Substance P-induced Trafficking of β-Arrestins

doi:10.1074/jbc.274.23.16257

Substance P-induced Trafficking of β-Arrestins

THE ROLE OF β-ARRESTINS IN ENDOCYTOSIS OF THE NEUROKININ-1 RECEPTOR*

From the Departments of ^‡Surgery and ^‖Physiology, University of California, San Francisco, California 94143-0660 and the ^¶CURE Digestive Diseases Research Center, West Los Angeles Veterans Affairs Medical Center, and Department of Medicine, UCLA Medical School, Los Angeles, California 90073-1792

Abstract

Agonist-induced redistribution of G-protein-coupled receptors (GPCRs) and β-arrestins determines the subsequent cellular responsiveness to agonists and is important for signal transduction. We examined substance P (SP)-induced trafficking of β-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in real time using green fluorescent protein. Green fluorescent protein did not alter function or localization of the NK1R or β-arrestin1. SP induced (a) striking and rapid (<1 min) translocation of β-arrestin1 from the cytosol to the plasma membrane, which preceded NK1R endocytosis; (b) redistribution of the NK1R and β-arrestin1 into the same endosomes containing SP and the transferrin receptor (2–10 min); (c) prolonged colocalization of the NK1R and β-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma membrane and β-arrestin1 in the cytosol (4–6 h). SP stimulated a similar redistribution of immunoreactive β-arrestin1 and β-arrestin2. In contrast, SP did not affect Gα_q/11distribution, which remained at the plasma membrane. Expression of the dominant negative β-arrestin^319–418 inhibited SP-induced endocytosis of the NK1R. Thus, SP induces rapid translocation of β-arrestins to the plasma membrane, where they participate in NK1R endocytosis. β-Arrestins colocalize with the NK1R in endosomes until the NK1R recycles and β-arrestins return to the cytosol.

Footnotes

↵* This work was supported by National Institutes of Health Grants DK39957, DK43207, NS21710 (to N. W. B.), DK52388 (to E. F. G.), and DK35740 (to J. H. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Both authors contributed equally to this paper.
↵** To whom correspondence should be addressed: University of California San Francisco, 521 Parnassus Ave., San Francisco, CA 94143-0660. Tel.: 415-476-0489; Fax: 415-476-0936; E-mail:nigelb{at}itsa.ucsf.edu.
Abbreviations:

GPCR

G-protein coupled receptor

ARR

arrestin

β₂AR

β₂-adrenergic receptor

Cy3-SP

Cyanine 3.18-labeled substance P

GFP

green fluorescent protein

EGFP

enhanced green fluorescent protein

GRK

G-coupled protein receptor kinase

NK1R

neurokinin 1 receptor

SP

substance P

KNRK cells

Kirsten murine sarcoma virus transformed rat kidney epithelial cells

DMEM

Dulbecco’s modified Eagle’s Medium

BSA

bovine serum albumin

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

LAMP-1

lysosomal-associated membrane protein-1

GST

glutathioneS-transferase
- Received November 6, 1998.
- Revision received March 17, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.