Receptor-Tethered Cytosolic Modulators Enable Spatial Control of Cell Signaling Specificity.

Membrane receptors sense external stimuli and transduce signals by recruiting cytosolic effector proteins to initiate downstream signaling cascades. Despite precise and distinct functional outcomes, diverse receptors often engage common cytosolic effectors, implying that signaling specificity is regulated by the spatial dynamics of effector activity within membrane-proximal cytoplasmic microdomains. Here, we engineered receptor-tethered cytosolic modulators for manipulating the activity of effectors within localized cytoplasmic regions adjacent to target receptors. These modulators comprise three modules: an extracellular receptor-targeting unit, a cytosolic effector inhibitor, and an intervening transmembrane linker. By using the modulator to locally inhibit Src family kinases (SFKs, effectors) within the cytoplasmic microdomain of PD1, we observed robust suppression of PD1 phosphorylation and subsequent restoration of T-cell activity. Conversely, inhibition of SFK activity in the proximity to the T-cell receptor/CD3 complex remarkably impaired T-cell activation. Collectively, our work establishes a versatile and modular nanoplatform for dissecting signaling networks with biologically compatible spatial precision, providing opportunities for targeted bioregulation and therapeutic interventions.