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

doi:10.1074/jbc.m603369200

Abstract

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.

Highlights

Agonist-induced trafficking of G-protein-coupled receptors (GPCRs)2 between the plasma membrane and organelles determines cellular responsiveness
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 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 cascade such as Src, Raf-1, MEKK, and activated ERK1/2 (14 –17)
NK1Rwt, neurokinin-1 receptor (NK1R)⌬5K/R, NK1R⌬10K/R, and NK1R⌬325 Are Expressed at the Plasma Membrane and Internalize after Binding substance P (SP)—The ubiquitination of intracellular lysine residues is required for the agonist-induced trafficking of certain GPCRs (29 –33, 39)

Summary

Introduction

Agonist-induced trafficking of G-protein-coupled receptors (GPCRs) between the plasma membrane and organelles determines cellular responsiveness. “Class B” receptors (e.g. NK1R, angiotensin II type 1A receptor, and neurotensin receptor-1) form high affinity and sustained interactions with both isoforms of ␤-arrestin and slowly recycle to the plasma membrane [18]. After stimulation with low concentrations (Ͻ1 nM) of SP, the NK1R is minimally phosphorylated, internalizes into endosomes immediately beneath the plasma membrane, and rapidly dissociates from ␤-arrestins to recycle by Rab4a- and Rab11a-dependent mechanisms [23,24,25]. Agonists induce ubiquitination of the ␤2-adrenergic receptor [31], chemokine (CXC motif) receptor-4 (CXCR4) [32] ␦ and ␮ opioid receptors [49], and PAR2 [33] This ubiquitination is necessary for postendocytic sorting and degradation rather than for endocytosis. To establish the role of intracellular lysine residues and ubiquitin, we engineered mutant receptors lacking either lysine residues in the C-terminal tail or on the entire intracellular face of the receptor and generated a chimeric molecule of the NK1R conjugated to ubiquitin

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Conclusion