Dynamic <i>in Situ</i> Confinement Triggers Ligand-Free Neuropeptide Receptor Signaling.

Membrane receptor clustering is fundamental to cell-cell communication; however, the physiological function of receptor clustering in cell signaling remains enigmatic. Here, we developed a dynamic platform to induce cluster formation of neuropeptide Y₂ hormone receptors (Y₂R) <i>in situ</i> by a chelator nanotool. The multivalent interaction enabled a dynamic exchange of histidine-tagged Y₂R within the clusters. Fast Y₂R enrichment in clustered areas triggered ligand-independent signaling as determined by an increase in cytosolic calcium and cell migration. Notably, the calcium and motility response to ligand-induced activation was amplified in preclustered cells, suggesting a key role of receptor clustering in sensitizing the dose response to lower ligand concentrations. Ligand-independent versus ligand-induced signaling differed in the binding of arrestin-3 as a downstream effector, which was recruited to the clusters only in the presence of the ligand. This approach allows <i>in situ</i> receptor clustering, raising the possibility to explore different receptor activation modalities.