Abstract 1829 Investigation into the Functions of MS4A4A and MS4A6A: Exploring Their Roles in Immune Cell Signaling

MS4A4A and MS4A6A are proteins within the MS4A family, with structural similarity to CD20 (MS4A1) and FceR1β (MS4A2). While MS4A1 and MS4A2 are recognized for their respective roles in the activation of B-cells and mast cells, MS4A4A and MS4A6A are expressed in various myeloid cells. Human genome-wide association studies have linked polymorphisms in MS4A6A and MS4A4A genes to Alzheimer's Disease (AD) risk. In the brain, MS4A4A and MS4A6A are specifically expressed by microglia, the brain's resident macrophages. However, neither the molecular functions of MS4A4A and MS4A6A proteins nor their impact on microglial functions are well understood. The objectives of our research are to define the molecular and cellular functions of MS4A4A and MS4A6A by 1) characterizing their subcellular localization patterns, 2) identifying novel protein-protein interactions, and 3) testing whether they can regulate immunoreceptor tyrosine-based activation motif (ITAM) signaling. To characterize the localization and protein interactomes of MS4A4A and MS4A6A, we have developed THP-1 cell lines that harbor gene expression cassettes for doxycycline-inducible expression of LAP-tagged MS4A4A or MS4A6A proteins. The LAP (localization and affinity purification) fusion construct encodes green fluorescent protein (GFP), TEV protease cleavage site, and the TwinStrepII peptide, fused to the N-terminus of an MS4A protein. The GFP will enable studies of MS4A protein localization in PMA-treated THP-1 (macrophage-like) cells, and the combined tags will be used for tandem affinity purification (TAP) and identification of co-purifying proteins by mass spectrometry. To investigate whether MS4A4A and MS4A6A can regulate ITAM signaling, we have used Jurkat NFAT-luciferase reporter cells to develop cell lines with doxycycline-inducible MS4A protein expression, and we have observed dose-dependent suppression of ITAM signaling. We expect our research to illuminate the mechanisms by which microglial MS4A4A and MS4A6A proteins impact AD pathogenesis and potentially identify future targets for AD therapy. Brigham Young University Department of Chemistry and Biochemistry.