Dynamic in Situ Confinement Triggers Ligand-Free Neuropeptide Receptor Signaling

Abstract

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) in situ 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 in situ receptor clustering, raising the possibility to explore different receptor activation modalities.

Ligand-Free Signaling of G-Protein-Coupled Receptors: Physiology, Pharmacology, and Genetics

“…Receptor states are stabilized by allosteric interactions with other membrane components such as cholesterol and proteins, generating receptor ensembles poised for ligand activation or already basally active in a ligand-free form. Conditions leading to ligand-free signaling include high receptor density [30] either by high expression or confinement in receptor clusters within cellular membrane compartments such as caveoli, shown with the neuropeptide Y2 receptor (Y 2 R) [31]. Also, GPCRs tend to dimerize or oligomerize with each other (homo-oligomers) or with other GPCR members (hetero-oligomers), forming higher order arrays that can account for biphasic and bell-shaped dose-response curves [3,4,25,[30][31][32].…”

“…Conditions leading to ligand-free signaling include high receptor density [30] either by high expression or confinement in receptor clusters within cellular membrane compartments such as caveoli, shown with the neuropeptide Y2 receptor (Y 2 R) [31]. Also, GPCRs tend to dimerize or oligomerize with each other (homo-oligomers) or with other GPCR members (hetero-oligomers), forming higher order arrays that can account for biphasic and bell-shaped dose-response curves [3,4,25,[30][31][32]. Activation of one receptor, either in ligand-free form or agonist stimulated, can allosterically interact with its neighbors, leading to a range of ligand-free signaling modes [3,4,30,[33][34][35] Demonstrated for metabotropic glutamate receptors (mGluRs), a variable region within transmembrane helix 4 (TM4) contributes to homo-and heterodimerization and modulates orthosteric, allosteric, and ligand-free activation [36].…”

“…Whether intracellular GPCRs display a substantial degree of ligand-free signaling has yet to be studied broadly. Upon agonist stimulation, GPCRs tend to aggregate in cellular membrane compartments, assuming a punctate pattern when stained with fluorescent antibodies; such aggregation alone can activate ligand-free signaling by R** [31]. It is hence conceivable that repeated agonist stimulation results in sustained, enhanced ligand-free signaling in various cellular compartments (R** in Figure 1).…”