Project Summary/Abstract The goal of this project is to understand how CD53 regulates B cell trafficking, and to determine the CD53- partner network underlying this process. CD53 is a member of the tetraspanin family of transmembrane proteins that organize multi-protein networks on the cell surface to regulate a wide variety of cellular processes such as proliferation, homing and survival. Loss of CD53 is associated with impaired immune system function. CD53 is highly expressed on both normal and malignant B cells, however its role in these cells is not clear. We previously reported that CD53 is required for normal B cell development in the bone marrow. In preliminary data, we now find that CD53 is essential to both normal and malignant B cell trafficking, with the loss of CD53 causing significant impairment in B cell adhesion, migration, bone marrow homing and antibody production. To understand the underlying molecular mechanisms, we have determined the crystal structure of CD53; in the entire tetraspanin family, this the first structure captured in an active conformation. We reveal how conformational changes influence CD53 partner interactions by mass spectrometry (MS) based footprinting. We have used proximity labeling to identify several candidate CD53 partners, whose functions link chemokine signaling to cell motility. These data support our hypothesis that CD53 coordinates a complex of adhesion, signaling and cell motility proteins that facilitate B cell trafficking. Both normal and malignant B cells rely on accurate trafficking to their niches in the bone marrow and secondary lymphoid organs to optimize their maturation and function. Thus, an understanding of the interactions that guide B cell trafficking are important not only for optimizing normal B cell function, but will also reveal potential therapeutic targets of malignant B cells. Using a combination of in vitro and vivo adhesion and migration studies in combination with proximity labeling, quantitative MS, live-cell MS- based footprinting, biochemical and electron microscopic analyses, this dual-PI proposal presents a 5-year plan to: 1) elucidate the CD53-partner interaction network regulating B cell trafficking, and 2) determine the functional consequences of disrupting the CD53-partner interactions. Armed with our newly developed tools, we will reveal a novel network of protein interactions coordinating cell signaling and motility to regulate B cell adhesion, migration, chemokine signaling and niche localization. We will uncover how this CD53-mediated network responds to chemokine signaling during B cell migration and how cross-cell interactions are established during B cell homing. Thus, the proposed studies will significantly advance our understanding of how B cell trafficking is coordinated and regulated. Given the conserved structure and functional redundancy of tetraspanin family members, this will lead to our long-term goal of elucidating tetraspanin/partner relationships t...