1. 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
- Author
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P.A. Shenoy, Michelle L. Halls, Luigi Aurelio, Tim Quach, Joshua W. Conner, Quynh N. Mai, Stephen J. Hill, Thomas P. Davis, Meritxell Canals, Christopher J.H. Porter, Nigel W. Bunnett, Arisbel B. Gondin, Cameron J. Nowell, Holly R. Yeatman, Bim Graham, Nicholas A. Veldhuis, Jeffri S. Retamal, Daniel P. Poole, and Stephen J. Briddon
- Subjects
0301 basic medicine ,Male ,OEt, ethyl ester ,pmEpac2, plasma membrane localized Epac2-camps FRET biosensor ,Substance P ,Cy5-OEt, cyanine 5 with an ethyl ester linked via PEG ,Biochemistry ,DMEM, Dulbecco’s modified Eagle’s medium ,chemistry.chemical_compound ,Neurokinin-1 Receptor Antagonists ,pain ,Internalization ,AC, adenylyl cyclase ,media_common ,Calcium signaling ,Analgesics ,cytoCKAR, cytosolic C kinase activity reporter FRET biosensor ,Chemistry ,InsP3, inositol trisphosphate ,Cy5, cyanine 5 ,tachykinin ,BACE-1, β-site amyloid precursor protein cleaving enzyme 1 ,Cell biology ,Cholestanol ,cAMP, cyclic adenosine monophosphate ,FCS, fluorescence correlation spectroscopy ,medicine.symptom ,Research Article ,Cell signaling ,G-protein-coupled receptor ,Endosome ,media_common.quotation_subject ,TAMRA, tetramethylrhodamine ,Endosomes ,Cy5-Chol, cyanine 5 with cholestanol linked via PEG ,Endocytosis ,cytoEpac2, cytosolic Epac2-camps FRET biosensor ,Span, Spantide I ,03 medical and health sciences ,CFP, cyan fluorescent protein ,FBS, fetal bovine serum ,PKC, protein kinase C ,medicine ,Animals ,Humans ,Pain Management ,cell signaling ,Span-Chol, Spantide I conjugated to cholestanol via PEG linker ,BRET, bioluminescence resonance energy transfer ,Molecular Biology ,endosome ,lipid conjugation ,GPCR, G protein-coupled receptor ,SP, substance P ,030102 biochemistry & molecular biology ,Beta-Arrestins ,Chol, biotin conjugated to cholestanol via a PEG linker ,Cell Membrane ,ERK, extracellular signal regulated kinase (mitogen activated protein kinase) ,Inositol trisphosphate ,Cell Biology ,YFP, yellow fluorescent protein ,EGFR, epidermal growth factor receptor ,Mice, Inbred C57BL ,RLuc8, Renilla luciferase ,030104 developmental biology ,HEK293 Cells ,Mechanism of action ,NK1R, neurokinin 1 receptor ,drug delivery ,PKA, protein kinase A ,DAG, diacylglycerol - Abstract
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
- Published
- 2020