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J. Biol. Chem., Vol. 281, Issue 38, 27773-27783, September 22, 2006

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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, and Nigel W. Bunnett¹

From the Departments of Surgery and Physiology, University of California, San Francisco, California 94143-0660 and the Department of Dermatology, Interdisciplinary Center for Clinical Research, and the Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, University of Münster, Von-Esmarch-Strasse 58, 48149 Münster, Germany

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 (NK₁R5K/R, C-terminal tail lysines; and NK₁R10K/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, NK₁R5K/R, and NK₁R10K/R. However, NK₁R5K/R and NK₁R10K/R resensitized 4–8-fold faster than NK₁Rwt by cycloheximide-independent mechanisms. NK₁R325 (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, NK₁R5K/R, and NK₁R10K/R. After 4 h in SP-free medium, NK₁R5K/R and NK₁R10K/R recycled to the plasma membrane, whereas NK₁Rwt remained internalized. SP induced ubiquitination of NK₁Rwt and NK₁R5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK₁R10K/R was not ubiquitinated. Whereas SP induced degradation of NK₁Rwt, NK₁R5K/R and NK₁R10K/R showed 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK₁R, which mediates its degradation and down-regulation.

Received for publication, April 7, 2006 , and in revised form, June 21, 2006.

^* This work was supported by National Institutes of Health Grants DK39957, DK43207, and DK57840 (to N. W. B.) and Grant DK52388 (to E. F. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: University of California, 521 Parnassus Ave., San Francisco, CA 94143-0660. Tel.: 415-476-0489; Fax: 415-476-0936; E-mail: nigel.bunnett{at}ucsf.edu.

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