SummaryRAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs.

Graphical Abstract Highlights d BTNL3 binds directly and specifically to Vg4 + TCRs via its IgV domain d The superantigen-like binding mode focuses on germlineencoded TCR regions d In contrast, gd TCR binding to a clonally restricted antigen is CDR3-mediated d Mutagenesis indicates parallels with BTN3A1-mediated activation of Vg9Vd2 T cells SUMMARY Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse gd T cell subsets, but considerable contention surrounds whether they represent direct gd T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vg4 + TCR, ''LES'' with an affinity ($15-25 mM) comparable to many ab TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vg4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3g and CDR3d loops mediated LES TCR bindingto endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the gd TCR can employ two discrete binding modalities: a non-clonotypic, superantigenlike interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive gd T cell biology. How these findings might broadly apply to gd T cell regulation is also examined.

The RET receptor tyrosine kinase is essential to vertebrate development and implicated in multiple human diseases. RET binds a cell surface bipartite ligand comprising a GDNF family ligand and a GFRα coreceptor, resulting in RET transmembrane signaling. We present a hybrid structural model, derived from electron microscopy (EM) and low-angle X-ray scattering (SAXS) data, of the RET extracellular domain (RET(ECD)), GDNF, and GFRα1 ternary complex, defining the basis for ligand recognition. RET(ECD) envelopes the dimeric ligand complex through a composite binding site comprising four discrete contact sites. The GFRα1-mediated contacts are crucial, particularly close to the invariant RET calcium-binding site, whereas few direct contacts are made by GDNF, explaining how distinct ligand/coreceptor pairs are accommodated. The RET(ECD) cysteine-rich domain (CRD) contacts both ligand components and makes homotypic membrane-proximal interactions occluding three different antibody epitopes. Coupling of these CRD-mediated interactions suggests models for ligand-induced RET activation and ligand-independent oncogenic deregulation.