Export from the Endoplasmic Reticulum Represents the Limiting Step in the Maturation and Cell Surface Expression of the Human δ Opioid Receptor*
- From the ‡Département de Biochimie, Université de Montréal, Montréal, Quebec H3C 3J7, Canada and the §AstraZeneca R & D Montréal, St. Laurent, Quebec H4S 1Z9, Canada
Abstract
Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes that include protein folding, post-translational modifications, and transport through distinct cellular compartments. Relatively little is known about the nature and kinetics of specific steps involved in these processes. Here, the human δ opioid receptor expressed in human embryonic kidney 293S cells is used as a model to delineate these steps and to establish the kinetics of receptor synthesis, glycosylation, and transport. We found that the receptor is synthesized as a core-glycosylatedM r 45,000 precursor that is converted to the fully mature M r 55,000 receptor with a half-time of about 120 min. In addition to trimming and processing of two N-linked oligosaccharides, maturation involves addition of O-glycans containing N-acetylgalactosamine, galactose, and sialic acid. In contrast to N-glycosylation, which is initiated co-translationally and is completed when the protein reaches the trans-Golgi network, O-glycosylation was found to occur only after the receptor exits from the endoplasmic reticulum (ER) and was terminated as early as thetrans-Golgi cisternae. Once the carbohydrates are fully processed and the receptor reaches the trans-Golgi network, it is transported to the cell surface in about 10 min. The exit from the ER was found to be the limiting step in overall processing of the receptor. This indicates that early events in the folding of the receptor are probably rate-limiting and that receptor folding intermediates are retained in the ER until they can adopt the correct conformation. The overall low efficiency of receptor maturation, less than 50% of the precursor being processed to the fully glycosylated protein, further suggests that only a fraction of the synthesized receptors attain properly folded conformation that allows exit from the ER. This indicates that folding and ER export are key events in control of receptor cell surface expression. Whether or not the low efficiency of the ER export is a general feature among G protein-coupled receptors remains to be investigated.
Footnotes
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↵* This work was supported by grants from the Medical Research Council of Canada (to M. B.) and from the Ella and Georg Ehrnrooth Foundation, the Helsinki University Pharmacy, and the Oulu University Scholarship Foundation (to U. P.-R.).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.
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↵¶ Scientist of the Medical Research Council of Canada. To whom correspondence should be addressed: Département de Biochimie, Faculté de Médicine, Université de Montréal, P.O. Box 6128, Succursale Centre-Ville, Montréal, Quebec H3C 3J7, Canada. Tel.: 514-343-6319; Fax: 514-343-2210; E-mail: Bouvier@bcm.umontreal.ca.
- Abbreviations:
- GPCR
-
G protein-coupled receptor
- BFA
-
brefeldin A
- BSA
-
bovine serum albumin
- DDM
-
n-dodecyl-β-d-maltoside: DMEM, Dulbecco's modified Earl's medium
- Endo H
-
endo-β-N-acetylglucosaminidase H
- ER
-
endoplasmic reticulum
- hδOR
-
human δ opioid receptor
- HEK-293S
-
human embryonic kidney 293S
- NHS
-
N-hydroxysuccinimide
- PBS
-
phosphate-buffered saline
- PMSF
-
phenylmethylsulfonyl fluoride
- PNGase F
-
peptide-N-glycosidase F
- PAGE
-
polyacrylamide gel electrophoresis
- SNC-80
-
((+)-4-[(αR)-α-((2S, 5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N, N,-diethylbenzamidin)
- STI
-
soybean trypsin inhibitor
- WGA
-
wheat germ agglutinin
-
- Received November 29, 1999.
- Revision received February 8, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
