A Genetically Encoded Biosensor Reveals Location Bias of Opioid Drug Action

Abstract

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.

Signalling in response to sub‐picomolar concentrations of active compounds: Pushing the boundaries of GPCR sensitivity

“…Recent advances in the understanding of compartmentalised GPCR signalling have illustrated the importance of receptor location (within different plasma membrane domains or within different subcellular compartments) for the control of distinct signalling outcomes (Halls et al, ; Jensen et al, ; Jimenez‐Vargas et al, ; Stoeber et al, ; Tsvetanova & von Zastrow, ; Yarwood et al, ). Cellular context also appears to be very important in order for GPCRs to respond to ultra‐low concentrations of ligand (Figure ).…”

When trafficking and signaling mix: How subcellular location shapes G protein‐coupled receptor activation of heterotrimeric G proteins

“…Indeed, early efforts to assess the potential of adrenergic receptors to initiate signaling from endosomes detected receptors and adenylyl cyclase activity in the same fraction but failed to detect functional coupling between them . Over the last decade, however, the hypothesis that GPCRs can initiate G protein‐coupled signaling from endosomes as well as the plasma membrane has gained considerable experimental support …”

“…Broadly considered, four experimental approaches have produced evidence supporting endosomal GPCR‐G protein signaling. In the first, agonist application followed by washout showed a persistent component of the cellular response after agonist removal from the extracellular medium . In a second approach, the ability of membrane‐permeant relative to membrane‐impermeant antagonists to reverse the GPCR signaling was assessed; incomplete reversal by the membrane‐impermeant antagonist was found .…”

“…In the first, agonist application followed by washout showed a persistent component of the cellular response after agonist removal from the extracellular medium . In a second approach, the ability of membrane‐permeant relative to membrane‐impermeant antagonists to reverse the GPCR signaling was assessed; incomplete reversal by the membrane‐impermeant antagonist was found . In a third approach, GPCR endocytosis was inhibited using genetic or chemical manipulations; endocytic blockade was found to reduce the strength and/or duration of downstream cellular responses .…”

“…In a third approach, GPCR endocytosis was inhibited using genetic or chemical manipulations; endocytic blockade was found to reduce the strength and/or duration of downstream cellular responses . In a fourth approach, biosensors derived from single‐domain antibodies (nanobodies) were used to localize active‐conformation GPCRs as well as detect conformational activation of G protein; these studies reported a second phase of GPCR and G protein activation in endosomes, with a brief (seconds to about a minute) refractory period separating the arrival of receptors in endosomes from the second activation phase …”