Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief

Abstract

Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)–coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK1R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK1R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane–targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and β-arrestin blocked SP-induced NK1R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal–regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK1R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK1R antagonists. These results reveal a critical role for endosomal signaling of the NK1R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.

Intracellular GPCRs Play Key Roles in Synaptic Plasticity

“…For example, to prevent NK1 receptor endosomal signaling, Jensen et al . (59) synthesized tripartite compounds composed of cholestanol to promote membrane insertion, a polyethylene linker for flexibility and a membrane impermeable NK1 receptor antagonist. This strategy successfully blocked further NK1 receptor endosomal signaling and promoted the desired behavioral response, in this case antinociception (59).…”

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

“…Another is to deliberately exploit the potential of conformationally selective nanobodies to block GPCR signaling reactions, specifically localizing them to a defined target membrane using chemical recruitment tools and assessing effect on the cellular response . A third approach, which may also have therapeutic potential with further development, is to generate antagonist ligands that accumulate in endosomes to inhibit activation in this compartment selectively …”

“…Ligand trapping in the endosome lumen is thought to prolong or enhance GPCR responses by favoring ligand rebinding after dissociation from the receptor, and G protein activation at endosomes may differ inherently from that at the plasma membrane because of location‐specific differences in activation or regulatory machineries engaged (see below). GPCR‐G protein activation in endosomes (or the trans‐Golgi network after endocytic delivery) can also preferentially promote a subset of downstream effects such as GPCR‐dependent transcriptional responses . Studies of GPCR‐dependent transcriptional induction identified an additional function of endosomal G protein activation by GPCRs in enhancing cellular discrimination between similar ligands and increasing reliability of the cellular response …”

“…How can location matter for signaling via diffusible second messengers? As noted above, endosomal activation of G proteins has been reported to confer selectivity on GPCR‐mediated transcriptional control . It is not clear how this is possible, particularly for cAMP‐dependent transcriptional control, because this second messenger chemical is thought to diffuse very rapidly in the cytoplasm.…”

Role of mast cells and basophils in pruritus

“…Mast cells themselves express the PAR family members PAR 1 and PAR 2 not only on the plasma membrane but also on the membrane of intracellular tryptase‐positive granules, indicating a potential autocrine regulation of tryptase‐mediated cutaneous inflammation and itch via PAR 2 activation . This observation may be consistent with the increasing appreciation of endosomal signaling by GPCRs …”