Photoinduced receptor confinement drives ligand-independent GPCR signaling

Laser-controlled receptor clustering A strategy that allows light-controlled local confinement of a heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor may help to elucidate signal transduction mechanisms. Sánchez et al. developed just such an approach using a poly-L-lysine-graft-polyethylene glycol copolymer with a photoactivatable head. Laser light could locally activate a chelator that rapidly (within seconds) captured polyhistidine-tagged neuropeptide Y receptors. Such receptor confinement enhanced calcium signaling and increased spreading and cell motility in cultured human cells. The ability to control the location, size, and density of receptor clusters may help to elucidate receptor signaling mechanisms. Science, this issue p. eabb7657 Laser control of receptor clustering enables studies of cell signaling. INTRODUCTION Lateral membrane organization and receptor cluster formation are implicated in signal transduction and regulation of cellular responses. The physiological relevance of clustering or confinement of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) is not well understood, and methods to study how the spatial distribution and location of receptor clusters affect cell responses are limited. However, the urgency of these questions is substantiated by the fact that several receptor families show cell responses that depend on the topology of the stimulus. Neuropeptide Y (NPY) hormone receptors, which belong to the class A GPCR rhodopsin family, contribute to a large variety of physiological processes, ranging from the regulation of cell migration to memory retention, fear extinction, and disorders such as epilepsy. Confinement of Y2 receptors (Y2Rs) is of high relevance because spatially restricted ligand-receptor interactions have been observed in vivo. Therefore, new approaches are needed to modulate receptor confinement in a rapid and noninvasive manner. RATIONALE To better understand how the location and dynamics of GPCR clustering influence cell responses, we developed a system to precisely control the lateral membrane organization of neuropeptide Y2Rs in living cells by light. Light triggers photoreactions within seconds and can in turn be modulated in time, space, and intensity. We used ultrasmall, high-affinity lock-and-key pairs; a light-controllable multivalent chelator on a two-dimensional matrix; and histidine-tagged Y2Rs. This system enables in situ induction of receptor clusters of variable size, location, and density upon photoactivation of the multivalent chelator, which is initially inactivated through an intramolecular histidine tag. RESULTS We generated a human cell line stably expressing small amounts of histidine-tagged neuropeptide Y2Rs at the cell surface, comparable to an in vivo setting. Cells over matrices modified with the photoactivatable chelator trivalent nitrilotriacetic acid were illuminated by in situ laser-scanning microscopy. By following the process of cluster formation in real time, we observed a fast receptor assembly within the first 60 s, which increased continuously over the first minutes. Photoactivation of circular regions covering the cell body, edge, and periphery showed an immediate effect on cell spreading and motility. Cells moved toward the photoactivated spots, which resulted in new receptor accumulations in regions that were previously distant from the cell periphery. Photoactivation restricted to the cell leading edge also modulated cell migration. Using live-cell calcium imaging, we found that light-induced receptor clustering provoked a global increase in cytosolic calcium concentration. Changes in cell motility and calcium concentration were also observed after stimulation with the canonical ligand NPY. Subsequent photoinduced receptor confinement in the same cell triggered further increases in cytosolic calcium and cell motility. By contrast, these effects could be selectively blocked by a Y2R antagonist. CONCLUSION By revealing ligand-independent receptor activation after cluster formation, our results demonstrate that Y2R clusters locally enhance constitutive receptor activity. High-affinity Y2R/G protein complexes have been observed in the absence of the ligand. In our system, the high local receptor density may increase the residence time of proximate G proteins and recruit further downstream effectors, which could boost the likelihood of activation. In this regard, our results highlight that confinement of GPCRs can bias their active state and initiate downstream signaling akin to the canonical agonist. This versatile approach, along with the nanotool used, creates new possibilities to study in situ the impact of membrane organization on cell signaling and mechanotransduction. In situ receptor confinement triggered by light. Input (light) activates multivalent chelators displayed on a generic matrix to generate fine-tunable receptor clusters at the cell membrane in a rapid, noninvasive, and reversible manner. Increased cell motility and calcium signals as output reveal ligand-independent receptor activation. Cell-cell communication relies on the assembly of receptor-ligand complexes at the plasma membrane. The spatiotemporal receptor organization has a pivotal role in evoking cellular responses. We studied the clustering of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) and established a photoinstructive matrix with ultrasmall lock-and-key interaction pairs to control lateral membrane organization of hormone neuropeptide Y2 receptors in living cells by light. Within seconds, receptor clustering was modulated in size, location, and density. After in situ confinement, changes in cellular morphology, motility, and calcium signaling revealed ligand-independent receptor activation. This approach may enhance the exploration of mechanisms in cell signaling and mechanotransduction.