Journal of Biological Chemistry
Volume 296, January–June 2021, 100345
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Research Article
A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes

https://doi.org/10.1016/j.jbc.2021.100345Get rights and content
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G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of β-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease

Keywords

drug delivery
endosome
G-protein-coupled receptor
cell signaling
pain
lipid conjugation
tachykinin

Abbreviations

AC
adenylyl cyclase
BACE-1
β-site amyloid precursor protein cleaving enzyme 1
BRET
bioluminescence resonance energy transfer
cAMP
cyclic adenosine monophosphate
Chol
biotin conjugated to cholestanol via a PEG linker
CFP
cyan fluorescent protein
Cy5
cyanine 5
Cy5-Chol
cyanine 5 with cholestanol linked via PEG
Cy5-OEt
cyanine 5 with an ethyl ester linked via PEG
cytoCKAR
cytosolic C kinase activity reporter FRET biosensor
cytoEpac2
cytosolic Epac2-camps FRET biosensor
DAG
diacylglycerol
DMEM
Dulbecco’s modified Eagle’s medium
EGFR
epidermal growth factor receptor
ERK
extracellular signal regulated kinase (mitogen activated protein kinase)
FBS
fetal bovine serum
FCS
fluorescence correlation spectroscopy
GPCR
G protein-coupled receptor
InsP3
inositol trisphosphate
NK1R
neurokinin 1 receptor
OEt
ethyl ester
PKA
protein kinase A
PKC
protein kinase C
pmEpac2
plasma membrane localized Epac2-camps FRET biosensor
RLuc8
Renilla luciferase
SP
substance P
Span
Spantide I
Span-Chol
Spantide I conjugated to cholestanol via PEG linker
TAMRA
tetramethylrhodamine
YFP
yellow fluorescent protein

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Present address for Quynh N. Mai: Cardiovascular Research Institute, Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158.

This article contains supporting information.