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Ubiquitin-dependent Down-regulation of the Neurokinin-1 Receptor

Graeme S. Cottrell, Benjamin Padilla, Stella Pikios, Dirk Roosterman, Martin Steinhoff, Daphne Gehringer, Eileen F. Grady, Nigel W. Bunnett
2006 Journal of Biological Chemistry  
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
more » ... 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 R⌬5K/R, C-terminal tail lysines; and NK 1 R⌬10K/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 R⌬5K/R, and NK 1 R⌬10K/R. However, NK 1 R⌬5K/R and NK 1 R⌬10K/R resensitized 4 -8-fold faster than NK 1 Rwt by cycloheximide-independent mechanisms. NK 1 R⌬325 (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 R⌬5K/R, and NK 1 R⌬10K/R. After 4 h in SP-free medium, NK 1 R⌬5K/R and NK 1 R⌬10K/R recycled to the plasma membrane, whereas NK 1 Rwt remained internalized. SP induced ubiquitination of NK 1 Rwt and NK 1 R⌬5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for monoand polyubiquitin. NK 1 R⌬10K/R was not ubiquitinated. Whereas SP induced degradation of NK 1 Rwt, NK 1 R⌬5K/R and NK 1 R⌬10K/R showed ϳ50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK 1 R, which mediates its degradation and down-regulation. Agonist-induced trafficking of G-protein-coupled receptors (GPCRs) 2 between the plasma membrane and organelles determines cellular responsiveness. The molecular mechanisms of receptor endocytosis have been thoroughly investigated. Many activated GPCRs exemplified by the angiotensin II type 1A receptor, ␤ 2 -adrenergic receptor, neurokinin-1 receptor (NK 1 R), and protease-activated receptor-1 and -2 (PAR 2 ) are phosphorylated by G-protein receptor kinases (1-5). This phosphorylation increases the affinity of receptors for ␤-arrestins, which translocate to the receptors at the plasma membrane (3, 6 -10). ␤-Arrestins (a) sterically hinder the interaction between GPCRs and heterotrimeric G-proteins to desensitize G-protein signaling (3, 11, 12); (b) are adaptor proteins for clathrin and activator protein-2 and are thus required for endocytosis of GPCRs (3, 7, 12, 13) ; and (c) serve as molecular scaffolds for the formation of signaling modules that include components of the MAPK (mitogen-activated protein kinase) cascade such as Src, Raf-1, MEKK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase), and activated ERK1/2 (extracellular signal-regulated kinase-1 and -2) (14 -17). However, in comparison with endocytosis, much less is known about the molecular mechanisms of post-endocytic sorting of GPCRs back to the plasma membrane (recycling) or to lysosomes or proteasomes (down-regulation). The nature of interaction between GPCRs and ␤-arrestins determines the rate of receptor recycling. GPCRs can be divided into two classes according to their interaction with ␤-arrestins (18). "Class A" receptors (e.g. ␤ 2 -adrenergic, -opioid, ␣ 1b -adrenergic, and neurokinin-3 receptors) show a preference for ␤-arrestin-2 over ␤-arrestin-1 and interact with low affinity and transiently with ␤-arrestin-2 to rapidly dissociate and recycle (18 -20). "Class B" receptors (e.g. NK 1 R, angiotensin II type 1A receptor, and neurotensin receptor-1) form high affinity and sustained interactions with both isoforms of ␤-arrestin and then slowly recycle to the plasma membrane (18). The existence of Ser and Thr residues within the C-terminal domains of these receptors, which are sites for phosphorylation by G-protein receptor kinases, specifies high affinity interactions with ␤-arrestins and therefore determines the rates of
doi:10.1074/jbc.m603369200 pmid:16849335 fatcat:2ssjrwd5frccnnpav37yf7kqxa