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Cottrell GS, Padilla B, Pikios S, Roosterman D, Steinhoff M, Gehringer D, Grady EF, Bunnett NW: Ubiquitin-dependent down-regulation of the neurokinin-1 receptor. . J Biol Chem. 2006 Sep 22;281(38):27773-83. Epub 2006 Jul 17.
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. |
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