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The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. This process underlies nearly every aspect of proper B cell function. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track B cell co-receptor signaling dynamics from 10 seconds to 2 hours after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 quantified phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the key signaling subunit of the co-receptor complex. We detail the recruitment kinetics of essential signaling effectors to CD19 following activation, and then identify new mediators of B cell activation. In particular, we show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming immediately downstream of BCR stimulation and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of the BCR signaling pathway and a rich resource for uncovering the complex signaling networks that regulate B cell activation.

SUMMARYCD81 and its binding partner CD19 are core subunits of the B cell co-receptor complex. While CD19 is a single-pass transmembrane protein belonging to the extensively studied Ig superfamily, CD81 belongs to a conserved but poorly understood family of four-pass transmembrane proteins called tetraspanins. These functionally diverse proteins play important roles in a wide variety of different organ systems by controlling protein trafficking and other cellular processes. Here, we show that CD81 relies on its ectodomain to control trafficking of CD19 to the cell surface. Moreover, the anti-CD81 antibody 5A6, which binds selectively to activated B cells, recognizes a conformational epitope on CD81 that is masked when CD81 is in complex with CD19. Mutations of CD81 in this contact interface suppress its CD19 surface-export activity. Taken together, these data indicate that the CD81 - CD19 interaction is dynamically regulated upon B cell activation, suggesting that this dynamism can be exploited to regulate B cell function. These results are not only important for understanding B cell biology, but also have important implications for understanding tetraspanin function more generally.