Ubiquitin-dependent down-regulation of the neurokinin-1 receptor.
Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.
Cottrell
Graeme S
GS
Departments of Surgery and Physiology, University of California, San Francisco, 94143-0660, USA.
Padilla
Benjamin
B
Pikios
Stella
S
Roosterman
Dirk
D
Steinhoff
Martin
M
Gehringer
Daphne
D
Grady
Eileen F
EF
Bunnett
Nigel W
NW
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D015536:Down-Regulation; D004705:Endocytosis; D021381:Protein Transport; D051381:Rats; D018040:Receptors, Neurokinin-1 / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013373:Substance P / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
D018040:Receptors, Neurokinin-1; D025801:Ubiquitin; D013373:Substance P; D002118:Calcium