Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK₁R). The effects of sustained stimulation by high concentrations of SP on NK₁R trafficking and Ca²⁺ signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nM, 3 h) completely desensitized Ca²⁺ signaling by wild-type NK₁R (NK₁Rwt). 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 (NK₁RΔ5K/R, C-terminal tail lysines; and NK₁RΔ10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca²⁺ ions. SP desensitized NK₁Rwt, NK₁RΔ5K/R, and NK ₁RΔ10K/R. However, NK₁RΔ5K/R and NK ₁RΔ10K/R resensitized 4-8-fold faster than NK₁Rwt by cycloheximide-independent mechanisms. NK₁RΔ325 (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 NK₁Rwt, NK₁RΔ5K/R, and NK₁RΔ10K/ R. After 4 h in SP-free medium, NK₁RΔ5K/R and NK ₁RΔ10K/R recycled to the plasma membrane, whereas NK ₁Rwt remained internalized. SP induced ubiquitination of NK ₁Rwt and NK₁RΔ5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK₁RΔ10K/R was not ubiquitinated. Whereas SP induced degradation of NK₁Rwt, NK ₁RΔ5K/R and NK₁RΔ10K/R showed ∼50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK₁R, which mediates its degradation and down-regulation.