Ligand-induced interactions between butyrophilin 2A1 and 3A1 internal domains in the HMBPP receptor complex.

The ligand-bound (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) receptor (BTN3A1 and BTN2A1) is detectable by the T cell receptor (TCR) of Vγ9Vδ2 T cells. Although BTN3A1 binds to phosphoantigens (pAgs), the mechanisms resulting in receptor activation are not clear. We used CRISPR-Cas9, ELISA, nano-bioluminescence resonance energy transfer (BRET), and isothermal titration calorimetry (ITC) to evaluate the role of BTN2A1. Depletion of BTN2A1 and rescue experiments demonstrate that its internal domain is essential for pAg detection. Internal hetero-BRET signals are observed between BTN2A1 and BTN3A1 that are increased by pAg. ITC detects a direct interaction between the intracellular domains of BTN3A1 and BTN2A1 only in the presence of pAg. This interaction is abrogated by removal of the BTN2A1 juxtamembrane (JM) region but not by removal of the BTN3A1 JM region. Regional mutations between BTN2A1 316-326 clearly affect the interferon γ (IFNγ) response and hetero-BRET signal. Mutations to amino acids L318, W320, and L325 indicate that these amino acids are crucial. This study demonstrates a pAg-inducible interaction between BTN2A1 and BTN3A1 internal domains.