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Abstract

Recycling of the mu opioid receptor to the plasma membrane after endocytosis promotes rapid resensitization of signal transduction, whereas targeting of the delta opioid receptor (DOR) to lysosomes causes proteolytic down-regulation. We identified a protein that binds preferentially to the cytoplasmic tail of the DOR as a candidate heterotrimeric GTP-binding protein (G protein)–coupled receptor-associated sorting protein (GASP). Disruption of the DOR-GASP interaction through receptor mutation or overexpression of a dominant negative fragment of GASP inhibited receptor trafficking to lysosomes and promoted recycling. The GASP family of proteins may modulate lysosomal sorting and functional down-regulation of a variety of G protein–coupled receptors.

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REFERENCES AND NOTES

1
Claing A., Laporte S. A., Caron M. G., Lefkowitz R. J., Prog. Neurobiol. 66, 61 (2002).
2
O. B. Goodman Jr. et al., Adv. Pharmacol. 42, 429 (1998).
3
Trejo J., Coughlin S. R., J. Biol. Chem. 274, 2216 (1999).
4
Lefkowitz R. J., Pitcher J., Krueger K., Daaka Y., Adv. Pharmacol. 42, 416 (1998).
5
Gagnon A. W., Kallal L., Benovic J. L., J. Biol. Chem. 273, 6976 (1998).
6
Tsao P. I., von Zastrow M., J. Biol. Chem. 275, 11130 (2000).
7
Cao T. T., Deacon H. W., Reczek D., Bretscher A., von Zastrow M., Nature 401, 286 (1999).
8
J. Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 95, 7157 (1998).
9
Zhang J., et al., J. Rec. Sig. Trans. Res. 19, 1 (1999).
10
Law P. Y., Loh H. H., J. Pharmacol. Exp. Ther. 289, 607 (1999).
11
T. Koch et al., J. Biol. Chem.273, 13652 (1998).
12
Finn A. K., Whistler J. L., Neuron 32, 829 (2001).
13
Evans C. J., Keith D. E., Morrison H., Magendzo K., Edwards R. H., Science 258, 1952 (1992).
14
HEK293 cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Cells were transfected using calcium phosphate coprecipitation. Stably transfected cells were isolated following selection on 0.5% geneticin or 0.2% Zeocin (Invitrogen, Carlsbad, CA).
15
D. L. Kaufman et al., J. Biol. Chem. 270, 15877 (1995).
16
Radioligand binding assays were carried out using 3H-diprenorphine as previously described (6, 12).
17
Intact cells were reacted with disulfide-linked biotin to label receptors in the plasma membrane. After the indicated period of agonist exposure, surface biotin was cleaved by exposure of cells to glutathione and the internalized (glutathione-resistant) pool of receptors was detected in immunoprecipitates. “100%” refers to the biotinylated receptor signal present in cells after initial labeling and without further manipulation. “strip” refers to biotinylated cells that were reacted with glutathione without other manipulations, demonstrating the efficiency with which biotin can be cleaved from surface receptors. Detailed methods are available as supporting materials on Science Online.
18
Whistler J. L., Tsao P., von Zastrow M., J. Biol. Chem. 276, 34331 (2001).
19
Receptors present in the plasma membrane of living cells were labeled with M1 mouse anti-FLAG IgG2b (Sigma) and then surface-labeled cells were incubated at 37°C as described in the materials and methods (17) and were fixed using paraformaldehyde. Labeled receptors were localized relative to LAMP1 and LAMP2 or HA-GASP using appropriate mouse IgG1 antibodies and dual-color fluorescence microscopy.
20
The R DOR was constructed by replacing the 30 COOH-terminal residues of the DOR with the 38 corresponding residues of the MOR.
21
Membrane adenylyl cyclase assays were performed as previously described (9) to measure the ability of opioid receptors to inhibit adenylyl cyclase activity upon agonist rechallenge of a membrane fraction prepared from receptor-expressing cells after the ligand incubations.
22
The COOH-terminal tail of murine DOR-1 (residues 337 to 391) was used to identify interacting clones, and the interacting clones were isolated from a 293 cell-derived cDNA library (Clontech, Palo Alto, CA) using the Gal4-based MATCHMAKER system (Clontech). A total of 2.5 × 106 recombinants were screened.
23
Suyama M., Nagase T., Ohara O., Nucleic Acids Res. 27, 338 (1999).
24
Klein U., Ramirez M. T., Kobilka B. K., von Zastrow M., J. Biol. Chem. 272, 19099 (1997).
25
In vitro translated cGASP bound to GST-tail constructs in the following order: DOR = D MOR » R DOR > MOR > GST. The same order of binding strength was obtained using binding of in vitro–translated full-length GASP.
26
Altschul S. F., et al., Nacl. Acids Res. 25, 3389 (1997).
27
The GASP antibody was prepared commercially by Zymed (South San Francisco, CA) to the 15 COOH-terminal residues of GASP using standard methods.
28
