Download PROTEASE SWITCHES: PATHWAYS TO INFLAMMATION AND PAIN

PROTEASE SWITCHES: PATHWAYS TO INFLAMMATION AND PAIN
Nigel Bunnett, University of California, San Francisco
Signal transduction must be tightly regulated to prevent uncontrolled stimulation and disease. By
cleaving agonists and receptors at the cell-surface and in endosomes, proteases play a major role in
regulation of signaling. My laboratory investigates how proteases regulate signaling of receptors present in
the nervous system that mediate neurogenic inflammation and pain.
Cell-surface metalloendopeptidases degrade neuropeptides in the extracellular fluid and terminate
signaling. Neprilysin (NEP) degrades substance P (SP) and calcitonin gene-related peptide (CGRP) and
limits their pro-inflammatory and pro-nociceptive actions. Deletion of NEP exacerbates neurogenic
inflammation in multiple tissues, due to diminished degradation of SP . Conversely, administration of
recombinant NEP ameliorates inflammation. Ongoing work in our laboratory explores the use of engineered
proteases as anti-inflammatory agents.
Injury activates extracellular protease cascades that amplify inflammation and cause pain. By using
activity-based probes, proteomics and whole animal imaging, these proteases can be cataloged and localized.
Serine proteases from the circulation, immune cells and epithelial tissues regulate cells by cleaving cellsurface protease-activated receptors (PARs). PAR2, a receptor for tryptic proteases, is expressed by primary
spinal and vagal afferent neurons. PAR2 stimulates release of SP and CGRP, causing neurogenic
inflammation. PAR2 also activates second messenger kinases, including protein kinases C, D1-3 and A,
which phosphorylate and thereby sensitize transient receptor potential (TRP) ion channels, including
TRPV1, TRPV4 and TRPA1. This sensitization results in hyperalgesia to mechanical and thermal stimuli,
major sequelae of inflammatory diseases. Ongoing work in our laboratory is defining the role of proteases
and PARs in inflammatory disease and pain using antagonists, blocking antibodies and gene deletion
approaches.
Activated neuropeptide receptors and PARs interact with -arrestins at the plasma membrane, which
uncouple receptors from heterotrimeric G-proteins to mediate desensitization, and couple receptors to
clathrin and AP2 to mediate endocytosis. -arrestins also recruit MAP kinases to receptors in endosomes,
allowing internalized receptors to continue to signal by G-protein independent mechanisms. Compared to
our understanding of mechanisms that regulate signaling and trafficking of receptors at the plasma
membrane, almost nothing is known about the mechanisms that control receptor signaling and trafficking in
endosomes. The membrane metalloendopeptidase endothelin-converting enzyme-1 (ECE-1) co-internalizes
with receptors for SP (NK1R) and CGRP (CLR/RAMP1) to early endosomes. ECE-1 degrades SP and
CGRP in acidified early endosomes, causing disassembly of the peptide/receptor/-arrestin/Src MAP kinase
signalosome. This novel mechanism allows receptors, freed of -arrestins, to recycle and resensitize, and
terminates -arrestin-mediated activation of ERK1/2 and p38. Disruption of this mechanism has major
functional consequences in the nervous system. ECE-1 inhibition/knockdown causes sustained ERK1/2
activation in spinal nociceptive neurons, which induces hyperalgesia, and activates the Nur77 death receptor
in enteric neurons, resulting in neurodegeneration. Thus, both central and peripheral neuronal signaling are
altered.
Proteases activate PARs by an irreversible mechanism. Thus, PARs cannot be reactivated and
internalize and traffic to degradatory, lysosomal pathways rather than recycle. Sorting of PAR2 from early
endosomes to lysosomes requires ubiquitination by the E3 ubiquitin ligase c-Cbl. However, ubiquitin is
removed by endosomal deubiquitinating proteases or DUBs prior to lysosomal sorting. Two DUBs, AMSH
and UBPY, deubiquitinate PAR2. Expression of dominant negative AMSH and UBPY mutants or siRNA
knockdown prevents PAR2 deubiquitination and causes retention of PAR2 in endosomes, thereby preventing
lysosomal sorting and degradation. Ongoing work will define the role of prolonged endosomal PAR2.
These findings illustrate the importance of proteases in regulating the signaling and trafficking of
diverse receptors both at the plasma membrane and in endosomes. By cleaving peptide agonists or receptors,
proteases can both initiate and terminate signal transduction by mediators of inflammation and pain. We
have shown that proteases can function as exquisitely sensitive “molecular switches”. Thus, proteases are
critical regulators that enable precise initiation or termination of signaling events that alter responses to
inflammatory and nociceptive stimuli.