A conserved basic motif mediates membrane anchoring and TRAF2 recruitment across costimulatory TNFRs.

Costimulatory receptors of the TNFR superfamily are key components in synthetic immunoreceptors, yet how their intracellular domains coordinate membrane anchoring with adaptor recruitment remains unresolved. Here, using solution-state NMR and membrane-reconstituted FRET assays, we identify a conserved basic-residue-rich sequence (BRS) in the juxtamembrane region of OX40 that binds anionic phospholipids and adopts a membrane-inserted conformation. This membrane engagement enables a dual-site interaction with TRAF2, combining canonical PxQxx binding with a secondary membrane-proximal interface, to promote robust NF-κB activation. Disruption of the BRS impairs adaptor docking without reducing receptor surface expression or ligand-induced clustering. This regulatory architecture is further supported in 4-1BB and CD40, indicating broader relevance across TNFR costimulatory receptors. Our findings refine current models of TNFR signaling and offer a generalizable framework for receptor design. Incorporating membrane-anchoring motifs into synthetic constructs may enhance spatial control of adaptor recruitment, thereby improving signal fidelity, sensitivity, and performance in CAR-T applications.