Background/Purpose: Human serum IgA can paradoxically inhibit immune cell activation. Inhibition of such activation, mediated by the inhibitory configuration of the immunoreceptor tyrosine-based activation motif (ITAM) in the Fc receptor common γ-chain (FcRγ), suggests a potential dual role for FcαRI (CD89)-FcRγ signaling pairs. However, in monocytes and neutrophils FcαRI is often expressed in the absence of FcRγ (Launay et al,1999). We previously reported a common FcaRI Ser248Gly variant in the cytoplasmic domain of CD89 that affects receptor function in neutrophils. In the absence of FcRg, the Ser248 allele does not initiate signaling while the Gly248 allele maintains robust signaling. To elucidate the basis for this α-chain functon, we sought allele-specific FcαRI-associated molecules

Methods: To isolate and identify cellular proteins binding to the FcaRI Ser248 allele we used an anti-FcαRI affinity column followed by mass spectrometry. We used pull down assays with His6-tagged proteins to confirm direct protein-protein interactions. To test the differential function of the FcaRI Ser248Gly alleles in mononuclear cells, we used freshly isolated human monocytes from donors homozygous for each allele. To investigate the molecular mechanisms underlying differential allele effects, we used the P388D1 murine monocytic cell line stably expressing FcaRI Ser248 or Gly248 with or without the engineered R209L point mutation in the transmembrane, which abrogates FcRg chain pairing. We also used the U937 human monocyte cell line, which endogenously expresses FcaRI Ser248 to test the effects of the dominant negative truncation of SH3BP5 (SabD31) which is incapable of inhibiting Btk.

Results: FcaRI Ser248 does not stimulate production of cytokines by human monocytes while FcaRI Gly248 consistently activates cytokine production (Figure 1). FcαRI Ser248 specifically recruited Sab (SH3-domain binding protein that preferentially associates with Bruton’s tyrosine kinase, Btk), a trans-inhibitor of Btk, in a phosphorylation-dependent fashion, whereas the Gly248 variant reciprocally recruited the protein tyrosine kinase Lyn (Figure 2B,C,D). Compared to FcαRI Gly248, FcαRI Ser248 recruitment of Sab results in inhibition of Btk phosphorylation/activation (Figure 3A) and suppression of effector functions (Figure 3B) independent of FcRγ-pairing. A dominant negative Sab incapable of binding to Btk fails to inhibit Btk phosphorylation/activation (Figure 3C) and releases FcαRI-mediated inhibition by the Ser248-allele (Figure 3D,E). Ser248-phosphorylation amplifies Sab association and disrupts Lyn binding through an overlapping region containing an unconventional SH3-domain binding motif.

Conclusion: These findings reveal a novel and reversible serine-based phosphorylation-dependent Sab/Lyn switch for regulation of receptor-mediated inhibition/activation that couples FcαRI α-chain to divergent inflammatory properties of human IgA. This genetically determined switch has important implications for the role of IgA in antibody-mediated autoimmune disease and host defense as well as implications for the use of IgA as the backbone for therapeutic antibodies.

Sequence variation of FcαRI (S248G) alters IL-6 and IL-1β production in human monocytes . Peripheral blood monocytes (MNC) (n = 4) homozygous for FcαRI Ser248 or Gly248 variants were stimulated with mAb A59 F(ab)’2 or heat aggregated IgA (HA IgA). IL-6 and IL-1β release were determined by ELISA at 24 hours following stimulation. * p < 0.05; ** p < 0.01.

Identification of Sab as an FcαRI-binding protein. (A) A workflow diagram showing steps for isolating and identifying FcαRI variant-specific binding proteins. (B) FcαRI was immuno-isolated from P388D1 transfectants stably expressing human FcαRI-Ser248 or Gly248 using anti-FcαRI A59 F(ab’)2-conjugated Sepharose column. The prominent 65KDa co-eluting protein band was subjected to MALDI-Mass Spectrometry analysis for identification. (C) Sab co-precipitates with FcαRI Ser248 independent of FcRγ pairing. (D) His6-Sab specifically pulls down FcαRI-Ser248. Input (1), the unbound flow through (2) and the bound (3) fractions are shown.

The FcαRI Ser248 variant attenuates Btk activation-dependent FcαRI-mediated functions. (A) P388D1 cells expressing FcαRI Ser248 or Gly248 variants with or without the Arg209Leu transmembrane change were stimulated for 3 minutes. Btk immunoprecipitates and whole cell lysates were blotted with anti-phosphotyrosine mAb, 4G10, and anti-pY223 Btk mAb, respectively. (B) The P388D1 transfectants were incubated with A59-opsonized FITC-conjugated Ox RBCs and phagocytosis of the RBCs measured by FITC fluorescence. C,D,E) Dominant negative Sab reverses FcαRI Ser248 failure to signal. C) The U937-SabΔ31 transfectants were opsonized with A59 F(ab’)2 and stimulated with GAM F(ab’)2 cross-linking for the indicated times. Whole cell lysates of the cells were immunoblotted for phospho-Btk, total Btk and total Syk. For phospho-Syk, phosphorylated proteins were immunoprecipitated using PY20 and immunoblotted for phospho-Syk. D) SabΔ31-expressing U937 cells were stimulated with LPS and A59 F(ab’)2 for 24 hours, and secreted IL-6 was measured by ELISA. E) SabΔ31-expressing U937 cells were opsonized for 30 minutes with A59 F(ab’)2 and incubated with human IgG-opsonized FITC-conjugated Ox RBC for 1 hour. Internalized RBCs were measured as intracellular FITC fluorescence. * p < 0.05; ** p < 0.01.

« Back to ACR Convergence 2025

ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-genetically-determined-serine-based-and-phosphorylation-dependent-molecular-switch-regulates-the-inflammatory-potential-of-human-iga/