

AS-FcRγ is an expressed protein that associates with partner alpha-chains but is not signaling competent. It may represent a novel contributory mechanism mitigating the ability of Fc receptors to stimulate cellular signaling in myeloid cells with important implications for host defense and antibody-mediated autoimmune diseases.