The GASP sequence encodes an acidic protein with a predicted mass of 156.8 kD. In vitro transcription and translation of this clone yielded a protein with an apparent mass of ∼190 kD when resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in tris-glycine buffer and ∼170 kD in Mops buffer. We have not investigated this observation in detail. However, differences between actual and apparent molecular weights estimated by SDS-PAGE are not uncommon. TATA-binding protein, for example, has a predicted molecular weight of 30 kD but electrophoreses with an apparent molecular weight of 45 kD (29).
29
Peterson M. G., Tanese N., Pugh B. F., Tjian R., Science 248, 1625 (1990).
30
cGASP was tagged at the NH2-terminus using pEGFP-C1 (Clontech). Coimmunoprecipitations were performed without crosslinking from lysates prepared using 0.1% Triton X-100; 150 mM NaCl; 25 mM KCl; and 10 mM tris-HCl (pH 7.4) containing 1 μM leupeptin, 1 μM pepstatin A, 1 μM aprotinin, and 2.5 μM Pefabloc SC.
31
Myelin basic protein (MBP)–DOR cytoplasmic tail and MBP-lacZ fusion proteins were expressed and purified on amylose resin. Full-length GASP probe was generated by in vitro transcription and translation as previously described (25) and incubated with 15 μg MBP-fusion protein. For competition, GST-cGASP was bacterially expressed and purified on glutathione agarose, and the purified protein was eluted with glutathione and quantified. Eluted GST-cGASP was added to the MBP-DOR slurry just before addition of in vitrotranslated full-length GASP probe.
32
Cells expressing only endogenous GASP and cells overexpressing GFP-cGASP (∼40 times the level of endogenous GASP as estimated by immunoblotting) were grown to 80% confluency and treated with 5 μM EGF in DMEM for the indicated times or left untreated. Cells were washed with phosphate-buffered saline (PBS), and the EGF receptor was immunoprecipitated with rabbit anti-EGFR–affinity resin (Santa Cruz Biotechnology, Santa Cruz, CA), separated by SDS-PAGE, and immunoblotted with goat antibodies to EGFR (Santa Cruz Biotechnology), followed by horseradish peroxidase (HRP)–conjugated secondary antibody to goat (Jackson Immunoresearch, Malvern, PA) and development with ECL reagents (Amersham, Piscataway, NJ).
33
Heck D. A., Bylund D. B., J. Pharmacol. Exp. Ther. 282, 1219 (1997).
34
Innamorati G., Sadeghi H. M., Tran N. T., Birnbaumer M., Proc. Natl. Acad. Sci. U.S.A. 95, 2222 (1998).
35
von Zastrow M., Kobilka B. K., J. Biol. Chem. 267, 3530 (1992).
36
M. Cong et al., J. Biol. Chem.276, 45145 (2001).
37
Gage R. M., Kim K. A., Cao T. T., von Zastrow M., J. Biol. Chem. 276, 44712 (2001).
38
Hicke L., Trends Cell Biol. 9, 107 (1999).
39
Marchese A., Benovic J.L., J. Biol. Chem. 276, 45509 (2001).
40
Shenoy S. K., McDonald P. H., Kohout T. A., Lefkowitz R. J., Science 294, 1307 (2001).
41
U. E. Petaja-Repo et al., J. Biol. Chem. 276, 4416 (2001).
42
Chaturvedi K., Bandari P., Chinen N., Howells R. D., J. Biol. Chem. 276, 12345 (2001).
43
We thank P. Chu for expert assistance in carrying out the yeast two-hybrid screen; U. Klein for valuable advice on the interaction cloning strategy and for performing initial GST binding assays; P. Tsao for insightful discussion and assistance in early studies of the GASP interaction; G. Vargas for providing the GST-D4 tail construct; and H. Bourne, H. Deacon, R. Edwards, A. Finn, R. Kelly, D. Ron, and M. Waldhoer for helpful discussion and critical reading of the manuscript. Supported by research grants from NIH (M.v.Z.) and funds provided by the state of California for medical research on alcohol and substance abuse (J.L.W.). J.L.W. is supported by a NARSAD young investigator award from the Staglin family and received an individual NRSA from NIH.
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Science
Volume 297 | Issue 5581
26 July 2002

Submission history

Received: 26 April 2002
Accepted: 13 June 2002
Published in print: 26 July 2002

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Authors

Affiliations

Jennifer L. Whistler*
Department of Neurology, Ernest Gallo Clinic and Research Center, University of California, San Francisco, Emeryville, CA 94608, USA.
Johan Enquist
Program in Molecular Medicine, University of Lund, Sweden.
Aaron Marley
Program in Cell Biology, Department of Psychiatry and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA.
Jamie Fong
Department of Neurology, Ernest Gallo Clinic and Research Center, University of California, San Francisco, Emeryville, CA 94608, USA.
Fredrik Gladher
Program in Molecular Medicine, University of Lund, Sweden.
Pamela Tsuruda
Program in Cell Biology, Department of Psychiatry and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA.
Stephen R. Murray
Program in Cell Biology, Department of Psychiatry and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA.
Mark von Zastrow*
Program in Cell Biology, Department of Psychiatry and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA.

Notes

*
To whom correspondence should be addressed. E-mail: [email protected] (J.L.W); [email protected] (M.v.Z.)

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