A spatiotemporal map of co-receptor signaling networks underlying B cell activation.

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track co-receptor signaling dynamics in Raji cells from 10 s to 2 h after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the signaling subunit of the co-receptor complex. We detail the recruitment kinetics of signaling effectors to CD19 and identify previously uncharacterized mediators of B cell activation. We show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of BCR signaling and a rich resource for uncovering the complex signaling networks that regulate activation.

Tetraspanins: structure, dynamics, and principles of partner-protein recognition.

Tetraspanins are a large, highly conserved family of four-pass transmembrane (TM) proteins that play critical roles in a variety of essential cellular functions, including cell migration, protein trafficking, maintenance of membrane integrity, and regulation of signal transduction. Tetraspanins carry out these biological functions primarily by interacting with partner proteins. Here, we summarize significant advances that have revealed fundamental principles underpinning structure-function relationships in tetraspanins. We first review the structural features of tetraspanin ectodomains and full-length apoproteins, and then discuss how recent structural studies of tetraspanin complexes have revealed plasticity in partner-protein recognition that enables tetraspanins to bind to remarkably different protein families, viral proteins, and antibody fragments. Finally, we discuss major questions and challenges that remain in studying tetraspanin complexes.

A Spatiotemporal Map of Co-Receptor Signaling Networks Underlying B Cell Activation.

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.

Cryo-EM structure of the B cell co-receptor CD19 bound to the tetraspanin CD81.

Signaling through the CD19-CD81 co-receptor complex, in combination with the B cell receptor, is a critical determinant of B cell development and activation. It is unknown how CD81 engages CD19 to enable co-receptor function. Here, we report a 3.8-angstrom structure of the CD19-CD81 complex bound to a therapeutic antigen-binding fragment, determined by cryo-electron microscopy (cryo-EM). The structure includes both the extracellular domains and the transmembrane helices of the complex, revealing a contact interface between the ectodomains that drives complex formation. Upon binding to CD19, CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude a cholesterol binding pocket present in the apoprotein. Our data reveal the structural basis for CD19-CD81 complex assembly, providing a foundation for rational design of therapies for B cell dysfunction.

A dynamic interaction between CD19 and the tetraspanin CD81 controls B cell co-receptor trafficking.

CD81 and its binding partner CD19 are core subunits of the B cell co-receptor complex. While CD19 belongs to the extensively studied Ig superfamily, CD81 belongs to a poorly understood family of four-pass transmembrane proteins called tetraspanins. Tetraspanins play important physiological roles by controlling protein trafficking and other processes. Here, we show that CD81 relies on its ectodomain to traffic 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 bound to CD19. Mutations of CD81 in this interface suppress its CD19 export activity. These data indicate that the CD81 - CD19 interaction is dynamically regulated upon B cell activation and this dynamism can be exploited to regulate B cell function. These results are not only valuable for understanding B cell biology, but also have important implications for understanding tetraspanin function generally.