0041: A Genetically Determined, Serine-based and Phosphorylation-dependent Molecular Switch Regulates the Inflammatory Potential of Human Iga

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.