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.

Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR₂) at R³⁶↓S³⁷ and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR₂ at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR₂ at E⁵⁶↓T⁵⁷, which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR₂ coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca²⁺, activate ERK1/2, recruit β-arrestins, or induce PAR₂ endocytosis. Cat-S caused PAR₂-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR₂ or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR₂ antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR₂ in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR₂ that causes PAR₂- and TRPV4-dependent inflammation and pain. They expand the role of PAR₂ as a mediator of protease-driven inflammatory pain.

The NMR-derived structural model of the dynamic interaction between synaptotagmin-1 and the SNARE complex contributes to addressing the longstanding problem of how synaptotagmin-1 triggers neurotransmitter release. Rapid neurotransmitter release depends on the Ca2+ sensor Synaptotagmin-1 (Syt1) and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Syt1 triggers release has been unclear, partly because elucidating high-resolution structures of Syt1–SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode in which basic residues in the concave side of the Syt1 C2B-domain β-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Syt1 that markedly impair SNARE-complex binding in vitro and Syt1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Syt1 function. Our results support a model whereby dynamic interaction facilitates cooperation between Syt1 and the SNAREs in inducing membrane fusion.

Proteases that cleave protease-activated receptor-2 (PAR2) at R36/S37 reveal a tethered ligand that binds to the cleaved receptor. PAR2 activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR2 at non-canonical sites can trigger distinct signaling cascades, the functional importance of PAR2 biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR2, causes inflammation and pain by activating PAR2 and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR2 at A66/S67 and S67/V68. Elastase stimulated PAR2-dependent cAMP accumulation and ERK1/2 activation, but not Ca2+ mobilization, in KNRK cells. Elastase induced PAR2 coupling to Gαs but not Gαq in HEK293 cells. Although elastase did not promote recruitment of G protein coupled receptor kinase-2 (GRK2) or β-arrestin to PAR2, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR2-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR2-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR2- and TRPV4-mediated influx of extracellular Ca2+ in mouse nociceptors. Adenylyl cyclase and PKA mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR2- and TRPV4-mediated mechanisms. Thus, elastase biased agonism of PAR2 causes Gαs-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR2 as a mediator of protease-driven inflammation and pain.